Studies on the secretion of some digestive enzymes in
certain insects with special reference to feeding.
by.
Mumtaz Ahmed Khan, M.Sc.
A thesis presented for the degree of Ph.D. in
entomology in the University of London.
Department of Zoology,
Imperial College of Science & Technology,
South Kensington,
London, S.W.7., 12th July 1961 ABSTRACT.
1. Newly emerged adult Locusta contains abundant proteinase in the gut lumen but both invertase and proteinase activity are negligible in the midgut and caeca tissue. Later, on ,starvation these enzymes are synthesized endogenously, showing maximal activity within two days after emergence.
Similar variations occur in tissue invertase in 5th instar Locusta hoppers and adult Dysdercus.
2. When Locusta adults are kept on food continuously after emergence, both proteinase and invertase, in the tissue, increase progressively for a few days and fluctuate afterwards. Similar changes have been observed for the invertase activity in adult Dysdercus. In 5th instar Locusta hoppers provided with food from emergence, tissues of midgut and caeca have makimal invertase in the intermoult period and very low quantity in the pre—moult stage.
3. In adult Locusta, starved or fed, proteinase concentration is always higher in the tissue of caeca than that of midgut but reverse is true for invertase in adults unfed from emergence. In the presence of food both tissues have nearly equal concentration of invertase after few days from emergence.
4. Feeding after starvation, in Locusta, stimulates the secretion of invertase in the midgut earlier than in caeca. Conversely proteinase secretion is first stimulated in caeca. Ingestion of water or cellulose powder did not demonstratethese changes. But in Dysdercus production of tissue invertase is stimulated by the intake of water alone. 5. The midgut and caeca epithelium of adult Locusta are histologically uniform without traces of cytoplasmic extrusions at the time of heightened secretory activity.
6. In Dysdercuso invertase is mainly produced in the 1st ventriculus of the midgut whereas amylase is mostly secreted from the salivary glands.
The pH optimum for invertase lies in the acid range while that for amylase is almost a neutral point.
TABLE OF CONTENTS. Page.
INTRODUCTION. 1
MATERIALS AND METHODS 14
PROTEINASE ACTIVITY IN LOCUSTA MIGRATORIA MIGRATOIDES.
(i) Serial experiments to relate proteinase activity
to colorimeter readings 20
(ii) Variation of proteinase activity in the whole
gut extract (except hindgut) of adult Locusta
migratoria migratoides, unfed from emergence 21
(iii) Variation in proteinase activity in crop,
midgut and caeca of adult Locusts migratoria
migratoides, unfed from emergence 24
(iv) Proteinase concentration in 5th instar feeding
hoppers, 5th instar non—feding (pre—moult)
hoppers and newly emerged adults of Locusta
migratoria migratoides 27
(v) Variation of proteinase activity in the midgut
and caeca tissue of adult Locusts, unfed and fed
from emergence 30
(vi) Effect of feeding on the secretion of proteinase
in adult Locusts m.m 33
INVERTASE ACTIVITY IN LOCUSTA MIGRATORIA MIGRATOIDES.
(i) Invertase activity in serial dilutions of midgut
tissue extracts of Locusts migratoria migratoides. 45 Page.
(ii) Variation in invertase activity and weight of midgut
and caeca tissue of 5th instar hoppers, unfed and
fed after moult 47
(iii)Effect of feeding on the weight and the invertase
activity in the midgut and caeca tissue of 5th instar
hoppers of Locusta, unfed for 3 days following moult 51
(iv) Variation in invertase activity and tissue weight in
the midgut and caeca tissue of adult Locusta, unfed and
fed following emergence 54
(v) Effect of feeding on the weight and invertase activity
in the midgut and caeca tissue of Locusta 58
(vi) Possible hormonal control of enzyme secretion 65
HISTOLOGICAL OBSERVATIONS ON THE MIDGUT AND CAECA OF LOCUSTA 72 MIGRATORIA MIGRPTOIDES
OBSERVATIONS ON THE DIGESTIV NZYMES OF DYSDEP.CUS FASCIATUS.
(i) pH optima for amylase and invertase activity in the 88 midgut extracts of adult Dysdercus fesciatus
(ii) Distribution of invertase and amylase in the midgut
tissue of adult Dysdercus fasciatus 93
(iii) Distribution of invertase and amylase in the salivary
gland of adult Dysdercus fasciatus 96
(iv) Invertase activity in serial dilutions of midgut
extract of adult Dysdercus fasciatus.. ... 100
(v) Variation in the invertase activity and weight of 1st ventriculus tissue of adult Dysdercus fasciatus, unfed
and fed following emergence 103 Page.
(vi) Effect of feeding on the weight and invertase activity
of 1st ventriculuc tissue of adult Dysdercus fasciatus 107
DIPEPTIDASE ACTIVITY IN LOCUSTP. AND DYSDERCUS 111
DISCUSSION 118
SUMMARY 134
REFERENCES 139
APPENDIX (Experimental data) ACKNOWLEDGMENTS.
I wish to express my gratitude to Professor O.W.Richards, F.R.S.
for providing me facilitics to work in this department and for his valuable
suggestions. I am highly indebted to Dr.D.R.P.Murray for his instructions
on biochemical techniques and most encouraging supervision throughout the
progress of this work. My sincere thanks are due to Dr.R.H.Dadd, Mr.R.G.
Davies and-Dr.N.Waloff for their valuable discussions, criticisms and
suggestions on various aspects of this problem. I am also thankful to
Mr.C.Meredith for his assistance in photography.
It is my pleasant duty to acknowledge the facilities provided by
the Anti—Locust Research Centre, London, and I am very grateful to Mr.P.
Hunter—Jones and his colleagues who always made available large stocks of
Locusta stages.
Finally* I record my sincere thanks to Aligarh Muslim University,
Aligarh, India (sponsoring authority) and the Ministry of Scientific Research
and Cultural Affairs, Government of India, New Delhi, for the award of a
Central Overseas Scholarship which made this study possible. 1.
INTRODUCTION.
Our knowledge of the physiology of digestion in insects is based on very fragmentary information, when contrasted with the position in mammals, which have been studied very extensively. Although vertebrate physiology and especially mammalian physiology has provided ample technique, the knowledge of digestion in insects made very little progress in the past.
Most of those investigations on insects include the identification of digestive enzymes, their characteristics, rate of food passage through the digestive tract end the pH conditions'in the regions of the gut. Uvarov
(1928) gave a comprehensive summary of various digestive enzymes observed in a variety of insects. Further advancement on the physiology of the digestive system with regard to Acrididae was also reviewed by Uvarov (1948).
More information is available on many aspects of digestive physiology of insects from the text book of Roeder (1953) and Wigglesworth (1953). Later progress on digestion in insects has been reviewed by Waterhouse (1957).
However, a detailed study on the secretion of digestive enzymes in relation to feeding was first undertaken by Schlottke (1937 a, b & c).
For this prupose he selected carnivorous Carabus species, two species of phytophagous grasshoppers, Stenobothrus and Tettigonia; a purely predatory grasshopper Decticus and omnivorous Periplaneta orieritalis. His obsery— . ations showed thit the enzymes occurring in highest concentration in the digestive juice of an insect are the ones which digest the substrates occurring in highest proportion in the normal food. Thus in the digestive juice of carnivorous carabids and predatory Decticus, the concentration of 2. proteolytic enzymes is higher than that of carbohydrases. On the other hand in phytophagous grasshoppers carbohydrates ere more dominant than
other enzymes. In omnivorous cockroaches the concentration of proteinase,
dipeptidase, lipase, amylase and also aminopolypeptidase, carboxypolypeptidase,
lichenase and Maltase are relatively less than in the other insects studied
by him.
The effect of feeding starved insects was always an initial
increase of enzyme concentration in the gut but as digestion proceeded
further the enzyme activity decreased. In carabids after the intake of
food, proteinase was pushed forward from the mid-gut to the crop which was
regarded as the main seat of proteolytic activity. Proteinase was also
emptied from the caeca into the crop in Decticus following feeding. The
stimulation of the secretion of digestive enzymes was also noted in phyto-
phagous grasshoppers following feeding. Similar results were also obtained
in Periplaneta in which the enzyme concentration increased in the crop and
most of the digestion probably occurred in this region. The experiments on
feeding different diets to cockroaches did not result in any alteration of
the relative concentration in the components of digestive juice in response
to the nature of diet.
A correlation between enzyme secretion and feeding both histologic-
ally and chemically was established by Duspiva (1939) in Dytiscus marginalis.
According to him feeding in starved adults leads in one minute to the mid-
gut epithelial cells discharging tryptase in the form of cytoplasmic globules
which are passed on to the crop where maximal enzyne concentration is recorded. Two hours after feeding the mid-gut tissue had only 1/12 of 3. enzyme before feeding. These observations led him to suggest that the mid— gut epithelial cells of Dytiscus were polyphasic involving secretory, restitution, absorption and resting phases.
Day and Powning (1949) investigated a number of factors involved in the process of digestion in insects. For this purpose they selected two species of cockroaches, Blattella germanica and Periplaneta americana but Tenebrio molifor was also used in certain experiments. They concluded that enzyme production is reduced on starvation. But subsequent feeding stimulates enzyme synthesis. The observations of Day and Powning further supports Schlottkels (1937 c) result that cockroaches are unable to vary the proportions of the components of digestive juice according to the chemical nature of the diet provided. The decrease in enzyme activity following feeding on its substrate, and later on very slow recovery to normal secretory condition, also confirmed Schlottke's result on Periplaneta.
The histological observations on the mid—gut and caeca of Blattella at different levels of proteinase and amylase secretion in relation to starvation and feeding revealed that the epithelium was cytologically uniform at the time of greatest secretory activity. The so—called merocrine secretion was attributable to different degrees of cell breakdown due to starvation.
Day and Powning also analysed the causes of immediate stimulus to secretion of digestive enzymes in an attempt to explain the mechanism of secretion in insects. Although they had some evidence in favour of secretogogue mechanism for the stimulation of digestive enzyme secretion they rejected this idea on the grounds that the stimulus originates first in 4. the caeca of Pcriplaneta (from Schlottke, 1937 c). Since in this species caeca are ill-fitted for a rapid diffusion of food material from the mid-gut therefore a secretogogue cannot explain the immediate stimulus for secretion in caeca. An application of special histological techniques could not demonstrate the innervation of epithelia of mid-gut and caeca in either
Slattella or Periplaneta. Thus a neural mechanism, involved in the secretion of digestive enzymes, was thought to be doubtful, a conclusion also supported by the lag of time between feeding and the response for secretion. In the absence of strong evidence for neural or secretogogue mechanism Day and ?owning resorted to the possibility of a hormonal factor in the haemolymph of cockroaches. The experiments were based on the criterion that increased rate of mitosis in the nidi was correlated to the. enzyme production. Periplaneta proved an unsuitable insect for such a study, since the regenerative cells are not conveniently distributed, and
Tenebrio was substituted since the regenerative cells are grouped in nidi in crypts of the mid-gut. The injection of blood from fed Tenebrio to the starved ones demonstrated a small increase in the number of mitoses in the recipients, within half an hour following injection. Later the rate of mitosis was one per hour for the next 4 hours. Blood from a starved adult did not cause such a change in starved recipients. These observations led them to suggest that a factor existed in the blood of normally feeding
Tenebrio to which the name "Mid-gut regeneration stimulating factor" (M.R.S.F.) was applied.
A survey of the various possible sources of "M.R.S.F." was also 5. discussed by Day and Powning. They considered corpora allata and corpora cardiaca involved in the production of this factor. In Blattella the mid— gut epithelium is completely renewed during moulting and metamorphosis, hence there is great increase in the mitosis of regenerative cells; the corpora allata, at that time believed to be central gland controlling moulting, might thus also control these mitoses and thereby the secretion of enzyme.
In the absence of a relationship between the secretory condition of corpora cardiaca and the feeding condition of Periplaneta, the possibility of
N.R.S.F." was related to Wigglesworth's (1948) observation that a hormone was involved in rapid digestion of food and maturation of ovary in female
Rhodnius prolixus.
But alternative hypothesis that "M.R.S.F." originated from either the salivary glands or the mid—gut epithelial cells were considered; supporting evidence for the latter is nil, for the former is based on the innervation and the existence of two types of cells in the salivary glands.
Fisk (1950) working on female Andes aegypti mosquitoes observed that the unfed females, when offered a partial blood meal developed a signif— icant increase in the proteolytic activity of the mid—gut. He tried to discover the possibilities of the presence of activators or precursors of this enzyme from the crop and the salivary glands of unfed mosquitoes but all these attempts gave negative results. Females fed on 5% sucrose solution did not show any significant increase. It was, therefore,suggested that the presence of blood in the stomach might be the stimulusoroteolyticproteolytic activity in female Aides. Using the determination of amino nitrogen content of whole mosquitoes at different intervals following a blood meal, and assuming the 6.
increase of amino nitrogen concentration Cs a consequence of digestion, it was shown that proteolytic activity commenced after 3 hours and thereafter followeo a uniform rate of increase for about 24 hours. The probable reason for this notable lag of time to get the increased proteolytic activity was suggested as due to autocetalysis of the secreted trypsinogen to increase trypsin concentration similar to the process known in mammals.
A more comprehensive work was done by Fisk and Shambaugh (1952) on the protease activity in adult Ades aegypti mosquitoes as related to feeding. The blood—starved female mosquitoes had a residual value of protease activity but if they were fed on a brief blood meal there was an immediate decrease in protease activity, below the residual value, which was followed after some time by a great increase with maximal activity after
18 hours but still evident at 48 hours after feeding. When unfed females were offered 5;',, sucrose solution the protease activity was moderately enhanced during 30 minutes following feeding but declined later and slowed down to the residual value after 4 hours. As regards these two different food stuffs it was noted that a blood meal directly reached the mid—gut whereas sugar solution entered the ventral diverticulum of the mid—gut.
These observations conclude that the stimulus to enzyme secretion was more likely based upon the full mid—gut condition than the effect of blood as a meal. The decrease of protease activity just after the ingestion of blood meal was considered due to the excess of substrate which caused depletion of enzyme as noted by Day and Pawning (1949) in cockroaches.
Fisk and Shambaugh also thought that the immediate stimulus for 7. the secretion of proteolytic enzyme in mosquitoes might be a factor in the haemolymph rather than a neural or secretogogue mechanism. But they obtained negative results by blood injection experiments and thus concluded that a secretogogue was more probable than hormonal factor involved to influence protease secretion.
Based upon the above conclusion Shambaugh (1954) analysed further the possibility of a secretogogue mechanism in relation to protease secretion in female Aedes aegypti. For this purpose protease activity was estimated following the feeding of separate blood fractions as well as their combinations.
It was observed that although the sheep erythrocytes in physiological saline stimulated protease secretion slightly, the addition of dialyzable plasma protein into the erythrocyte solution caused further tecretion. %ben dialyzable plasma protein was substituted by non—dialyzable plasma protein the of effect was more than double4that caused by the former fraction. An equal concentration of the three plasma proteins, viz. fibrinogen, albumin and gamma globulin, were separately fed and it was found that albumin stimulated the secretion of protease more than fibrinogen but gamma globulin had greater effect than the sum of the activity caused by the other two fractions. The mixture of these plasma proteins had greater enzyme increase than any of these fractions fed separately but not higher than the sum effect of' individual fractions. These results were considered very conclusive to support the secretogogue hypothesis suggested earlier (Fisk and Shambaugh,
1952). Shambaugh also pointed out a correlation between the amount of blood ingested by the females and the subsequent protease activity of their mid— guts, suggesting a graded stimulus for secretion. 6.
Latee, Fiek and Shomhzu,;.h (1951) observed that in the yut of female mosquitoes adcs eegypti„ invereese activity was very little stimulated by feeding on its proper substrate, viz. cane sugar, by comparison with the effect of the normal blood diet. Therefore, in conjunction with their previous result, that proteese activity Was greatly increased by its proper substrate, i.e. blood and less affected by sugar solution, they concluded that both invertese and protease were probably stimulated by E similar factor present in the blood meal. This idea strengthened the Leecretogogue hypo— thesis' suggested by them earlier (Fisk & Shembaugh, 1952).
Dadd (1954} undertook a detailed investigation on the proteolytic activity in the mid—gut of Tenebrio molitor in relation to feeding. The possibility of an endogenous rhythm in secretion related to age and develop— ment of an insect, hitherto unattempted by chemicel methods, was first examined by him in Tenebrio. Newly emerged Tenehrio adults hod practically no protease activity in the total mid—yuL extracts. On subsequent starvation some protease was built up which showed maximal activity on 5th or 6th post— emergence dey and them decreased until death. Eut the reLL of enzyme production decreased on 4th or 5th day following emergencelwhich was observed to coincide with the normal resumption of feeding. This endogenously initiated enzyme synthesis was regarded as one of the pre—requisites to active adult life. Protease activity remained unaffected by feeding at the time of maXimal activity developed endogenously (between 5th and 6th post—emergence day) but later enzyme activity mae:edly increased following feeding. This 9. change was also noticeable by feeding the starved adults on moist cellulose powder or water, suggesting that the stimulus for enzyme secretion was mechanical rather than chemical.
On the hypothesis developed by Duspiva (1939) in Dytiscus and
Needham (1E97) in ()donate nymphs (both discontinuous feeders) that digestive enzyme mainly accumulated in the tissue and discharged into the gut after feeding, Dadd studied the protease activity of mid—gut tissue of unfed adult
Tenebrio at a different ages. At no stage during his experiments could he find the storage of protease in the tissue equal to the total protease of the mid—gut. This led him to suggest that in Tenebrio the protease was in a state of constant discharge from the tissue to the mid—gut lumen, and the increased protease activity observed in the tissue might be regarded as an index of the ret of enzyme synthesis.
Discussing the relationship between the weight and the protease activity in the mid—gut of adult Tenebrio, Dadd pointed out that on starvation following emergence the increasing protease activity of the total mid—gut extract was not correlated with similar change in the total mid—gut weight during the first five post—emergence days. It was thought that the enzyme was prdbably secreted in a very concentrated form into a pre—existent inert
"carrier" in the gut lumen and while the carrier would gradually increase in activity its weight would remain almost unchanged. But during the continued starvation after 6th post—emergence day, although the tissue activity was still high total protease activity followed a decrease parallel with the loss of total mid—gut weight, probably caused by evacuation of gut contents. Similar correlation was obtained when adult beetles, starved for 10.
9 or 14 days following emergence, were fed on flour.
However, the variations in mid—gut tissue weight and corresponding
tissue protease activity in unfed Tenebrio were parallel following emergence.
Both tissue weight and tissue protease rose together and this was followed by
gradual decline. The possible reason for the increase of tissue weight was
suggested to be in accordance with Day's (1949) observation, that secretion
would be accompanied with mitotic acceleration in the epithelium which would result in an increase of epithelial bulk. From the available data, increase
in tissue weight at a time when total mid—gut weight was almost constant
(during first five post—emergence days) could not be correspondingly related
with the expected decrease in the weight of mid—gut contents.
In contrast to the adult Tenebrio, newly moulted larvae possessed
considerable protease activity in the whole mid—gut extract. It was ascribed to the partial carry—over of residual gut contents from the previous inster. Some protease was secreted within 24 hours following moulting in unfed larvae. Further, when the larvae were subjected to continuous starvation, protease activity remained almost unaffected for about 4 days end then later (between 5th and 6th post—moult days) it had a second peak of maximal activity which declined subsequently. The second peak of protease activity was observed to coincide with a time shortly before the resumption of normal feeding following a moult. Vhen larvae were provided with food from the moult, a linear relationship was observed between the mid—gut weight and the corresponding protease activity. An abrupt decrease in protease activity was observed at the end of the inster in preparation to next moult which was accompanied by empty gut. 11.
ts i susidiaoe e"-oey ye-exetlinee, a part of Duspivals (1939) result (mostly M.rtologicel cl'servations) on preteolytic activity in the gut of the carnivorous beetle, Zytiscus marginalis (a discontinuous feeder) in relation to feeding. In beetles starved for a sufficiently long time, proteese wes accumuleeted in the mid-gut tissue. Immediately after feeding enzyme activity ties greatly reduced in the tissue and after an hour both mid-gut tissue and lumen had almost negligible protease activity. On the other hand crop contained 90% of the total Protease activity of the gut.
Tissue protease was again produced within 3 hours after feeding but did not accumulate in the tissue as long as food was retained in the crop. When
the gut was completely empty protease was stored in the tissue and the crop ceased to receive enzyme from the mid-gut. This demonstrated a mechanism of secretion different from that of Tenebrio, a continuous feeder.
Pedd made an attempt to demonstrate the immediate cause of endo- genously initiated' post-emergence secretion in Tenebrio. blood injected into starved beetles from fed beetles shored an enhancement of protease activity and this occurred lehether the vied,' beetles had received flour or cellulose powder. His observations by ligaturing the pupae one day before the emergence and later 1, 2 and 3 days after the emergence of adult and then measuring the protease activity over a period of 3 to 14 days proved that some factor in the head influenced the secretion of protease after emergence. It was further demonstroted that the immediate cause of enzyme production following emergence wns a 'olcod factor which was active in the presence of head. But its source of origin was not discovered. The endocrine organs, brain, neuro-secretory cells and the pro-thoracic gland involved in the control of metamo::phosis, were considered as possibly controll—
ing the secretion of digestive enzymes in relation to moulting.
The foregoing reeumt reveals that although feeding stimulates the
secretion of digestive enzymes, the mechanism by which it does so depends upon the feeding behaviour of the insects. Moreover the secretory activity
of mid—gut appears to vary according to age and developmental stages in a
continuous feeder like Tenebrio which undergoes complete metamorphosis. It
is very well known that insects present a variety of anatomical structure of
alimentary tract, different feeding behavior and also difference in the
control of growth and metamorphosis. These factors may be involved to
affect the physiology of digtstion and may be responsible for known or still unknown modes of digestion in insects. Thus detailed studies, on various
aspects of digestive physiology in a variety of insect species, would be useful to formulate a theory on digestion in insects. In this context
Locuste migratoria migratoides was selected as a representative type of hemimetabolous insect with phytophagous and continuous feeding habits. The main line of work was proposed to examine the secretion of some digestive enzymes in relation to feeding and non—feeding conditions. It was expected
that information available from the observations on Locusta might resemble those of Tenebrio in many respects.
As a subsidiary insect Dysdercus fasciatus was also included which respresented a sap sucking phytophagous insect with almost continuous feeding habit. In Dysdercus in addition to the information on enzyme secretion in 13.
relation to feeding certain preliminary experiments also contributed some useful observation on its digestive physiology which were not recorded before.
Throughout the present investigation insects have keen taken singly and the enzyme activities measured, thus concentrating attention on individual variations, whether or not a reason for such variation was known. Useful correlations with other physiological observations have thereby been obtained specially in the case of Dysdercus. 14.
MATERIALS AND METHODS.
A stock of 5th instar hoppers of Locusta migratoria migratoides
was obtained at frequent intervals from the Anti—Locust Research Centre.
These hoppers were subsequently kept in a 31 x 31 x 31 cage with continuous
illumination by a 60 watt bulb, fixed in the centre of its roof. This
cage was kept in a constant temperature room at 28 ± 1°C. and a relative humidity 50 I 5%, also continuously illuminated. The temperature inside
the cage was approximately 2°C. higher than the room temperature. These hoppers were fed on wheat bran which was always present in the cage. Some
green grass (obtained from the Anti—Locust Research Centre) was also provided twice a day in morning and evening only. Newly emerged adults
were sorted out several times during 24 hours,10 a.m. to 10 a.m. Subsequent references to age are based on the definition that zero age counted from
the end of the 24 hour period. For experiments on unfed adults, each
individual was isolated in a glass jar (with 4 lb. capacity) covered with a
lid of which the centre had been cut out and replaced by a plastic mesh.
Adults feeding continuously following emergence, were kept in a Watkins and
Doncaster cage in a group of 12 to 14 individuals. These cages were cleaned
on alternate days.
Later, due to increased requirement of Locusta hoppers for the
investigation of invertase activity, 10 to 15 egg pods, almost ready to hatch, were obtained from the Anti—Locust Research centre every week. The newly hatched hoppers were maintained in a constant temperature room with
conditions approximately similar to those at the above mentioned centre.
The method of breeding was that of Hunter—Jones (1956). From this culture 15.
5th instar hoppers were transferred to the same constant temperature room as
used for experimental adults in the determination of proteinase activity.
For the experiments on 5th instar hoppers, recently moulted 4th instar hoppers
were also transferred to the above mentioned conditions. Then newly moulted
5th instar hoppers were sampled for different experimental groups similar to
the newly emerged adults.
For the studies on Dysdercus fasciatus, a stock of last instar
nymphs was obtained from the insectary of Imperial College Field Station,
Silwood Park, Berks. These nymphs were then placed in glass jars (with
7 lb. capacity) with a damp peat at the bottom, in the above mentioned const—
ant temperature room. These were fed on germinating cotton seeds provided
on alternate days. Newly emerged adults were sorted out from the main
culture and aged in the same way as for Locusts. Unfed adults and adults
continuously fed following emergence were kept in separate glass jars (with
4 lb. capacity) without damp peat. The continuously fed insects were
provided with fresh stock of germinating cotton seeds every day.
Enzyme determinations.
In adult Locusta proteinase activity was determined in the extracts
of total gut (excluding hindgut), whole midgut and caeca with their contents,
crop with contents, midgut and caeca tissue. For dissection,the appendages, head and the posterior end of the abdomen of a live insect were cut off.
Then the entire digestive tract was pulled out from the posterior end and the adhering tissue and malpighian tubules were removed. According to the nature of the experiment the entire gut or its separate regions were weighed on an Oertling balance recording to the nearest milligramme. The 16. entire gut or its region was homoganized singly in a hard glass tube measuring
9 X 1.5 cm. with a little quantity of acid washed sand by crushing the tissue with the rounded end of a stout glass rod and then 4 ml. of a phosphate buffer solution adjusted to pH 8.0 was added in each. tube. Since Powning et al. (1951) observed that an optimal pH range was between 8.0 to 8.5 for the proteinase activity in Locusta therefore phosphate buffer at pH 8.0 was considered suitable for the investigation of proteinase activity. The extraction tubes containing the gut portions or their extracts were placed in a small ice bath to minimise deterioration of the activity of enzyme.
Four at a time of these tubes were centrifuged at a speed of 1,000 r.p.m. for 15 minutes. This time and speed was sufficient to get a clear super— natant.
For the midgut and caeca tissue extracts, the dissected gut was transected into midgut and caeca regions which were slit open longitudinally and washed rapidly into Ringer's solution to remove the gut contents. Then each tissue was put on a filter paper for E. little while to get rid of any adhering solution from its surface and weighed on the Oertling balance to the nearest milligramme. Finally each tissue was extracted separately in
1 ml. phosphate buffer with a pinch of Kieselgurh in an extraction tube measuring 7.5 x 0.7 cm. Twelve such tubes at a time were centrifuged for the same duration and speed as extracts of total gut. These tubes either with the tissue or with the extracts were also placed in a small ice—bath.
The proteinase activity was measured quantitatively by the colorimetric method of Charney and Tomarelli (1947) employed for the determination of proteolytic activity in the duodenal juice and later 17. modified by Tomarelli et al. (1949). According to this method a solution of a red protein, sulphanilic acid azo—albumin was prepared, but, since a freeze—drying plant was not available, only small quantities at a time were made and stored in solution in a refrigerator at 40C. To avoid the deterioration of the substrate the solution was exposed to room temperature only for a minimum possible time and in this way it was found quite reliable to use for a week or so.
From each extract a sample of 0.5 ml. was mixed with 2.0 ml. of substrate, sulphanilic acid azo—albumin and then incubated et 37°C. The incubation period was only an hour for samples obtained from total gut extracts or from the gut regions with their contents. Whereas the samples from the tissue extracts were incubated for 24 hours because a period less than this time was not enough to get measurable activity. A reagent control containing 0.5 ml. phosphate buffer and 2 ml. substrate was also incubated with the other samples. After incubation, undigested protein was precipit— ated by adding 6 ml. of 51 Trichloroacetic acid solution in each sample including the reagent control. These samples were then filtered and 5 ml. of each filtrate was thoroughly mixed with equal quantity of 0.5 N Na off in a tube of E.E.L. Photoelectric colorimeter to develop the colour.
The colorimeter was adjusted to read zero for the final solution of the reagent control with a blue filter and then the colour density of different samples was compared in terms of colorimeter scale divisions. These divisions were taken to represent proteinase activity of different samples.
For this purpose a dilution curve was obtained before the assessment of proteinase activity in samples of different experimental insects. The le. procedure for a dilution cuive is explained in the section of proteinase activity in Locust, m. m.
The invcrtase activity was studied in the nicyut and caeca tissue of 5th instar hoppers and adult Locusta. The tissue was cleared and washed of the gut contents and then weighed as described before. Each tissue was extracted in the same way.as for the protainase activity except that the phosphate buffer was pH 6.0. The quantitative determination of invertase activity was based on the coloeimetric method used by Sumner (1925) to detect sugars in urine. Walker and heisinger (1933) have further demonstrated the use of Sumner's reagent (3, 5—dinitrosalicylic acid) for the detection of as low as one microgram reducing sugars in a 0.2 ul. sample of glomerular urine. • A sample of 0.25 ml. was obtained from each extract and mixed with
2 ml. of 5;; sucrose solution as substrate and then incubated at 40°C. for one hour. A reagent control containing 0.25 ml. phosphate buffer pH 6.0 and
2 ml. of substrate solution was also incubated. Immediately after incubation enzyme reaction was stopped by adding 3 ml. of Dinitrosalicylic acid reagent and by heating the samples for 5 minutes in boiling water. Then these samples were cooled down in running cold water for half an hour. Aliquot of 1 ml. from each sample was diluted to 10 ml. by distilled water. The diluted solution of reagent control was used to adjust the colorimeter at zero by using a green filter and the colour density of other samples was recorded in terms of colorimeter scale divisions. A dilution curve for invertase activity has been included in the section of invertase activity on
Locusta.
In Dysdercus fasciatus also the midgut was dissected out from a 19.
live insect. The 1st ventriculus was transected from other parts and slit
open to wash away the contents, then dried briefly on a filter paper and
weighed. Each tissue was extracted in 1 ml. of phosphate buffer at pH
6.0. From each extract 0.5 ml. was used as a sample. Each sample was
given 2 ml. of 5' sucrose solution as substrate and incubated at 400C. for 3
hours. Later, the rest of the procedures was similar to that described for
Locusta. The dilution curve for invertase activity in the 1st ventriculus
is mentioned in the relevant section.
The above mentioned method and procedure was fundamental to all
experiments. Wherever special methods have been used, reference has been
made in the relevant experiments. The data was collected from sufficiently
large numbers of individuals using mostly 4 to 6 individuals from different (t3441 experimental groups4at a time. Although, sometimes, great variations
occurred in the enzyme activity between the individuals of the same experim—
ental group, yet between individuals in different experimental groups, as
well as between mean values for such groups, marked differences occurred
which were regarded as significant. On account of the variations in an
experimental group standard error of its mean value was calculated by using
the following equation.
Standard deviation of the individual Standard error = x 2 Square root of number of observations
The figures of standard error are represented with the sign of +.
Since the data was mostly recorded in whole numbers, all the figures of mean
values and the standard errors were rounded off to the next higher or
preceeding lower whole number if the fractions were higher or lower than 0.5 respectively. 20.
PROT:HUSE ACTIVIT7 IN LOCUSTA nIcT?AronIA 7aCRATOIDES.
i) Serial experiments to relate proteinase activity to colorimeter readings.
The quantitative determination of proteinase activity by the
colorimetric method required calibration checking of the E.E.L. Photoelectric
colorimeter under standardized conditions. For this purpose considerably
strong extract of whole midget and caeca of Locusta was serially diluted into
a number of samples and their activity was recorded in colorimeter units.
From each concentrated extract two samples of 0.5 ml. each were
obtained. One of them was serially diluted to a number of samples: thus
the last solution was 1/32 of the strength of the 1st sample. Therefore
their enzyme activity would be expected to maintain the same ratio with each
other. A reagent control consisting of 0.5 ml. buffer and 2.0 ml. distilled
water was also included. The colour was developed as described before.
The colour of each sample was compared with respect to the reagent control.
This experiment was repeated with different preparations of the enzyme
extract to get more data for conclusion.
The data mentioned in Table 1 shows that the extract concentration
and colorimeter units fairly correspond up—to 80 units (colorimeter scale
divisions). Moreover in weaker dilutions, as low as 2 units mean
significant difference in concentration and curve is almost a straight line
(Fig.1). But in higher concentrations representing more than 40 units a
fluctuation of 2 to 5 units is possible for the same concentration. It was therefore resolved to ecord the colorimeter units as direct measure of ft+zfrat+Lta trA":"4•44- 14/^A proteinase activity/provided they did not exceed 80 scale divisions and to FIG. 1
PROTEINASE DILUTION C UR VES FOR LOCUSTA
12 4 8 16 32 EX TRACT CONCENTRATION 21. assess the comparative values based upon the present data.
Table 1. Proteinase activity in serial dilutions of total gut extracts
of Locusta migratoria migratoides.
Treatment Proteinase activity in colorimeter units No. Relative enzyme concentrations in serial dilutions
1 2 4 8 16 32
1 3.5 7.0 14.5 29.0 58.0 110.0 2 0.0 0.5 2.0 4.5 10.0 20.0
3 1.0 3.0 6.0 12.0 24.0 53.0
4 1.5 3.0 6.0 12.0 22.5 48.0 5 2.6 5.0 10.0 20.0 40.0 78.0 6 2.5 5.0 10.0 20.0 39.0 76.0 7 2.0 4.0 8.0 16.0 33.0 60.0 8 3.0 6.0 12.0 26.0 57.0 90.0
ii) Variation of Proteinase activity in the whole gut extract (except h4ndgut) of adult Locusta migratoria migratoides, unfed from emergence.
Total proteinase activity in the gut extract of Locusta was studied to observe the changes in secretion due to starvation from emergence. Total gut except the hindgut was used to include also the digestive juice very often observed in the crop. As the rate of mortality in unfed adults was quite high between 6 to 9 days after emergence, therefore from these age groups only healthy and active adults were used for extracts. At a time 4 to 6 adults from each age group were included in one treatment. 22.
The individual de to for verieua t.cje groups is givsn in the
respective experimental categories and Tel:le ' represents total mean values
for either sex. The individual data eithin the same ege group show
.appreciable variations e.re more mired in femeles then the males. The
comparison of total gut v:eight and the corrseonding preteinasc activity
either within the same age group or from different age groups does not show
any correlation between the tsio. floth gut weight and the proteinase
activity are generally higher in female then male. The total mean value
for proteinase activity in hoth sexes goes down during the first three post—emergent days and then temporarily enhances, continuing longer in
females, and finally then gradually decreases later. rut the total mean
of the gut '.-:eight increases on the following day after emergence and there—
after gradually falls down (Fig.2). The initial decrease of proteinase
may be accounted for the loss of enzyme during the exit of accumulated
products during moulting and possibly some digestive juice is also pushed
out when inhaled air, during moulting, is expelled out after emergence.
This idea can be supported from the observation that all individuals had no
tir on the following day after emergence end that during the first three
days the glass containers of unfed adults had thread—like mass of excreta
often with dark brown spots. On exemination the excreta as found to
contain peritrophic membrane and larval intima of the crop. Sometimes
loss of digestive juice nigh also occur due to vomiting when the insect
was caught for dissection but this was avoided as far as possible by pipetting
the vomited drops from the gnathel appendages to the extraction tube.
The temporary increase of the total gut weight is due to the
23.
Table 2. Mean value showing variation in weight and proteinase activity
in the whole gut (except hindgut) of Locusta migratoria migratoides.
Days after Both Semis Males Females Experimental Emergence Wt. Proteinase Wt. Proteinase Wt. Proteinase Category (mg.) units (mg.) units (mg.) units
0 79 47 74 43 94 58 1 + 7 + 6 + 6 4. 6 +_ 9 +_ 4 1 97 31 81 30 114 32 2 9 _ 9 5± 5 + 8 ±6 ±8 ...t 2 92 20 77 20 108 20 3 + + + +_ 9 _ 4 .+ 8 _ 6 — 9 +_ 4 3 81 19 73 20 96 19 4 7 + +_ 3 + 3 ±4 +10 ±6 4 77 31 68 30 89 32 5 + + ! 7 4. 6 _ 6 _ 6 ±12 ±12 5 66 27 59 26 90 31 6 ±10 + 6 +— 7 I: 5 ±23 ±17 6 66 30 57 26 76 36 7 3 +— 7 1.10 + + 7 +12 ±17 7 67 29 50 19 76 34 8 + + +_ 9 + 6 + 8 _ 8 —17 +_ 4 8 60 16 51 10 74 25 9 3 + +± 8 + + 5 +_ 3 ±12 _ a 9 59 14 55 10 63 17 10 +— 5 ± 3 + 5 + 3 + 8 + 3 FIG. 2 VARIATION IN WEIGHT AND PROTEINASE ACTIVITY IN THE TOTAL GUT (EXCLUDING HINDGUT) OF LOCUSTA, UNFED FROM EMERGENCE. so 120 ._s 4...... proteinase activity in male —o—o— „ female 70 ...0—_0--weight of gut in male 110 I. .. I. lemale
60 . 100 ui • • S 50 \ 0 so IT o ••••••.... N 0
SE U 40 .80 ra A 5 TEIN 30 70 0 PRO
20 .60
10 50
1 2 3 4 5 6 7 8 9 AGE ( DAYS) AFTER EMERGENCE
24.
similar increase in thole caeca es observed later. The effect of prolonged
starvation on the midgut and caeca is externally marked by the shrinkage
in their shape and size, so much so, that the posterior caeca are reduced to
very small knobs, and their lumen contains dark brown paste instead of brown
juice. This suggests some chemical changes, in the juicy contents of the
gut, which result in either some enzyme inhibitor or enzyme destruction.
In contrast to Tenebrio adult (Dadd, 1954) Locusta adults are
provided with enzyme ready for its use shortly after emergence.
iii) Variation in proteinase activity in crop, midgut and caeca of adult
Locusta migratoria migratoides, unfed from emergence.
A preliminary observation on the digestive tract of starved Locusts
showed the presence of some amylase, invertase and proteinase in the contents
of midgutp caeca as well as crop. It was resolved to investigate the
quantitative distribution of proteinase in the regions of the gut. This
was studied in adults unfed from emergence to avoid any source of enzyme
from the salivary gland and also to observe the regional variations which
might occur due to post—emergence starvation.
From groups of adults 1 to 5 days old, 3 to 6 were used at a time.
Their digestive tracts were transected at the cardiac and pyloric ends of
the midgut and the entire group of caeca was separated from the midgut wall.
Crop, midgut and caeca of individual adult were separately weighed and
extracted. Newly emerged adults were found to have air bubbles in the
lumen of the caeca and midgut. The individual data is given in appropriate
category. Table 3 shows the total mean value for both sexes as well as
males and females separately. 25.
Proteinase activity is present in the extracts of all three regions. The caeca hes higher proteinese concentration than midgut and crop which show almost equal values. The enzyme activity in the crop changes little during 5 days starvation Lut in the midgut of the female the enzyme activity falls immediately on the first day following emergence.
There is a decrease in activity in the male caeca on the 5th day.
In both sexes starvation leads to faster decrease in total midgut
weight then weight in caeca:. Although the data give large deviations in
the caeca weight the time of emergence, there appears song increase on
1st and 2nd post—emergent days end then further, decline later. The crop
weight remains more or less steady except that it markedly drops on the day
following emergence. The decrease in the crop and midgut weight after
emergence is mainly due to the passage of cast—off intima in the crop es
well as certain breakdom products accumulated in the midgut during moulting.
The initial increase in caeca weight may be a nomal grotth erocess at the
expel-Ise of the reserve food material present in the heemolymph. On
examination the size and shape of the caeca were rauch better in 1 and 2 days
adults than those of the neely emerged adults.
Both the individual date as well as total mean values do not show relationship between the weight and the eroteinese activity of the
respectiva regions. The f,resenct_ of preteinese, in the crop, is obviously
due' to reguigitation which hes also been observed in cachroech (Abbott, 1926,
Schlottkc, 1.937).
26.
Table 3. Mean Values showing variation in total weight end proteinase activity in crop, midgut and caeca of adult Locusta unfed from emergence. Regions Sexes Days after 0 1 2 3 4 5 Emergence
Experimental 11 12 13 14 15 16 Category Weight 21± 2 12 + 015±2 16 + 2 18 I 2 17 5 (mg.) Male Proteinase 4± 3 3 4" 3 2± 1 4± 2 4± 1 4± 5 units Crop Female Weight 22 ± 6 24 # 4 18 t 4 24 4. 3 23 ± 3 21 4. 2 (mg.) Proteinase 2± 1 5t 1 3± 1 4 4' 3 2 4. 1 4 t 1 units Male Weight 19 ±8 16 ±6 16 ± 3 18 I- 4 14 ± 2 13 ± 1 (mg.) Midgut Proteinase 2 0 4± 6 3± 1 6± 4 3± 2 3± 4 units Female Weight 36 ±6 22 7 20 ± 3 17 ± 6 23 ± 6 15 ± 2 (mg.) Proteinase 16 + 10 3± 2 3 2 6± 4 4± 2 3± 3 units Male Weight 43 ± 24 44 4. 5 42 ± 7 35 5 36 ± 5 28 ± 13 (mg.) Protein ase 17 ± 15 16 "± 2 12 ±4 14 ± 3 14 + 3 8 I 3 units Caeca Female Weight 37 ± 12 52 ±5 57 ± 7 39 4. 7 46 ± 9 44 — 8 (mg.) Proteinase 16 ± 8 16 ± 5 16 ± 3 14 ± 8 12 .1." 7 16 + 5 units 27.
Day and Powning (1949) observed the regurgitation of proteinase in the crop of Periplaneta but not in Blaliella. The mechanism of regurgitat— ion was ascribed to the complex oesophageal invagination which was also considered responsible for preventing the passage of air from the distended crop to the midgut during moulting. Although the posterior part of the crop is much narrower the anatomical structure between crop and midgut is simple in Locusta and there is no oesophageal invagination (Hodge, 1939;
Albrecht, 1953) as compared to a complex nature in Blattidae (Snodgrass,
1935). Moreover, in Locusta during the moulting, air is not confined only to the crop but it is extended into the entire length of the midgut and also to the caeca. This means a free passage between the crop and the midgut.
Since most of the digestive juice of the midgut lumen is located anteriorly in the vicinity of the caeca and crop therefore the presence of a fraction of this juice in the crop of Locusta is very likely due to uncontrolled forward flow of the juice. iv) Proteinase concentration in 5th instar feeding hoppers, 5th instar non—
feeding '(pre—moult) hoppers end newly emerged adults of Locusta
eigretoria migratoides.
The presence of proteinas4 concentration in the gut at emergence was thought either due to a transfer of similar quantity from the feeding hoppers of the last stage or a fresh addition related to moulting. This idea was briefly examined by estimating the proteinase concentration in feeding hoppers almost in the middle of the 5th instar (i.e. 4 or 5 days old), non—feeding 28. hoppers in the pre—moult period of 5th instar nnd the newly emerged adults.
In connection with hreeding it tens observed that before moulting to adults, seven or eight days old 5th instar hoppers did not eat for the next
4P, hours ap:,roxilately. Normallhonpers prefer fresh grass over bran.
Therrore 6 dasold hoppers were intermittently offerer grass at every 6 hours hetween 10 a.m. to 10 p.m. and those hoppers which took their last meal in the night WCIC used rs aamoles for Pre—moult stage. Total proteinase activity of the individual gut (oxcent hindgut) vas concurrently measured in 4 to 6 individuals from, each ceteecry. The complete date is given in Table 4.
The feeding heppere of both sexes have heavier Cut which becomes lighter in the pre—moult hoppers and much reduced in the newly emerged adults.
This change is most likely duo to evacuation of the gut contents before moult. The e.iengcs in proteinoso concentration are more pronounced in females than the males. It €ppecrs that the clearance of the cut is also eiccompanied by the loss of some en7yme %blob is again coxpensEted by fresh production eithel hefore rr at the time of the moult. 29. Table 4. Proteinase concentration in 5th instar feeding hoppers, 5th instar non—feeding (pre—moult) hoppers and newly emerged adults of Locusta migratoria migratoides. 5th instar hopper 5th instar hopper Newly emerged adult (feeding) (pre—moulting) Experimental Category 17 Experimental Category 18 Experimental Category 19
Sex Weight Proteinase Sex Weight Proteinase Sex Weight Proteinase (mg.) unit (mg.) unit (mg.) unit
7 310 66 V 135 47 9 75 68 te 90 40 04 104 20 di 65 40 9 213 66 drl 80 23 9 103 60
9 249 58 q 110 27 dr 48 23 7 103 58 dl 74 31 9 73 50 is 232 64 9 122 60 9 84 39 ? 234 68 9 125 58 or 58 27 i d 73 13 or 76 36 9 82 46 i d 150 38 9 75 31 9 83 76 d 80 29 di 76 21 9 106 72 234 68 9 117 28 t? 60 43 6" 206 36 (1 96 35 9 83 39 ? 223 72 9 93 .12 136 60 dr' 79 25 Mean Values
44 181 ± 40 53±' 9 97 }11 32'1 7 77}4 9 9} 10 a 120 + 16 31 ± 10 84 +— 8 27 }4+ 58 _ 5 33 } 10 + 99 215}41 64 + 3 111 _ 15 35 _ 986+_ 9 56 }10 0 30. v) Variation of proteinase activity in the midgut and caeca tissue of adult
Locusts, unfed and fed from emergence.
From the observations on the total proteinase variation in the gut of unfed adults it is clear that the increase after 3 days from emergence is due to more enzyme production which is either stored in the epithelium or the gut lumen. To eeemins this is ee midgut and caeca tissue only of unfed adults at various post—emergent days were studied. But fox comparison a similar experiment was also performed for the adults which were continuously fed from emergence. bt a time 4 to 6 adults from fed and unfed category of similar age were dissected to obtain midgut and caeca tissue extracts for each Individual. The comple'Lo data is based upon 12 to lE1 adults from each category. The individual data is listed in the eeeperir.tental section and the moan values for each category is given in Table 5.
The tissue proteinase activity does not shot a difference between the sexes. Therefore conclusions from the date are based upon the total mean values including both sexes.
A corparison of total gut proteinase activity, as previously ascertained, with the tissue peoteinese activity of the corresponding unfed age group shows very negligible enzyme in the tissue which means that the enzyme is stored in the gut lumen and the quantity present in the tissue may be only an index of its production. In the continuously fed adult Locusts, though the enzyme
concentration is little higher than those of unfed adults, the tissue enzyme is still very low in comparison to total proteinase of unfed adults, which also supports the abovs view.
It was thought that proteinese activity might be inhibited due to certain Table 5. Mean Values showing variation in weight and proteinase activity of midgut and caeca tissue of adult Locusta migratoria migratoides, unfed and fed after emergence. Days Unfed from Emergence Fed from Emergence. after Emerg— Experi— Midgut Tissue Caeca Tissue , Experi— Midgut Tissue Caeca Tissue ence . .mental mental Category Weight Proteinase Weight Proteinase Category Weight Proteinase Weight Proteinase (mg.) units (mg.) units . (mg.) units (mg.) units
0 20 7 ±1 0 14 + 2 0 1 21 9 ! 1 1 + 1 18 I 2 1± 0 22 10 + 3 14. 1 21 4. 2 2 + 1 2 23 10 + 1 3 t 1 23 + 3 4 + 2 24 11 + 1 6 + 2 27±._3 10 + 3 . c•I .•-• 3 25 9 _+ 1 1 +_ 0 18±3 1 + 0 26 12±1 3±1 32 + 5 4 + 1
4 27 9±1 2 + 0 15 + 22 +_ 1 28 11 + 1 3 + 1 36 + 5 6 t 2
5 29 8 ±1 1± 0 15± 2 2± 1 30 11± 2 4 + 1 33± 5 5 ± 1 6 31 6 + 1 1 4. 0 11 + 1 1 + 1 32 12± 1 1 + 0 39 + 3 6 + 2 7 33 7 + 1 1 + 0 12 + 2 1 It 1 34 12 + 1 2 4' 1 35 + 3 5 + 1 8 35 6 + 1 1 + 1 9 ! 1 I + 0 36 11 ± 1 4 + 1 34 + 4 7 + 1 9 37 6 +_ I 1 + 1 10 ± 2 1 + 1 38 11 + 1 3 17 2 35 + 6 9 + 3 10 39 5 it 1 1 t 0 8 + 1 0 + 0 40 11 + 1 3 + 1 37 + 5 6 + 1 32. factors in the gut tissue tut activated in the gut contents. This hypothesis was checked by a simple experiment. This included 0.5 ml. of gut contents extract mixed with equal quantity of tissue extract and two 0.5 ml. control samples containing contents extract and tissue extract respectively. These were incubated at 37 0C. for 24 hours. The comparison of their proteinase activity did not show any appreciable difference and thus the possibility of activator in the gut contents was ruled out. It is therefore obvious that tissue enzyme must be discharged continuously into the gut lumen in both fed and unfed conditions and the process of synthesis and discharge goes together.
Although the values for tissue proteinase are very small and thus liable to much experimental error, certain results are clear. The values are generally higher for caeca tissue than those of the midgut. In the newly emerged adults proteinase activity is zero in both tissues but even in unfed condition some enzyme is built up on the following days with a maximal concentration on the 2nd post—emergent day. Furthers the activity drops on 3rd day and remains fairly low in continued starvation (Fig.3).
In contrast, fed adults have higher concentration on the 2nd dayl which is followed by a decrease and then almost steady in the midgut tissue while caeca tissue have a general increasing trend with little variations.
It is,therefore, very much possible that after emergence some initial enzyme is synthesized even in the unfed condition but if food is available this function becomes more enhanced. The decrease of tissue enzyme in fed adults, on the 3rd post—emergent day signifies slower production after a sufficient initial supply which may be required in the beginning of
FIG. 3 VARIATION IN PROTEINASE ACTIVITY IN MIDGUT AND CAECA TISSUE OF ADULT LOCUSTA FOLLOWING EMERGENCE proleinase activity in midgut, unfed
11 II „ caec a it „ midgui, fedre 1 0 n ,. caeca,.
0 S T I SE UN EINA O T PR
1 2 3 4 5 6 7 8 9 10 AGE ( DAYS) AFTER EMERGENCE 23. active adult life. Later,a little increase in synthesis may be enough for further reinfortement. In unfed cdult cleo,initial procedure is exhibited on smaller scale folloeing emerg,elce w'eich continues for sometime at c.—lower level if food is not available, and finally drops down.
Fig.4 shows the variation of tissue weight in fed and unfed adults following emergence. In unfed adults both midgut and caeca tissue arc initially increased to a maximum on the 2nd post—emergent dey but continued starvation decreases it further. Continuous feeding from emergence enhances tissue weight to a maximum on 3rd or 4th post—emergent day but later on considerable fluctuations occur almost along a straight line. Such fluct— uations may be due to different physiological conditions in the tissues at various times. The midgue tissue has proportionally very small addition
in weight than the caeca tissue. The progress of increase in tissue weight
in fed adults up to 4 days is probably due to initial growth requirement which must be obtained after moulting. The changes in tissue weight are not parallel with corresponding proteinase activity in all insectaltherefore
enzyme concentration cannot account for the changes in tissue weight. The
increase of tissue weight in ceece of unfed adults explains the total weight increaee in caece as previously observed. vi) Effect of feeding on the secretion of proteinase in adult Locusts m.m.
Enzyme secretion is generally stimulated by feeding after starvation in such insects as have been. studied (Schlottke, 1937 a, b and c; Day and
Powning, 1949; and Dadd, 1954). In the previous section it was observed that in unfed conditions some tissue proteinase was initially synthesized in
W EIGHT ( MGS) OF ADULTLOCUSTAFOLLOWINGEMERGENCE VARIATION INTISSUEWEIGHTOFMIDGUTANDCAECA 3 AGE( DAYS)AFTEREMERGENCE
r 4
FIG. 4 --w----.4--p, --x----m---4 _.A___A.__•_caeca of 5 A A
A
6 a A ,
-midgut ofunfe&adult caecaof midgut ofunfedadult 7 A
A fed adult ...... —...... y...... 4C ed \ adult
a A A 34. adults after emer;ence ;:ut :-.roduction could not continue further. This suggested that if such insecte are provided with food et a time when initial factor is incompetent to work it mey act Es a stimulus to accelerate enzyme synthesis in the ti ssue. For this purpose adults stervcd 3 days after eeergence wars good samples lecceuse in this cetegory tissue enzyme had recently drce,ped end the stervz Lion effect on the epithelium could be expected as minimun). In the first instance:5.0 wee planned to study the effect of only one real so as to know about the imediate changes in the tissue. It was observed that almost all adults, starved 3 days after emergence when offered with grass immediately started eating, but mostly did so for less than half an hour. Preference of grass was always present over bran, therefore in the following experiments these adults were allowed to eat grass for half En hour and then isolated from the food. The individual midgut and caeca tissue were extracted at various intervals after the feeding period. The control included 3 day old unfed adults. During the dissection it was observed that soon after the feeding period food travelled up to the half length of the midgut; Within 4 hours food occupied the entire midgut and later some faecal matter was passed out but the midgut still retained some food after 6 hours. The gut was completely empty at 12 hours. An examination of Table 6 shows only a small increase in enzyme content in the ceeca at 4 hours but an inspection of the individual data in the respective experimental category reveals only one high value out of 6 individuals so that this could be an experimental error., very likely due to incomplete washing of the tissue. But the tissue weight especially the 25. caeca have Ilef::edly incrc:s(d sor'n the f(,-dng ,'cried. This change
also corresponds to th,2 morphelogical nj_:setrEnccs of the ort-s which look mre dilat:6 end sx?ended then thosc of the cantrols. This tissue increase
reDarded as due to absorption of metotolic ,rod,..cts end water from
the digested food z-cter4 al.
Frov.! tile above dote, it was also possible to thin!, thet the enzyme
discharge into the gut lUMON at cer4i,in after reeding rnd
thus concentration might incrcose in the Therfor:I thc total gut
l)reteinase was determlned in e foY sLcioles at varirl:s after one real. The mean values (Table 7) es yell as the individuel drzto do not show
mer::ed increase from the control in either The proheble cause of a
consiC,erable decrecsO after the fr,eding do,.iod is the ol)s,f:cc of females in
that category which always have higher vol.tles than the -ialec. Howevc;a
general smell decline in the total gut 'droteinase after f^edinj is nerhaps
from the loss of enzyme during the oassogrl of gut contents or the individual
variations. From these observations ..t is clear that a single meal has no
narked enhancement in proteinase secretion aftt:r 2 days :;ost—emergent
starvation.
Thareforelthese insects wer allowed to 'E.:0d continuously on the
normal diet which consisted of :'Neat ::an and grern grass. The total
proteinase activ:ty as measurez on the following days aft:.r the commence—
ment of feeding. The mean values (Table 5) as Well as the individual
observations reveal that significant increase occurs in total gut proteinase
on the 3rd day after the 1st intake of food. Ageln,a low ,:roteinase activity after 24 hours in fe.c.alcs oLvipusly ;leans some unrluo loss through
Title 6. Mar vr,luE, s CL-,cvsln ;.::otcinasc zctivILy and weigilt of midgut and
car7cF Eftr f.F..,:ding for 20 minut es 'co adult Locusts, 3 days
unfed zft.(71. cmcv;cnce.
Experimental Time after Midgut tissue Caeca tissue Frseding Category Weight Proteinase Weight Proteinase (mc.) zctivIty ( 0g.) activity
41 Control 8± I. 2 + 1 17 + 2 2± 1
42 Zero 9 + 1 2 + 2 22 +_ 33 +_ 1 43 1 hour 10 + 1 2 + 1 22 +_ 3 3 +_ 1 44 4 hours 10 + 3 3 + 1 23 +_ 3 6 + 3 45 6 hours 10 4' 2 2 + 1 22 + 5 2 + 1 46 12 hours 9 + 1 3 4. 1 18± 4 2± 1 47 24 hours 11 + 2 0 + 0 23 + 91 +_ 1 37.
Table 7. Mean values showing total ;_roteinace activity and total gut
weight, after feeding for 30 minutes to adult Lccusta, 3 days
unfed after emergence.
Experimental Time after Total gut weight Total proteinase (except hindgut) Category Feeding (mg.) activity.
48 Control 85 ± 7 32 + 9
49 Zero 203 ± 40 23 ± 10
50 6 hours 136 ' 19 29 ..+ 8
51 12 hours 76 +_ 17 27 4- 10
52 18 hours 77 + 3 22 ± 11
53 24 hours 72 ± 11 26 + 5 38.
Table 8. Mean values showing total proteinase activity and total gut
weight after the commencement of continuous feeding in adult
Locusta, 3 days unfed after emergence.
Experimental Time from Commence— Sex Total gut weight Total Proteinase ment of feeding (except hindgut) Category (days) (mg.) Activity
54 Control 79 _+ 10 25 + 7
110 + 12 33 + 9
55 1 256 + 12 22 t 2 351 + 38 24 +_ 5 56 2 256 + 18 25± 10 352 + 27 25 + 7 57 2 243 ± 60 35 2
361 120 46 6
58 4 131 4- 24 4—tof)P-, 7
315 ±" 45 4 39. the passage of gut contents or individual variations.
Since enhanced total proteinase activity was observed after 3 days of the commencement of feeding, it was expected that the tissue must synthesise enzyme during this period and demonstrate some changes in the tissue protein— ase concentration. As observed before, individual tissue extracts give very small activity for comparison. Therefore, in the following experiment, tissue from 3 individuals of one sex composed one tissue unit to obtain higher values. The experimental adults were fed on bran and grass as for the previous experiment. Some individuals were kept on tap water only.
The complete data is listed in Tables 9 and 10 for females and males respectively. The values are always higher in females than the males. The caeca tissue in both sexes produce some proteinase within 48 hours after the commencement of feeding but the increase is very much significant on the
3rd day. Similar process occurs in the midgut tissue but on a small scale.
Tap water alone does not produce this effect. Thus, the tissue enzyme concentration corresponds to the previously observed total gut proteinase.
Fig. 5 shows the changes occurring in the tissues and the total gut proteinase activity in the two sexes in relation to time after the commencement of feeding. But these curves do not show any relative value between tissue and total gut enzyme activity because their strength in extract as well as incubation period differ as mentioned in technique. Considering these factors, the present observations suggest that bulk of enzyme activity is from the gut lumen and support the idea that in continuous feeding insects synthesis and discharge of enzyme go on simultaneously and the digestive 40. juice is mainly stored in the gut lumen (Daddy 1954).
Since the quantity of food in the gut remains almost the same prior to the observed proteinase increase, it is very likely that full gut conditions with food, and consequently, an adequate supply of nutrients is a pre—requisite for enzyme secretion after prolonged starvation.
In Fig. 6 tissue proteinase activity has been plotted against the corresponding tissue weight of midgut and caeca separately from the data of Tables 9 and 10. Whereas these scatter give a general impression of a correlation between weight and proteinase activity, the latter becomes very much variable in the higher weight ranges. This may mean that enzyme concentration does not entirely depend upon the tissue weight. 41. Table 9. Data showing the effect of feeding on weight and proteinase activity in the midgut and caeca tissue of female adult Locusta, 3 days unfed from emergence.
Days from the Commencement of 1 2 3 4 Feeding
Experimental 60 61 62 63 Category Control 59 Midgut Caeca Midgut Caeca Midgut Caeca Midgut Caeca Midgut Caeca
C) a, a) a) a) C) a) a) a) a) 0 4i) U) 0 0 U) 0 0 CI) 1.0 011 (0 CU (U CU (13 ezi co 0 co C C 0 c C C c C c c C 4.2 ..--.. ni 01 4.3 ..--...r.1 -1.3 +3 .0.--.. *-4 (J) 4..) ...... •r4 In 4.1 ...... 1-1 In .1.3 ...... -1 C1 .1.1 ...1.1-1 in +3 ---... .,-1 w +3 ..-... .11 0 +3 ..--..ri to 4 .. C' 4.) C • • C) ..r.1 ..0 • . 0 4.1 .C. • • 0 •4•4 .0 * . CU 4- .= • • 0 4.4 ..0 • • 0 .4-./ .. • . Cll 4.) C • • 0 +4 ..0 • • 0 4.0' CP DI ED .4-3 C 131 1:11 .1-1 *el C:11 01 42 ..1-1 1100 131 4.3 ...-1 .0 01 4.1 or.. CP CA .1-1 .1-1 CD 01 4-.1 ..-I 01 01 4-1 ..-1 0) 0l 4-)..-4 Hoc-4E00-4oc•Heac.4Eo C..--1E0C.AEOC..-1E0C -420C.AEOC •Q) ...... li z co ...... 6.4 C) ..._... $.1 U '— 1-1 0 0.1'....0 1.1 0 C) —. /4 0 CD `...• 1.1 0 41'— 1-1 0 11) '1.-.• • $4 C C) V- lk c RC la, 'X fa. o. .i.:7 a. i.L.- a. Bk..-• R. --. R. '...• a. "4.7. R. :..T. a. 21 0 72 7.5 27 4 83 7 40 5 125 20 32 15 125 65 38 6 140 40
27 1 90 8 35 6 103 12 39 2 116 19 33 4 130 37 30 6 120 21 28 3 87 8 38 2 •104 14 37 7 102 16 32 3.5 138 43 42 3 158 28 24 2 70 9 40 2 135 25 36 6 183 50 33 7 140 40 24 5 66 9 30 5 128 37 33 13 117 42 33 15 105 24 28 4 71 14 30 12 145 57 Means S.E.
25 3 76 9 33 4 106 11 37 4 121 23 33 9 140 49 35 7 133 31 ±2 ±2 ±9 ±2 ±6 ±2 ±27 ±4 ±4 ±2 ±11 ±7 +16 ±4 ±18 ±8 ±4 ±4 ±18 ±8
Continued on next page.
42.
Table 9 cont.
Fed on Tap Water only Experimental 64 65 66 Category (a.W., 2 Amp ) (4464" 3 ( Wit LI 414) 28 1 70 6 22 0 41 2 18 1 40 5 22 5 52 7 16 0 45 5 17 1 35 5
Means 25 3 61 6.5 19 0 43 3.5 17.5 1 37.5 5 S.E. ±6 ±3 ±18 +1 ±4 ±0 ±4 ±3 ±1 ±0 +±5 ±0
43. Table 10. Data showing the effect on weight and proteinase activity in the midgut and caeca tissue of male adult Locusta, 3 days unfed after
emergence. Days from the Commencement of 1 2 3 4 Feeding Experimental Category 68 69 70 71 Control (67) Midgut Caeca Midgut Caeca Midgut Caeca Midgut Caeca Midgut Caeca
a) a) 0 0 C) C) C) C) 0 4) U) U) U) U) U) U) U) U) 0 U) co 0 co ro co 0 0 ca co 0 c C c c c c c c c 4.) , .4.1 en 4.) ...... •444 0 44 44.-4..44 V) 44 ---.. NA 0 4-1 ••-.4. •4-1 0 4-1 ••••••.•1-4 CO 44 4.--.4 or-I (111 4.) ...--.. •r4 V) 4-1 — 44-4 IA -$-) .--•,r4 0 ..0 * C) 4-) C • 0 4-3 C • 0 4.) ...0 • W 4-) .0 60 +) 4 • 0 4-3 4 • C) 4.4 ,10 a 41) 44 „C • 434 44 .c • 0 4-) 01 01 44 .4-4 CA 01 4-) erl C71 CT 4-1 *4-4 01 01 44 •4-I 01 01 4-1 *4-1 C7) at 44 •4•4 01 cn +3 .4-1 CP 431 44 .4-4 04 01 44) •r4 01 C711+) .4-4 .44 0 C 24-4 E 0 0 -r) ! o c ..--1 E 0 0 •4-4 E o c ..-1 CO C .44 CO C •4-4 CO C •rt P.: 0 C 01-4 CO C C) •--- )4 0 4) •--- )4 0 4) •••••-• ii a 0 •••-. k C) ••••••• k 0 •••••-' Ii Cl ••—•• Ii O-- 1-1 0 ...... $.1 (1) ...... 0 k 0 .ir.. al .i...: 04 BC 0. 'a".:, O. ...'; 0. •••• 0. '4; 0.'.,:'; a. :72: fa. :6.: a. 16 1 38 0 27 0 54 3 22 4 62 7 22 3 90 16 28 12 81 34 20 1 49 5 26 3 100 14 28 2 73 10 31 3 96 14 25 9 108 36 20 2 51 9 25 2 81 10 22 0 100 10 32 16 113 62 25 1 95 25 19 2 49 10 30 2 82 17 20 4 71 10 28 10 93 44 26 10 85 18 18 6 60 7 19 3 41 6 Mean S.E.
19 3 48 6 27 2 79 11 23 3 77 9 28 8 98 34 26 8 92 28 +1 ±2 ±6 ±3 ±2 +1 ±19 ±5 ±3 ±2 ±16 ±2 ±5 ±6 ±10 ±23 ±1 ±5 +11 t8
Continued on next page. 44.
Table 10 cont.
Fed on Tap v'ater only Experimental 72 73 Category (404 2. dam) (4044- 30615) 18 1 44 3 15 0 36 1
19 1 56 5 18 0 33 1. Mean 18.5 1 50 4 16.5 0 34.5 1
S.E. ,.1. _12 +2 _3 0 _3 ASE LOCUSTA, UNFEDFOR3DAYSFOLLOWINGEMERGENCE. PROTEIN EFFECT OFFEEDINGONPROTEINASEACTIVITYADULT ar...... TIME (DAYS) FROMCOMMENCEMENT OFFEEDING
...... _proteinase activityinwholegutofmale. ""'''11.- 1 I
...... FIG. 5 It II /I II 11
. 4r, I 2
II .. ii
II II .,
caeca tissue.,male mi gut II d
3 I II
II tissue ofmale II
II II
••••••••• female II IS female female
FIG. 6 SCATTER DIAGRAM SHOWING TISSUE PROTEINASE PLOTTED AGAINST CORRESPONDING TISSUE WEIGHT OF LOCUSTA GUT
70 ( a ) CAECA TISSUE
•
•
• • tol • • • ••
30 0 a_ • • • 20 • • • • • • • • • • • 10 •• 00 • • • • 0 e • • • •• •• • 04 • • • • • •
20 40 60 0 109 1 0 140 160 180 200 WEIGHT ( MGS.
(b) MIDGUT TISSUE • • •
• • •
• • •
• • • • • • • • • • • • • • 0 • • • • • • • • • • • • • • • • • • • • • • • • • 1 I I C 1 0 20 30 40 50 WEIGHT (•MGS.) 45.
INVERTASE ACTIVITY IN LOCUSTA MIGRATORIA MIGRATOIDES. i) Invertase activity in serial dilutions of midgut tissue extracts of
Locusts migratoria migratoides.
For the assessment of invertase activity in the midgut and caeca tissue extracts the calibration of E.E.L. Colorimeter was regarded as essential. Therefore fromthe midgut tissue extracts of different strength*
0.5 ml. sample was serially diluted to a number of samples as for proteinase dilution curve. The further procedures for developing the colour in these samples were the same as described in Section 2. The invertase activity was recorded in colorimeter units by comparing the colour density of the final solutions with similarly treated reagent control.
As observed from Table 11 the values between 1 to 39 scale divisions of the colorimeter fall into the linear part of the curve (Fig.7). This suggests that a comparison of invertase activity from different extracts can be fairly appreciated within this range of the colorimeter scale and the divisions on the latter will represent the invertase units. 46.
Table 11. Invertase activity in serial dilutions of midsut tissue extract
of Locusta migratoria migratoides. Invertase activity in colorimeter units Treatment Relative enzyme concentration in serial dilutions
Numbers 1 2 4 8 16 32
1 2 4 9 16 30 50 2 2 4.5 8 16.5 30 52
3 1 1 2.5 5 10 19 4 0 1 2 4 8 17 5 1 2 4 10 '19 39 FIG. 7
INVERTASE DILUTION CURVES FOR LOCUSTA
50
45-
Li-) 40 _ I- 2 D m 35_ w I- w M }2 30 - 0 - J 0 ,...o 2 5- >-. I- 5: 20., U Q w a 15 _ Nt-- x w >. z 10 _
5-
1 2 4 8 16 32
' EXTRACT CONCENTRATION 47. ii) Variation in invertese activity and eelght of midgut and caeca tissue
of 5th instar hoppers, unfed and fed after moult.
From the previous observations it vias concluded that proteinase concentration in the tissue indicated its rate of discharge into the lumen.
Any change in the tissue enzyme is,in fact,a demonstration of total change occurring in the whole caeca or midgut. Since digestive juice contains a number of ferments it is expected that all enzymes follow more or less similar changes in the tissue and lumen. The study on the tissue enzyme activity affords more reliable measure of any particular region than the respective region including its contents because the enzyme discharge in one region may be easily carried away to the other by regurgitation. Therefore, midgut and caeca tissue were used to investigate the secretory changes in different conditions.
The tissue proteinase activity was found very negligible at the time of emergence in adults, which suggested that probably concentration of enzyme in the tissue is affected before moulting. If this is the case,
5th instar hoppers must show the changes in tissue enzyme activity prior to the moulting to adults. To examine this idea invertase activity in the tissue was investigated in fed hoppers from the time of their moulting to
5th instar to the emergence of the adult. Unfed hcypers were also examined so as to know the changes in invertase activity due to starvation.
This experiment was carried out in two steps using unfed and fed categories separately. Starvation did not affect the survival of hoppers for 3 post—moult days. But most of them suffered mortality later and could not survive more than 6 days after moulting. Feeding stops approximately 48.
2 days prior to the moult to adult. Thus 8 or 9 days old hoppers had no trace of food in the gut and in the crop, the intim had almost detached from the entire surface except the posterior end where it was loosely attached to the crop wall. The lumen of caeca and more specially that of midgut had a mass of brown paste—like substance instead of brown fluid of normally fed hoppers. These changes in the gut signify the conditions for the next moult.
The data given in Tables 12 and 13 show the mean values for unfed A41'444;41/ and fed categoriesi,. The comparison of values from individual observations as well as the averages of the groups shows slightly higher figures in females which are more significant in the fed categories. The variations in invertase activity and the weight of midgut and caeca tissue of unfed and fed hoppers following moult are graphically represented by plotting the total mean values including both sexes in Fig.6. Starved hoppers have weaker invertase activity in caeca than the midgut. As observed in tissue proteinase activity for adult Locusta newly moulted hoppers have also negligible invertase activity and on starvation,there is an initial increase in activity which significantly declines after four days. The hoppers in the presence of food have remarkable invertase synthesis following moulting.
During this change the rate of synthesis appears higher in the caeca than the midgut. In both tissue maximal activity occurs on the 4th post—moult day and thereafter it gradually declines to almost negligible at the end of nymphal period. The increasing enzyme activity in the first half of the inter—moult period may be suggested as related to the higher metabolic 49.
Table 12. Mean values showing variation in weight and invertase activity in the midgut and caeca tissue of 5th instar Locusta hoppers,
unfed following moult.
Time after Experimental Sex Midgut Tissue Caeca Tissue moulting Category (days) Weight Invertase Weight (mg.) units (mg.) Invertase units.
Newly 74 4 4 _ 1 1 + 0 12 + 1 0.0 + 0.0 moult + + die 4 _ 1 1 _ 0 10 ± 1 0 4' 0
il 5 + 0 1 + 0 13 + 1 0 +_ 0
1 75 d'T 6 + 1 4 _+ 1 13± 1 1 +_ 1
ecr 5 + 1 4 +_ 1 13 + 2 1 _+ 0
egii. 7±1 5 _ 2 14± 2 2 +_ 1
,.2 76 c? 5 + 1 4 +_ 1 13 + 2 2 + 1
de 4 + 0 4 + 2 12±1 1 ±1
q 6 + 1 5±1 15 + 5 2 +— 1
3 77 ,4 5 + 1 4 + 1 11± 2 2 _+ 1 cPe 4 + 1 4 + 2 8 + 1 2 +_ 1
5.? 6 + 1 5 + 2 12 + 1 1 + 1
4 78 ee 5 + 1 4± 1 10± 1 1 + 0
al?' 4± 1 4 + 1 9 + 11± 1
q? 5 + 1 4± 2 11± 2 1 + I
5 79 d? 4 I 1 3.±1 9±1 1 + 1 dlp 3 + 1 2 + 1 7±'2 1 + 1 Y? 5 + 1 5 4' 1 10±'1 1 ± 1 6 80 d' q 3 4' 1 2 J.: 1 6±1 1 + 1 cy 3 + 0 2 + 1 5±1 0 + 0 cq 4 + 1 3 + 1 8±2 '1 ± 1
50.
Table 13. Mean values showing variation in weight and invertase activity in the midgut and caeca tissue of 5th i.nstar Locusta hoppers, fed following moult.
Time after Experimental Sex Midgut Tissue Caeca Tissue moult Category (days) Weight Invertase Weight Invertase (mg.) units (mg.) units
81 df 9 ± 1 14 2 26 3 7 ± 1 ad 7 + 1 14 3 20 + 2 6 + 1 10 ± 1 14 2 29 4 8 + 2 2 82 9 — 1 15 4 33 1- 5 14 +— 3 9 + 2 14 2 28 + 9 10 + 3 9 + 2 15 7 36 + 7 16 ± 5 3 83 c9 8 + 2 15 3 34 + 5 17 ± 2 dd 7 + 1 15 3 28 + 6 16 + 5 8 + 2 15 4 38 + 4 17 + 2 4 84 9 + 2 23 4 40 + 9 23 ± 5 dIP 7 ± 2 20 8 33 ± 10 21±"9 99 11 ± 1 25 5 46 + 12 25+7 5 85 9 + 1 17 2 35± 5 15 + 4 g + 2 17 7 30 + 5 14 ± 7 9 ± 1 17 2 38± 6 16 — 4 6 86 c7T 9 + 2 16 5 31 + 5 14+4 8 + 2 13 6 27 + 4 11 ± 3 99 11 ± 1 22 7 38 + 3 20 7 7 87 c?'? 10 1: 1 9 4 22 5 11 ± 5 04? 10 ± 1 10 3 23 ± 4 13 ± 4 10 + 1 ±• 99 9 6 22± 9 10 8 8 88 9 ± 1 4 3 20 + 4 4 + 2 dd 9 4. 1 7 10 21 5 5 — 3 -7- 9 ± 1 3 1 19 5 4± 2 9 89 8 + 1 1 1 17 2 1 + o cei • 7 + 1 1 1 15 1 1 — 1 9? 10 ± 1 2 1 19 3 1 + 1 Fla 8 VARIATION OF WEIGHT AND INVERTASE ACTIVITY IN THE MIDGUT AND CAECA TISSUE OF 5IH INSTAR HOPPERS OF LOCUSTA AFTER MOULTING.
Invert:se activitY in midgut,unled caeca i• midget; fed caeca
40 weight of midgut unfed •A • e \ caeca / midgut; led . caeca
30
N■ "-- 10 A
2 3 t 5 6 8 9 AGE ( DAYS ) AFTER EMERGENLI. 51. activity for the optimal growth requirements before the conditions for the next moult ensue.
Tissue weights follow a course i7.arallel to the respective invertase activity in unfed hoppers. Whereas midgut tissue becomes heavier on the day following moult and continues to be almost unaffected later, the changes in caeca tissue weight are parallel to the invert-Ise activity. But prior to the emergence of adults caeca tissue weight of hoppers is still higher than its initial weight just after moulting to 5th instar. This evidently supports that some growth,in the midgut and caeca tissue,occurs during this instar period. But as far as higher weights of caeca tissue during the early periods of this instar are concerned, these may be ascribed to the absorptive function and the accumulation of the products of intermediary metabolism in the cells in the most actively feeding phase.
These observations clearly suggest that enzyme activity in the tissue is reduced to almost zero before moulting to adult. iii) Effect of feeding on the weight and the invertase activity in the
midgut and caeca tissue of 5th instar hoppers of Locusts, unfed for
3 days following moult.
Like proteinase activity in the tissue of adult Locusta the invert— ase activity in the 5th instar hoppers unfed from moulting is also increased initially on the day following moult and then slowly declines after 3 days on prolonged starvation. Therefore lthe assessment of feeding was done on
3 days old unfed hoppers which were unable to show further improvement in invertase activity.
These hoppers were allowed to feed continuously on bran and grass.
The tissue extracts were obtained from 4 to 6 individuals at different times 52. after the commencement of feeding. Almost equal nwibers of males and
females were used in each group. The hoppers had always food in the gut.
The control consists of 3 day old unfed hoppers. Table 14 includes the mean values of total number of adults as well as for males and females in each group.
The individual data and the mean values of the respective groups indicate some improvement in the invertase activity of the midgut tissue within 3 hours from the commencement of feeding. Both midgut and caeca tissue have very significant rise in invertase activity between 12 to 24 hours. Afterwards, both tissues have almost equal level of enzyme activity.
It is clear that invertase activity is more quickly stimulated in the tissue of unfed hoppers than the proteinase activity in unfed adults. The changes in invertase activity and the tissue weight are graphically shown in Fig.11.
The addition of weight in the midgut tissue is comparatively much less in the midgut than the caeca. The latter becomes nearly double in weight between 12 to 24 hours after the start of feeding.
The relationship between tissue weight and invertase activity in the midgut and caeca respectively are not similar. After 24 hours of feeding midgut and caeca have almost equal invertase activity but the proportional increase of weight in midgut is only 1/5th and in caeca about 3 times with respect to those of the control. This supports the idea, as previously suggested, that higher weight increase in the caeca tissue is due to its more additional function of absorption of digested food and accumulation of the products of intermediary metabolism. 53. Table 14.. Mean values showing changes in weight and invertase activity in the midgut and caeca tissue of 5th instar hoppers of Locusts,
(3 days unfed following moult) following feeding.
Time from Experimental Sex tvlidgut Tissue Caeca Tissue commencement Category of feeding Weight Invertase Weight Invertase (hours) (mg.) units (mg..) units Control 90 ei? 5 +.. 14± 1 10 ± 12} 0
crld 5 1." 1 4 I 2 10 + 2 2 + 0
?? 5 + 1 4 + 1 9 + 2 2 1: 1
.3 91 cf? 5 + 1 6.± 2 12 + 2 3 + 1 4? 5 + 1 6± 211 +— 2 3 + 1 + IT 6 4- 1 5 + 2 12 — 4 3 + 1 6 92 cilt? 6 :. 0 7 ± 2 15 + 2 3 + 1
c?cfl 6 + 1 5 + 2 13 4. 3 3 "1" 1
'?9 7 + 1 10 ±4 16 + '' 3 + 1 12 93 cfci• 6 + 1 7 + ,) 15 + 3 4 + 1 ed 6 ± 1 6 + 1 13 + 3 3 + 3
. ?? 7 + 1 9 + 2 16,± 2 4 + 1 24 94 j' 6 + 1 12 4. 2 25 + 4 12 :1. 2 Se 6 + 1 12 + 4 24± 4 11 + 2 99 7 + 1 12 + 3 25 + 7 13 + 3 48 95 e9 8 + 1 10 + 3 26 + 3 10 ± 2 de 7± 1 10 + 4 23 + 3 10 + 2 y9 9 ± 1 10 ± 4 29i 3 10 ± 3 72 96 et? 11 ± 2 11 ± 3 35 ± 6 13 ± 4 cfa 9 + 1 6 + 1 29 ± 4 8 + 2 li''? 12 + 2 14 4. 3 39 I 8 17 + 5 54. iv) Variation in invertase activity and tissue weight in the midgut and
caeca tissue of adult Locusta, unfed and fed following emergence.
Investigations on the tissue proteinase activity in adults and invertase activity in the tissue of 5th instar hoppers have provided the evidence for the fact that enzyme activity in the tissue is stimulated after moulting even in unfed conditions. Further investigation on the tissue invertase activity was pursued on adult Locusta unfed and fed following emergence.
The procedure was the same as for hoppers. The data for each individual of a group is listed under experimental groups. The total mean values for each group of unfed and fed adults is given in Tables 15 and 16, respectively. As regards the differences between males and females the values for invertase activity do not differ appreciably in unfed groups.
But the categories feeding continuously following emergence generally show higher enzyme activity in females than the males. Similarly their tissue weights also differ.
The initial changes in invertase activity are similar to proteinase activity in adults on starvation following emergence, except that the maximal invertase activity occurs earlier than proteinase activity. The effect of starvation results in slow decline in the activity which is very much reduced at the end of 10 dayslrhen very small percentage of population can survive. Similar to the observations mentioned before,feeding after emergence accelerates the invertase production in both midgut and caeca. It 55. appears that after achieving some essential enzyme requirementl within 2 to
3 post—emergent days considerablefluctuations may occur in the concentration of enzyme of the tissue with en overall increasing trend as seen in 10 days observation. Almost similar results are available from the observations on tissue proteinase activity. The caeca tissue finally has invertase activity nearly equal to the midgut tissue.
Variation in invertase activity and the tissue weight in unfed and fed adults following emergence is represented in Fig.9. As regards the changes in the tissue weight following emergencet the curves do not differ much from those obtained previously (Fig.4). Whereas curve for midgut tissue weight has no relationship with that of its enzyme activity the curve for caeca tissue weight is aeearently parallel with its invertase activity.
To examine the relationship between the tissue weight and the invert— ase activity individual enzyme activity wee elotted against the corresponding tissue weight of midgut and caeca separetely from the data of Tables 15 and
16 (Fig.10). On inspection) the scatter diagrams show that in both unfed or fed groups invertase activity is very much variable for any selected value of tissue weight. Moreovexl as the weight increases variation in enzyme activity becomes remarkably scattered. Therefor%the increasing tissue weight has no corresponding relationship with the enzyme activity. Thus changesin the tissue weight of midgut and caeca are not controlled by enzyme production. 56.
Table 15. Mean values showing variation in weight and invertase activity of
midgut and caeca tissue of adult Locusta, unfed following
emergence.
Time after Experimental Sex Midgut Tissue Caeca Tissue Emergence Category (days) Weight Invertase Weight Invertase (mg.) units (mg.) units
Newly 97 A 8 ± 1 0± 0 15 1+ 3 0 — 0 emerged die 7± 0 0 4" 0 11 2 0 + 0 *9 9 + 1 1 + 0 19 2 1 + 0 1 98 e9 9± 1 9 I 2 18 3 4±1 d'e 8 + 1 9 4" 2 14 +1+1+1+ 2 4 + 1 W 10 + 1 9 ± 3 21 +1 4 5 + 2
2 99 ar9 10 i" 1 7 + 2 21 1+1 4 2 ± 1
cry 8 + 1 6 ±2 18 1+ 2 2 + 1 94 12 + 1. 9 4- 1 26 7 3 4" 1 3 100 d'9 8 + 1 6 + 2 16 +1+ 3 3 — 1 lee 7 + 0 5 ± 2 12 1 2 + 1 ''i' 9 + 1 6 + 2 20 3 3 —+ 1
4 101 d9 6 + 1 5 i 1 15 1+ 1+1+1 3 3 ± 1 ec? 7 ."1: 1 4 ± 2 13 4 2 ± 1 +1+ 9? 9 + 1 7 + 1 18 1 4 4 ± 2 5 102 cri) 6 + 1 4 + 1 13 2 2 + 1 de 6 + 1 3 + 1 11 2 2± 1 99 7 + 1 6 + 1 16 +1÷ 1+ 3 3 + 3 6 103 dic 7 + 1 4 4" 1 Il 1+1 2 1 + 0 ea 6 ± 1 3 I 2 10 1 1 + 1 4i!4 8 + 1 4 t 2 12 2 1 + 0
7 104 &9 6± 1 3 4" 1 11 1+1+1+ 1 1 + &O 6 4" 1 2 ±'1 9 2 1 + 1
?9 7 1. 1 3 4. 1 12 1+1+ 1 2 ± 0 8 105 e? 6 + 1 3 + 1 8 1 1 + 0 ee 5 ±1 2 it 1 7 1 1 + 1 + 99+7 2 3 1 9 1+1+1+ 1 1 + 1 9 106 0'? 5 7 1 3 + 1 7 1 1 1: 1 ea 4 'I 1 2 + 1 7 1 1 + 1
+1+1+ iN 6 ± 1 3 ± 1 E 1 1 + 0
10 107 d 5 I 1 2 + 1 8 t+1 1 1 4' 0 d' a' 4 4. 1 2 4- 0 6 1 1 ± 1 9 6 + 2 2 4" 1 9 1÷1+ 1 1±0
57.
Table 16. Mean values showing variation in weight and invertese activity of midgut and caeca tissue of adult Locusta, fed foll&wing emergence.
Time after Experimental Sex Midgut Tissue Caeca Tissue Emergence Category (days) Weight Invertase Weight Invertase (mg.) units (mg.) units 1 108 11 ± 2 16 ± 2 23±3 7 t 1 d'd' 9 t 3 14 ± 2 21±5 6±2 99 13 ± 1 17 ± 3 27 ± 5 7±2 2 109 dg ± 19 ± 3 31±4 . 15 ± 3 11 ± 1 19 ± 7 25 4- 3 11 t7 951 12 ± 2 19 ± 3 36 i 5 17 ± 3 3 110 dq 12 ± 1 21±'3 32±3 15 t 3 ddi 11 ± 2 19 ± 3 29 ±4 13±2 T4 14± 1 24 t 3 36 ±4 18 t 5 4 111 dQ 11 t. 1 19 t 3 36 '±4 16± 4 d'cr 10 ± 1 16 ± 2 31 t 6 14 t 6 4i19 13 ± 2 24 ± 3 42 '4' 2 21 3 5 112 11 ± 2 19 t 2 34±'5 16 ± 6 ere 10 ± 4 18 ± 3 30 ± 2 15 ± 2 q4 14± 6 23 t 2 47 t 10 21 — 1 6 113 12 1 23±4 42 t 5 23 4- 4 10 ± 1 20± 4 34±3 19 t 3 cz9 14± 1 26 ± 5 50±4 27±' 6 7 114 are 13 ± 2 23 t 3 37 4. 6 22 t 5 ecf 10 ± 1 21 ± 3 32 4' 8 16 t 4 92 16 ± 1 26 t 4 42±9 28± 7 8 115 dg 10 ± 2 26 ±5 33±4 28± 7 ed, 8± 0 20 ± 4 30 t 5 20 t 5 99 13 ± 2 31 t 6 36 t 6 36 t 8 9 116 dg 11 ±1 24±4 24±4 22 ± 4 as 9±1 21 t 5 30 ± 5 19 ± 5 ?4? 12 1 27 t 5 37 t 8 24 t 6 10 117 cf? 11 t 2 27±5 37±7 23 t 7 al? 10 t 3 23±"9 32 t 15 20 t 11 '?q 12 2 31 ± 6 41 t 6 25 t 10 FIG. 9 VARIATION CF WEIGHT AND INVERTASE ACTIVITY IN THE MIDGPT AND CAECA TISSUE OF ADULT LOCUSTA FOLLOWING EMERGENCE. • ib--invertase activity in mid ut, unfed midgut, fed .. caeca, ..
0
0
0
•
, weight of midgut unfed „ caeca midgut, fed a__ caeca,
•
40
a • A-- -- „' A A
ui
20. hi
a
0
g lb AGE( DAYS) AFTER EMERGENCE FIG.I0 SCATTER DIAGRAMS SHOWING TISSUE INVERTASE ACTIVITY PLOTTED AGAINST CORRESPONDING TISSUE WEIGHT
7.0 (a) in midgift,unied alley emergence 15
•tl
10 20 30 540 60 vEEHT( 14(15.)
(b) in caecapied after emergence 101 • • 5 • . • •• • • • :•• •• • - - I 10 20 50 60 WEIGHT( 1.4G5)
40- (c) inr midgut, fed after emergence S
T 35- I N
U 30-
25- SE
TA 20- R E
V 15 IN 10
5
110 20 30 40 50 60 WEIGHT( MG 53
50—, (d)in caeca led after emergence
45-
40-
35- •
• •• • • •• •• • • 25 - • • • • • 20:
15-
5-
10 20 30 40 50 60 WEIGHT ( MGS ) 58. v) Effect of feeding on the weight and invertase activity in the midgut
and caeca tissue of Locusts.
The tissue proteinase activity is stimulated in approximately
24 hours from the commencement of feeding in 3 days unfed adults following emergence. But in 3 days starved hoppers following moulting:the invertase activity in the tissue is stimulated within 3 hours after the food is provid— ed. It means that either the hoppers have more ability for an immediate response to the stimulus involved or the latter may cause discrimination in the synthesis of these enzymes. This idea was put to test by invest— igating the changes in invertase activity of the tissue of adult Locusta unfed from emergence. Although,on post—emergent starvation, endogenously increased tissue activity appears to decline on the 2nd day following emergence,3 daysold unfed adults were selected to compare the changes with similar age group used for the investigation of effect of feeding on the proteinase activity of the tissue in adult Locusts.
As investigated for proteinase activity in the tissue:first attempt was made to observe the effect of a single meal of 30 minutes on the tissue invertase activity. The tissue extracts were individually obtained from a group of 4 to 6 adults at 0, 1, 4, 6, 9 and 12 hours after the meal.
The control included 3 days old and unfed adults. On examining the individual data of various groups and their mean values (Table 17)1 a very small increase in both tissues was found at 6 hours following the feeding period. Laters it falls down. The changes in tissue weight following feeding remains the same as observed before for proteinase activity. The present observation coupled with the fact that midgut retains very little food at 6 hours after one meal, as mentioned before, suggests that further 59.
intake of food within this period may be necessary for more effective
stimulus.
Therefore in the next experiment 3 day old but unfed adults were
continuously fed on bran and grass. The tissue invertase activity vas
measured after 3, 6, 12, 24, 48,Land q6 hours from the commencement of feeding.
The midgut was always full of food,which means that between 3 to 6 hours
from the first intake of food one or more meals are taken as the crop and
midgut become partially empty.
The data for individual insects is listed in the different categor—
ies. Table 18 shows the total mean values including both sexes as well as
for either sex of the different groups. Between 3 to 6 hours from the start
of feeding, only midgut tissue indicates more than double concentration of
invertase than that of control. In the next 6 hours,while activity in the
midgut tissue enhances further, caeca tissue has also a small increase.
Laterl the invertase activity is progressively stimulated in the caeca tissue
for a period of 96 hours of investigation. On the other hand,midgut tissue
has a maximal activity in 24 hours and thereafter small fluctuations occur
below the maximal activity.
There is a small addition of weight in midgut tissue within 3 hours
after the start of feeding,which remains almost unaffected later. The
weight in caeca tissue is significantly added shortly after the commencement
of feeding,which increases further after 24 hours.
These changes are gmerally more marked in females than the males. 60.
Table 17. Mean values showing changes in invertase activity and weight of midgut and caeca tissues, after feeding for 30 minutes to adult Locusta, 3 days unfed following emergence.
Time after Experimental Sex Midgut Tissue Caeca Tissue Feeding Category (hours) Weight Invertase Weight Invertase (mg.) units (mg.) units
Control 118 dg 8± 1. 8± 1 15 ± 1 3± 1. cre 7 + 1 8 ± 1 14 ± 1 3 + 1
gg 8 + 1 8 + 2 17 + 2 3 1: 1 0 119 cf9 10 1" 1 7 + 2 20 + 3 2 + 1 cc? 8 + 1 6 + 2 21 + 4 3 + 1 9 11 + 1 9 ÷ 2 19 + 4 1 ± 4 1 120 o 9 + 1 8 + 2 19 + 2 2 + 1 ecci 8 4' 2 6 + 2 17 + 4 1 + 1 99 10 + 1 10 + 2 21 + 2 2 ÷ 1 4 121 dig 9 + 1 9 1: 1 20 + 3 3 + 1 es a + 1 11 + 2 17 + 6 4 + 2
(il 9 1. 1 8 ± 1 22 + 1 3 ÷ 1 6 122 o 9 + 1 10 + 2 18 + 2 4 + 1 ee 7 + 1 8 + 2 15 + 2 4 + 3 99 10 + 1 12 4" 3 21 + 2 5 + 1 9 123 dig 9 + 3 9 + 2 16 + 3 2 4. 1 cre 6 + 1 9 4. 3 13 + 3 2 + 1 + 4+ (?4? 10 9 3 17 ±5 2 4. 1 12 124 cog 7 ..-1: 1 8 + 2 19 + 3 3 + 1 ee 7 + 1 7 + 3+19 6 3 + 2 9.9 8 + 1 10 4. 1 19 4. 5 3 + 2
61.
Table 18. Mean values showing changes in invertase activity and weight of
midgut and caeca tissue, after the commencement of feeding in
unfed Locusts, 3 days unfed following emergence.
Time after the Experimental Sex Midgut Tissue Caeca Tissue Commencement of Category Feeding Weight Invertase Weight Invertzse (hours) (mg.) units (mg.) units
Control 125 es) 8± 1 7 1 17±' 2 3± 1 7 ± 1 5±'2 15 ± 2 3 ± 1 99 10 ± 1 9 1 20±2 4±1 3 126 es? 10± 1 7 + 1 26 ±4 3±1 did 9 ± 0 6±'2 20 ± 5 3±'1 99 12 ± 1 8± 2 31 ± 5 4± 1 6 127 cr 9 10 ± 1 14±2 23±3 3±1 9 ± 1 13 ± 3 19 ± 4 2±1 99 12 ± 1 15 ± 2 26 ± 3 3 4. 1 12 128 11 ± 1 18 ± 3 25 ± 3 5 ± 2 9 ± 1 13 ± 2 19 ± 1 2±1 99 12 — 0 22 ± 2 29±3 6 + 2 24 129 11± 1 14 ± 2 29 ± 2 10 4- 2 d'e 10 ± 1 13 ± 2 26 ± 4 8±'2 9? 12 ± 2 16 ± 2 33± 5 14 4. 3 48 130 11 ± 1 18± 1 33 + 5 17±3 ea 11 ± 1 17 ± 4 29 ± 2 15±'2 12±1 19±5 37±8 18±5 72 131 10 ± 1 13±3 38±6 19'±6 10 ± 1 12± 3 33 ± 7 19±4 11 — 3 15 8 44±5 18±5 96 132 10± 3 16± 7 32±' 14 20'±9 ee 8±3 16 4" 12 20±4 13±'1 q9 12±1 17 + 4 44±9 28±3 62.
From these observationspit is clear that a single mealt which is mostly passed
out from the midgut within 6 hours after the intake cannotcause sufficient
stimulus for invertase production;but if the gut is full all the time within
this period some marked changes occur in midgut tissue. This may be explain—
ed due to either the mechanical effect of the food on the epithelium in
stretched condition of the midgut or the chemical nature of the food and its
hydrolysed products or a combination of both factors.
Fig.l1 shows the relative changes in the invertase activity of
midgut and caeca tissue after the commencement of feeding in similarly
starved 5th instar hoppers and adults. It appears that in both stages
the secretory response for invertase arises first in the midgut and also
that it is initiated a little earlier in the hoppers than the adults. But
in the latter the rate of production is comparatively higher than the former
within 12 post—feeding hours. Howevert in both cases,caeca tissue shows a
marked response for invertase activity between 12 to 24 hours after the
first intake of food. The slow response by the caeca may be suggested
due to greater damage to its secretory epithelium than that of midgut during
starvation.
Thuslif the lapse of time between the commencement of feeding and
the appearance of increased proteinase and invertase activity respectively
in the tissue of adult Locusta is taken into consideration;it is clear that
feeding stimulates secretion of invertase before proteinase. This can also
be supported by the fact that in unfed adults following emergence the
initially produced enzymes in the tissue show maximal activity for invertase
a day earlier than for the proteinase activity. In other wordsIsecretion
may be called "preferential". The obvious reason for this discrimination INVERTASE UNITS EFFECT OFFEEDINGONTHETISSUEINVERTASE ACTIVITY IN5THINSTARHOPPERSANDADULTLOCUSTA. 3 6 I ( UNFEDFOR3DAYSFOLLOWINGMOULT.)
TIME (HRS)FROMCOMMENCEMENT OFFEEDING
12 I
24. I
FIG. ii invertase activitYinmidgutofhoppers ” .0 II 4S
I
11 II
„ caeca., ll „ 72 I
caeca midgut ofadults
"
96 I " 63. may be the higher percentage of sugars than the protein in the normal diet.
If this is so,quicker production of invertase will be necessary to supplement
the quantity, already present in the gut contents during starvation on continuous feeding.
If the chemical nature of food was an important factor to stimulate enzyme secretiorqsubstances like distilled water or damped cellulose powder, which are regarded chemically inert,would not increase the invertase production. The mechanical effect of water oh the epithelium will not be like solid foodIbut if cellulose powder is ingested in bulk it may cause similar effect as solid food.
Since 3 days unfed adults after emergence had significant rise in the invertase activity of midgut and caeca tissue after 24 hours from the
commencement of feeding. Therefore 3 days starved adults from emergence were kept on normal diet, moist cellulose powder and water. The control had 3 days unfed adults following emergence. Four individuals were dissected at a time from each category after 24 hours were passed on food.
It was observed that adults drank some water frequently but they did not eat cellulose powder in bulk. However some traces of cellulose powder were seen as fine suspension in the digestive juice of the gut.
The individual data and the mean values for the respective groups are listed in Table 19.
Only the group fed on normal diet shows increased invertase activity.
No change occurs in categories kept on water or cellulose powder. This suggests that although these substances have been ingested to a very small extent, they do not cause any stimulus for secretion.
64.
Table 19. Invertase activity in the midgut and caeca tissue of adult Locusta migratoria migratoides, 3 days unfed following emergence and then fed on normal diet, moist cellulose powder and water for 24 hours.
3 days starved from Fed on normal diet Fed on moist Fed on water emergence (control) Experimental cellulose powder Experimental Experimental Category 134 Experimental Category 136 Category 133 Category 135 Midgut Caeca Midgut Caeca Midgut Caeca Midgut Caeca
a cu a) a) e) cu , a) ID N u) in u) u) u) u) in as a) a) to a) as al al 4.1 .-. 4.) ua +3 .--. 4., el +3 ...-.. 4-1 ilt +3 e•-... 4-, In 4) .---. 4-3 (() 43 .-----. 43 01 43 ..---.. 4+ 1/) •4-, ..--... 4-, U) 4 P f+ 4-3 4 ,; k 4-).- 4 op k 4-.) 4 .. k 4-1 4 .. Po +1 4 .• f-i +.1 .0 .• k +.1 4 .• k +3 al CA 4) n-I en ca a) •r4 al al W .43 al 0) CO •ri al 0) 4) *el 0) 01 (U .1-1 CS) CA CI) .1-1 tri 01 CD .1-1 4 c."4 x -rtE>c«-i E>c x.rie>c,-PE>c x •r-IE>C•ri e> C x ,.... a> E> c0 w 4)---0 o w---c z 4) 41 ,-.0CZG).....000 C) 11).-„, g , d...... - c 6 • c 7 43) ,..... a U) .1.-. I-4 l'-", U) ?... " ..''' " u) 1.1 0...1 Ca ).-) 1-4
01 7 11. 16 2 ell 22'.31 10 ce 7 8 14 1.5 e-8 10 15 2.5 c0 7 7. 17 2. 9. 7 10 39 16 9 12.5 16 , 2 9- 8 7.5 14 3 di 7 17 3: 0' 7 4 20 8 c* 4 3.5 11 2 e' 6 6 12 1.5 ? 8 11 24 5 5 8 10 29 10 e 6 5 18 2 .? 7 10 15 2.5 61 7 9 11 2 T 8 17 25 8 04 8 10 10 3 ' oe 5 5 8 3
c1 6 11 10' 4- e 6 12 18 7 e 8 9 10 2.5 ,? 10 15 26 6 8 7 12 4 le 6 15 16 5 y 8 11.,5 21 6 10 12 14 2
i? 9 9 18 5 - 4i 8 14 29 17 I. 5 9 14 3.5 00.6 10 14 5 d 7 8 11 5 d' 8 12 21 4 dr/ 8 11 19 3.5 00. 7 3.5 15 2 e 6. 5 8 1.5 0'1.10 18 30 7 8 9 23 4 0A3 11 17 5 T., 9 9 15 2.5 c. 9 9 40 16 00 9 10 15 4 cz13 14 20 5 c?. 11 11 201 2.5 5. 9 10 34 15 10 10 15 3 5 9 12 18 6 Means 6 4 41 4 1: 2 13 28 10 8 9 16 3 8 10 16 4 4 14 '1'3 +5 +3 .1.1 ±1 +2 +1 ±1 ±2 +2 +1 1 7 7 13 3 8 14 23 7' 7 8 14 3 7 8 14 3 r ±2 °d ±0 ±2 ±3 +1 ±5 ±5 ±2 ±1 +2 +3 +1 ±1 +2 +3 +1 9 9 17 4 8 12 33 14 8 10 18 4 10 1 ±1 ±2 12 18 4 4 ±1 ±1 ±4 ± ±2 16 +3 +1 ±3 ±1 ±2 ±2 ±4 ±1 65,
vi) Possible hormonal control of enzyme secretion. a) Effect of ligaturing the unfed adults of Locusta on the invertase
activity of the midgut and caeca tissue.
As concluded from the observations on the proteinase and invertase activity of hoppers as well as adult Locusta lit is very likely that the initial enzyme synthesislin unfed condition after emergencelis stimulated by endocrine secretions related with normal growth processes after moulting.
Thomsen and Moller (1958) demonstrated that the removal of medial neuro— secretory cells from the brain of adult Calliphora erythrocephala decreased proteinase secretion in the gut. Thus they substantiated that a neurohormone secreted from the medial neurosecretory cells controls the synthesis of intestinal proteinase. Earlier? it was demonstrated in adult Calliphora
(Thomsen, 1954), that neurosecretory material was transported to the nervi oesophagii via corpus ce.rdiacum and ganglion hypocerebrale and probably released into the body cavity. Whether the same process would account for the initial increase of tissue enzyme in Locustalunfed following emergence, was tested but only by ligaturing. For this purposer it was thought that by ligaturing the adult behind the head the neurosecretory material would not be transported farther into the oesophageal nerves. Since the initial increase in the tissue invertase activity was found maximum between 24 to
48 hours after emergence;the ligaturing was performed in adults just after shedding the larval cuticle, 24 and 48 hours old adults. The use of 66. anaesthesia was avoided. Each group of ligatured adults included a set of control adults of similar age which were unfed and unligatured. The invert— ase activity and weight of midgut and caeca tissue for individuals is listed in the experimental sections. These results are summarized in Table 20.
The changes in males and females of each group are almost similar.
From the individual data as well as the mean values of different groupslit is clear that adults ligatured immediately after emergence and also those which are ligatured after 24 hours have invertase activity in the tissue almost half of their respective controls. But those which are ligatured after 48 hours following emergence4 hageprectically no difference from its control, proving also that ligaturing has no adverse effect on the enzyme activity. The tissue weight is also influenced by ligaturing and males appear to be affected earlier than females.
These observations suggest that a factor located in the head,most likely similar to that found in Calliphora,influences the synthesis of invertase in unfed adults for sometime following emergence but during continued starvation this factor becomes gradually ineffective.
The adults ligatured immediately after emergence produced some invertase within the next 24 hours. This may be either due to an earlier release of this factor before ligaturing or a consequence of imperfect ligaturing which may allow some flow of this material through the nervi oesophagi/. Thomsen (1954) pointed out that Calliphora adults deprived of medial neurosecretory cells of brain utilize larval protein of the fat body for an initial growth of organs. In other words,a restricted metabolism can occur in the absence of neurosecretory material. This may also explain the small production of invertase in the adults ligatured immediately after
67.
Table 20. Showing mean values on the effect of ligaturing in the weight and invertase activity of midgut and caeca tissue of adult Locusts,
unfed from emergence. Age (days) Condition of Experimental Sex Midgut Tissue Caeca Tissue from unfed adults Category emergence Weight Invertase Weight Invertase (mg.) units (mg.) units 1 Unligatured 137 dt, 10 + 1 11 + 3 21± 1 4 + 1 ciAd, 10 t 1 11 ± 2 21 — 1 4 — 2 ?? 11 ± 3 12 t 6 20 + 2 4 t 1 Ligatured 138 d? 9 + 1 5 + 2 16 + 3 2 ± 1 soon after emergence es 8±1 5 + 2 15 t 4 2 + 1 ?/ 11 4' 2 7 + 4 19 3 3 + 1 2 Unligatured 139 err? 10 ± 2 9 + 2 17 ± 4 3 + 1 cq 9 + 2 8 + 2 12± 4 2 + 1 r? 11 '± 1 9 + 1 20 + 3 3 + 1 Ligatured 140 8 + 1 5 + 1 14 ± 2 2± 0.0 one day after 2 emergence 7 2 4 + 1 11 + 1 + 1.0
99 9±1 5 + 2 16 I 3 2 ± 1.0 3 Unligatured 141 cr? 7 + 1 7 + 1 17 ± 1 3 + 1 dd 6 + 1 5 +3 15 + 3 3 + 1 + 3 9 8 ± 1 8 + 1 19 3 + 1 Ligatured 142 St? 7 + 1 7 + 1 15 + 2 3 ± 1 2 days after emergence edt 7±1 6 I 2 13 ± 2 3 + 1 + 1 9 It 2 16±2 3 + 1 68. emergence. An attempt was made to ligature the 5th instar hoppers shortly before moulting to adults and to measure the enzyme activity after emergence.
Such hoppers moulted imperfectly and could not survive longer than a couple of hours. Therefore further investigation was not followed. b) Effect of blood injections on the invertase activity of midgut and
caeca tissue.
Day and Powning (1949) suggested a hormone—like factor in the haemolymph of fed insects which causes stimulus for secretion. They based their observation on the increase of mitosis in the nidi of epithelial cells of starved Tenebrio when the latter was injected with blood from fed Tenebrio.
Injection of blood from starved Tenebrio to similar recipients had no effect.
But a similar correlation based directly on chemical investigations was not found. Blood injections in Aedes were unable to demonstrate a hormonal factor to stimulate protease secretion (Fisk and Shambaugh, 1952). However the hormone hypothesis of Day and Powning gained support from the investig— ations of Dadd (1954) on protease activity following a series of blood injection experiments on Tenebrio.
As observed previously both proteinase and invertase activity were higher in the tissue of Locusta which were continuously eating than in those unfed after emergence. If a hormonal factor in the blood is concerned with secretion of digestive enzymes, then the blood of actively secreting insect must influence the enzyme production in unfed insects. An attempt was made 69.
to observe the possibility of this factor in the haemolymph of Locusts. It
was observed that fed hoppers of the last instar provided more blood than fed
adults for the injection technique. Therefore only 5th instar hoppers were used in this experiment. Moreover.3 daysold and continuously fed hoppers
show increasing invertase activity in the tissue which may be very well
considered with sufficient stimulating factor in the blood. Furthert 3 days
old hoppers unfed from moulting have also demonstrated quicker response to
secretion than adults of similar age when food is offered to them. It is,
therefore, expected that these unfed hoppers are physiologically suitable to
respond to any stimulus caused by the injected blood.
The following technique was used to obtain the blood from the donor
and then injecting into the recipient. The syringe was a narrow glass
pipette with one end drawn into a fine capillary and the other connected with
a narrow rubber tubing. Neither the donor nor recipient was anaesthesized.
The individuals for operation were held by a rubber band around a tile.
Drops of blood oozed out when the middle pair of leggs of donor was cut at
the coxo-femoral joint. 3y holding the free end of rubber tubing blood drops
were sucked into the capillary of pipette which was roughly calibrated for a
liquid column of 0.02 ml. When this quantity was roughly obtained the
terminal end of the syringe was slightly inserted in the body cavity of the
recipient through an intertergal region approximately in the middle of the
abdomen and then blood was gently pushed in. The unfed hoppers did not prov-
ide sufficient blood to inject into similar hoppers. The injected hoppers
were able to survive for more than 2 days.
70.
Table 21. Effect of blood injection, from 3 days old and actively feeding hoppers to 3 days old but unfed hoppers, on the invertase activity of midgut and caeca tissue. 3 day old 1 hour after 3 hours after 6 hours after 24 hours after fed hoppers injection of injection of injection of injection of (control) blood blood blood blood Experimenttl Experimental Experimental Experimental Experimental Category 143 Category 144 Category 145 Category 146 Category 147 0 4.1 0 0 0 g ro (13 0 M U U w U CP 0 0 10 U) U 4,0 CD 0 0 U) 0 (A 111 (1) C) •(-1 (0 .1-1 co ..-1 .1-1 .1-4 0 .r1 4'3 (-.1 4-3 C.)
M 0 4-3 4-, ts i its
, C n C n • • u
at u 0) 01
C4' 0) e e e 0 E a e E E d E a E s s
s CI) rn ...... 0 s N v w (13 ta ta
ta 4-1 ta 4) 4) 4.) 4-) 4.) ..0 4 14 r e
er 01 0 0/ 01 er coo 0.) a)I4 01 v
ver •r-1 v .r1 > v •,•1 > U C U C U C) 0 C U (21 C In In In I-1 a; 1.04 U) In tri j= 1-4 (4)
c? 3 3 8 1 e 4 2 16 1 2 5 4 14 2 6 4 12 5 ce 4 3 8 1 9 5 4 10 3 ? 4 5 13 2 2 4 5 12 2 5 4 9 14 2 2 4 6 9 2 7 610 2 cp 3 4 8 . 2 cei 4 3 11 2 4 2 11 1 cr 3 5 8 2 9 5 4 12 2 e 4 4 9 1 d' 3 3 9 1 3 2 6 1 7 8 14 3 d 4 4 9 4 7 6 9 3 5 6 12 3 cr 5 7 11 3 5 5 13 2 61 3 8 1 4 3 11 2 9 3 3 10 2 d' 4 4 12 2 3 4 10 0
6 8 14 2 di 5 8 6 1 e 4 3 13 6 04 4 5 91 a 4 3 7 1 e 4 411 4 7 616 1. d' 4 4 10 1 6 8 12 3 (54 5 3 9 4 6 6 13 4 ,? 6 4 9 3 Mean
4 4 9 1 4 5 10 2 4 411 2 4 4 10 2 4 5 10 1 .5 .3 .7 .8 .7 .0 .8 .3 .6 .0 .5 .6 .4 .0 .7.1 .3 .1 .3 .6 71.
The invertase activity of midgut and caeca tissue of injected hoppers was measured after 1, 3, 6 and 24 hours following injection. 3 days old and unfed hoppers were used as controls. The experiment included males as well as females and each treatment had 3 to 6 individuals at a time.
The individual result is listed in Table 21. An inspection of these values does not show any change in the invertase activity of any group of hoppers.
This leads to the conclusion that if a hormone in the blood of Locusta is responsible to stimulate secretion of digestive enzymes,it perhaps works only under certain favourable physiological conditionst most likely related to nutrition. 72.
HISTOLOGICAL OBSERVATIONS ON THE MIDGUT AND CAECA
OF LOCUSTA MIGRATORIA MIGRATOIDES.
Introduction.
Many forms of histological change, such as the extrusion of cyto— plasmic contents, formation of large vacuoles and various degrees of cell breakdown etc., in the midgut epithelium of insects have usually been related to the process of secretion. Such observations have been discussed by Wigglesworth (1953), who concludes that although some cytological changes are often visible in fresh material most of them result from the mechanical or chemical effects of fixation. From cytological observations on Melanooltas differentialis and Melanoplus femur—rubrum, Woodruff (1933) concluded that the appearance of large vacuoles in the epithelial cells of the midgut and caeca were probably due to faulty fixation or to intracellular stages in the life cycle of gregarines. The actively secreting epithelium does not show cytoplasmic extrusions or cell breakdown.
Duspiva (1939) correlated increased proteolytic activity in
Dytiscus, an intermittent feeder, with the concurrent appearance of cyto— plasmic extrusions from the midgut epithelium. On the other hand, in
Blattella germanica, a continuous feeder, Day and Pawning (1949) demonstrated the greatest enzyme concentration in the midgut lumen when the epithelium was cytologically uniform. They also concluded that discharge of cyto— plasmic globules generally supposed to indicate secretion were probably indicative of cell breakdown.
Dadd (1954) studied extensively the histological changes in the midgut epithelia of the continuously feeding Tenebrio larva and adult and 73. of an intermittent feeder, Dytiscus marginalis adult in relation to protease secretion in different conditions of feeding and non—feeding. He used a number of *elected fixatives in an attempt to distinguish the various artefacts due to fixation, but materials fixed in alcoholic Bouin supplied most information. In the newly moulted larva and recently emerged adults of Tenebrio the midgut epithelial cells were not uniform and the striated border was not developed. In the adults the lumen margin of the epithelium was irregular and globules of cytoplasm were constricted off at the free border. In both stages regularization of the epithelium was marked by the appearance of striated border and the delamination of concentric peritrophic membranes within 3 days of the last moult even when the insects were unfed.
These changes were related to the enzyme build up in similar conditions.
While recent feeding emphasized these features markedly starvation affected them adversely. In unfed mature larva as well as mature adult, cytoplasmic vesicles were extruded from the posterior region of the midgut and were more pronounced after feeding.
Dadd1 s observation on Dytiscus were in agreement with those of
Duspiva as regards the extrusion of cytoplasmic contents and cell breakdown in the anterior region of the midgut and in relation to increased protease activity in the gut lumen following a large meal after starvation. The posterior region of the gut was not involved in the histological breakdown to the same extent.
The observations made in this thesis on proteinase activity in the gut of Locusts demonstrate the continuous secretion of enzyme and its accum— 74. ,
ulation in the gut lumen. The concentration of enzyme in the tissue of
both unfed and fed individuals may be regarded as an index of continuous
synthesis and consequently of its rate of discharge into the lumen. On these
grounds changes in the invertase concentration of the midgut and caeca tissue
will also represent the different degrees of enzyme discharge into the lumen.
It was, therefore, proposed to examine the histological appearance of midgut
and caeca under certain low and high levels of enzyme synthesis.
This was carried out by a simple histological technique. The
tissue (from females only) was fixed in alcoholic Bouin as used by Day and
Powning (1949) and Dadd (1954) and the procedure and technique followed from
Pantin (1959). Sections were cut at 6 u and stained in Malory's triple stain
or Ehrlich's haematoxylin and eosin or Heidenhain's Iron haematoxylin. All
the photo—micrographs are based upon the latter stain. The sections of
midgut were obtained from the anterior region and those of caeca were cut from
the middle part of the anterior caeca.
Histology of the midgut and caeca of fed adult Locusta.
A brief account of the anatomy and histology of the alimentary tract
of Locusts has been given by Hodge (1939). The present description is an
addition to that account. The observations were made on a continuously fed
female about 7 days old, but at the time of dissection the midgut was only partly full of food. In this condition the midgut epithelial layer appears
to be folded probably due to the elastic nature of the underlying connective 75. tissue which has an expanded appearance. In transverse section (Fig.12) the connective tissue helow the epithelium has reticular arrangement of membranes between which lie certain structures very likely muscle fibres.
The epithelium has an arrangement of alternating furrows and folded lobes projecting into the lumen. However, the lumen margin of the epithelium is completely stretched. The epithelial cells are tall and columner with indistinct cell boundaries. Staining by Mallory's triple stain shows that the basement membrane is folded and sometimes forms tubular structures directed towards the inner margin and containing some epithelial nuclei.
The shape of the epithelial nuclei is also not uniform. Towards the inner margin of the lobes they are elongated and laterally compressed measuring
3 to 8 u in width and 15 to 18 u in length. The outer portion of the epithelium has oval nuclei which measure 8 to 10 u in width and 12 to 14 u in length. In the furrows there are almost centrally placed spherical nuclei only, and these measure 10 to 12 u in diameter. The regenerative cells are in groups which are scattered irregularly close to the basement membrane and are more clearly distinct in the furrows. Each furrow has one or more groups of such cells. Their nuclei are almost elliptical and measure 4 to 5 u in width and 7 to 9 u in length. The nuclei other than the regenerative cells are packed with deeply bastiphiLigranules. The cytoplasm of the epithelial cells of the lobes is more finely granular towards the inner margin than the outer or basal part of the cells. The cells in the furrows FIG.12
(a) T.S. OF MIDGUT OF LOCUSTA MIGRATORIA MIGRATOIDES
peritrophic lamella ciliated border unstained spaces In the striated border folded lobe of epithelium
compressed nuclei of epithelial cells
basement membrane
egenerative nuclei connective tissue circular muscle peritoneum
longitudinal muscle
0.2 mm
(b) T. 5. OF CAECA OF LOCUSTA MIGRATORIA MIGRATOIDES
—striated border
epithelial nuclei
regenerative nuclei
vIllus
connective tissue
basement membrane circular muscle peritoneum longitudinal muscle 76. show little granulation. The inner margin of the epithelial cells has a well defined striated border measuring 4 to 9 u in thickness and showing delamination of the peritrophic membrane. At certain places the striations are clumped together and some non—stainable vesicular secretion is also visible which may represent secretion from the cells.
Although an anatomical account of the caeca of Locusts has been given by Hodge, their histological structure has not been dealt. with. As in the midgut the muscularis layer of the caeca consists of an outer group of uniformly spaced longitudinal muscle bundles but the circular muscle fibres form a very narrow band measuring 12 to 14 u in thickness. A connective tissue layer like that of the midgut is absent, but a deep blue staining membrane by Mallory's triple stain is evident below the epithelium.
The latter is thrown into a number of alternating large and small
(Woodruff, 1933). The epithelial cells are columner in shape and their size gradually increases from the top of the villus (14 u by 44 u) to the adjacent crypt (20 u by 70 u). Each cell is provided with a single centrally placed nucleus. The nuclei in the villi are ovoidal and their dimensions also vary between the top of the villus and the adjacent part of the furrow from 15 to
20 u in width and 22 to 36 u in length. The cells in the furrow have spherical nuclei measuring from 21 to 28 u in diameter. These nuclei are packed with deeply basaphil material. The epithelial cells of the villi contain numerous cytoplasmic inclusions similar to those which Woodruff (1933) described in Melanoplus. He interprets them there as mitochondria but in 77.
Locusts the appropriate cytological technique for mitochondria were not employed and the subject requires further study. At all events, the basal region of the cells contain inclusions arranged in the form of longitudinal streaks. The regenerative cells form compact nidi which lie scattered inner to the basement membrane. Their cell boundaries are indistinct and their cytoplasm is less granular than the epithelial cells. Their nuclei are ellipsoidal and measure approximately 8 u by 16 u. The border shows fine striations with very fine granules within it but with no evidence of large cytoplasmic extrusions from the sides and apex of the villus. However, the striated border is not uniform between the villi and very often cytoplasmic contents are extruded from that region. The striated border is approximately
8 u in thickness in the ridge portion but sometimes it is wider in the crypts.
In generall Heidenhain iron haematoxylin stains the caeca more deeply than the midgut and in the caeca the projecting regions of the villi are more deeply basiphil than the furrows.
The arrangement of cytoplasmic inclusions in the cells of the villus is more or less the same as that described by Woodruff (1933) in the fed
Melanoplus as indicating secretory activity.
Histological changes in midgut and caeca after emergence.
In transverse section, the midgut epithelium of the newly emerged adult is more regularly folded (Plate la) and the cells of the folds are not so strongly compressed as they appear in the fed adult. The nuclei in these lobes are mostly ovoidal and measure 6 to 9 u by 18 to 22 u. Between the 78. lobes the cells have almost spherical nuclei measuring 8 to 14 u in diameter.
The cytoplasm of furrow cells show some scattered mitoses. The striated border is almost absent from the epithelial cells and the peritrophic membrane is absent. The epithelial cells in the caeca have an irregular free margin in and between the basal parts of the villi (Plate lb). The inner half of the villi normally show an uniformally developed striated border. The ovoidal nuclei in the cells of the villi measure 8 u by g u while the spherical nuclei between the villi are 10 u to 14 u in diameterw'
In the apical villar cells the cytoplasm is more granular than in the irregular basal cells.
After one day's starvation following emergence the epithelial lining of the midgut is better organized than at the time of emergence (Plate lc).
The striated border is quite uniformly developed and generally contains small and unstainable droplets. Some nuclei are being extruded from the cells.
The cytoplasmic contents are more strongly granular towards the free margin of the cells. One or two mitoses are visible in the regenerative cells of each section. The ovoidal nuclei of the caecal epithelium measure 10 u by
20 u and the spherical ones 9 u to 14 u in diameter. The peritrophic lamellae are not visible within the striated border but a number of concentric membranes are present in the lumen. These stain deep blue with Mallory's triple stain.
The caecal epithelium has an almost uniform striated border with no trace of extruding cytoplasmic material, (Plate ld). The regenerative cells are more compact but mitoses are not visible. The nuclei do not differ in size from ‘b) 79. those of the newly emerged adult.
In 3 day old adults wilich were continuously starved after emergence* the midgut epithelial cells (Platelta) show the uniform presence of small vacuoles below the striated border and the latter has some droplets within it. Some nuclei are also seen being extruded from the cells. The cyto— plasm of the cells is more granular towards the free margin and the nuclei are slightly smaller than before. No formation of new peritrophic membranes is seen within the striated border and those present in the lumen are much less clearly defined than the ones seen earlier. The caecal epithelium is marked with an indistinct striated border and extrusion of cytoplasmic contents* often accompanied by nuclei* from the free margin of the cells
(Plate 11b). Small vacuoles are also visible in the cytoplasm but the nuclei are not affected in size. Regenerative cells do not show mitoses. These changes may be regarded as an indication of cell breakdown due to starvation.
After continuous starvation for 8 days following emergence) more pronounced breakdown of epithelial cells is visible. The midgut epithelial lining becomes taller due to general contraction in the length of the midgut
(Platelic). The inner margin of these cells is losing its striated border* and the entire contour of the margin shows loss of cytoplasmic fragments or discharge of nuclei from the cells. The cytoplasm lacks granules and no mitoses occur in the regenerative cells. The epithelium of the caeca has also an indistinct striated border (Plate Id), and the discharge of cytoplasmic fragments and nuclei are also visible, but here the nuclei are also reduced in size. •
(b )
(e ) d )
2. 0 ,AA
(4. • 80.
The absence of a striated border has also been mentioned in the newly moulted larva and newly emerged adult of Tenebrio (Daddy 1954). Similar observations in the newly emerged Locusta suggests reformation of this structure following moulting. As observed in Tenebrio the appearance of the striated border and the formation of the peritrophic membrane in unfed adult Locusta 24 hours after emergence coincides with the enzyme build up in the tissue.
Histology of the midgut and caeca following continuous feeding for 24 hours after 3•days starvation from emergence.
The adult dissected at this time had its midgut full of food and consequently very much stretched in length. In such a condition the midgut epithelium is very much expanded in transverse section. The depth of the cells is reduced and the connective tissue layer is very narrow. The epithelium is still divided into lobes separated by shallow furrows. The free margin of the epithelium has an uniform striated border through which lamellae of the developing peritrophic membranes are passing into the lumen (Plate nro. At some places striations are not very distinct owing to the presence of peritrophic lamellae but elsewhere they are very clear. The cytoplasm of the epithelial cells shows relatively clear spaces around the nuclei but this may be an artefact of fixation. Otherwise the epithelium is uniform. Scattered granules are mostly present towards the free margin and internal to the nuclei but in the basal region of the celli these granules generally form long streaks. The nuclei of the epithelial cells are mostly placed in the centre. They are ovoidal or spherical, packed with densely 81. basophilic granules, and measure 8 to 12 u by 14 to 16 u. The regenerative cells are massed into groups very close to the muscle layer. Their nuclei are also full of strongly basophilacgranules and measure about 6 u by 10 u.
Mitotic stages are generally not visible but the nidi appear to have more nuclei than in the 3 day old unfed adult. There is no sign of cytoplasmic vesicles or any breakdown of the epithelium.
Although grass fibres never enter the caeca, they are dilated through the accumulation of liquid food materials, after feeding the starved adult for 24 hours. As a result, the villi are more widely spaced but the epithelial cells are deeper. The cytoplasm of almost all cells is densely packed with deeply staining granules vtlich generally form longitudinal filaments in the basal part of each cell. Small clear regions are present around the nuclei. Both regenerative and epithelial nuclei are packed with granular material. In the regenerative cells mitotic figures were not observed, and the nuclei are generally massed together. The striated border is a distinct zone but the striations are not easily observed because the surface may be covered with fine granules or the border contains small vacuoles. At certain places tiny vacuoles are visible beneath the striated border, but the cells do not show discharge of cytoplasmic vesicles or nuclei
into the lumen(PW4111.4).
Since the histological picture described above coincides with
increasingly active secretion of proteinase and, more especially, of invertase, Plate III.
(:3 82.
it can be concluded that in Locusta secretion of digestive enzymes is not accompanied by cellular breakdown or loss of nuclei from the epithelial cells.
If the discharge of cytoplasmic granules through the border is part of the normal process of secretion, then merocrine secretion may occur as a normal digestive process in this species.
Midgut epithelial regeneration in 5th instar hopper of Locusta migratoria migratoides.
Midgut epithelial regeneration has been studied in many insects and reviewed by Braun (1912) who also discussed various modes of renovation of midgut epithelium in relation to the larval moults of endopterygotes. In many endopterygote larvae there is a complete loss of the old epithelium before moulting and subsequent large scale renewal. Willer (1927), too, describes the shedding of the larval midgut epithelium and formation of an imaginal midgut epithelium in the pupal stage of Thysanopteri. Further information is available from Tiegs (1945) who says that comparable epithelial. renovation coincides with moulting in Diplopoda, a similar process occurs in
Collembola (Boelitz, 1933 etc.).
In the Exopterygota, midgut epithelial regeneration is known in very few insects, and is usually assumed to be the slow continuous type of small—scale renewal studied by Hollande (1927) in adults of Orthacanthacris
(Acridium) aegyptia L. 'However, the midgut epithelium is abruptly and completely reformed by the regenerative cells in termites (Weyer, 1935), where it coincides with moults And occurs in all stages and castes of a 83.
number of species, A similar process occurs in cockroaches before moulting
(Weyer, 1936). This was further confirmed by Day and Powning (1949) in
Elattella germanica in which nymphal midgut epithelium is completely replaced
by a new epithelium at the time of the last ecdysis.
The above information suggests that although a replacement of midgut
epithelium at a moult is not uncommon in endopterygote larvae and also occurs
in some archaic forms like Collembela some hemimetabolous Exopterygota also undergo comparable changes. The observations on invertase ectivity in the midgut end caeca tissue of 5th instar hoppers of Locusts showed very little
activity for sometime before the emergence of the adults. This may be
regarded either as due to some factor controlling enzyme synthesis in the
tissue, or to a removal of the midgut epithelium related to moulting. It
was the latter possibility which at first seemed most likely in the light of the above information on Blattella.
It has already been pointed out that the last stage hopper stops
feeding approximately 48 hours prior to moulting and enters a pre—moult condition. Midgut end cceca were, therefore, fixed at approximately every
12 hours after the cessation of feeding to observe any change in the epithelium during the pre—moult period. Only female hoppers were used in this invest— igation. For a.comparison histological observations were also made on the midgut and caeca of an actively feeding last instar hopper.
In the actively feeding hopper the midgut epithelium is almOst like that of the adult except that the depth of epithelial cells is variable 84.
according to the quantity of food present in the lumen. The nuclei are regularly arranged (Plate ara), and the cytoplasm is almost uniformly granular.
The striated border is covered with fine granules but gives no indication of
cell breakdown. Although the peritrophic lamellae were not seen in the border the membrane encircles the food. The caecal epithelium is uniformly provided with a striated border in the villi but between the latter the cells have cytoplasmic extrusions often with nuclei, and thus the striation is indistinct (Plate !3b). The regenerative cells do not show mitotic figures.
During 12 hours after the last meal the midgut becomes completely empty and the depth of the epithelial cells thus increases. The number of nuclei appears to increase in the nidi (Plate Nt) and the cytoplasm of the cells show streaks of fine granules towards the free margin of the cells.
The striated border becomes indistinct due to the appearance of fine granules or to small spaces within the striations. The midgut lumen does not contain a peritrophic membrane. In the caeca, the cells between the villi show an indistinct striated border and their inner margin is stretched and shows extrusion of cytoplasmic droplets (Plate 14). The cells in the villi have a very narrow striated border. Although mitotic figures were not seen in the few sections examined their nidi are more crowded with nuclei. The lumen of caeca contains some fine granules.
Twenty four hours after the last intake of food the midgut epithelium continues to discharge the cytoplasmic granules and the striated border is almost covered with such granules. Further dark staining particles accumulate (P 4a). all round the lumen periphery In the caeca the cells between the villi br
e
V.
yy ot AidA
(P) ( 0)
(q) (e) A1 Id 85.
show the breakdown of their inner margin, and the number of nuclei in the nidi
increases so much as to cover some areas of the adjacent cells. Mitotic
figures are also visible in certain nidi (Plate 10).
Within the next 12 hours the midgut epithelial cells are reduced in depth because the lumen is full of a yellow substance (Plategc). The striated border is packed with granules. There is no trace of a peritrophic membrane either in the cell border or the midgut lumen, but the latter is encircles by layers of yellow granules (Plate lid). The lumen of the caeca has also some masses of yellow granules. The striated border is very narrow and indistinct in the villi and between the latter it is not seen (Plateiaa).
The nidi still contain large numbers of nuclei.
Approximately 6 hours before moulting the midgut epithelium is more stretched longitudinally, and the striated border is not uniform. The midgut lumen is occupied by the yellow: substance (Plate $b). In the caeca the striated border appears to be loosely attached to the cell margin, though it is possible that this is a fixation artefact (Plate lc), the lumen contains the yellow substance which is amorphous in the central part, granular else— where.
Within 24 hours of the last meal more than half of the midgut lumen is full of some yellow substance which looks amorphous in the central part but granular on the periphery. This substance is unaffected by Mallery's triple stain and Ehrlich's heematoxylin eosin but Heidenhain's haematoxylin stains it very lightly. In a longitudinal section covering the anterior part of the midgut, posterior part of the crop and anterior caeca this material is also seen to extend into the crop. The caeca contains less material than cz) (C)
• 86.
the midgut or the crop; The longitudinal section of posterior midgut showed
only the concentration of granular yellow material. The significance of this
material is not clear, though it seems to be a product of the midgut and caeca
cells. The cast off midgut epithelium in the pupa of Dermestes and some
other insects has been referred to as the "yellow body" (see Braun, 1912) but
in Locusta the entire midgut or caeca epithelium is not 'cast off to form such
a mass:
The main conclusion of this section then, is that the reduction of
enzyme activity in the tissue towards the last moult is not due to massive
shedding of the old epithelium. The latter, in fact, persists largely or
entirely into the adult and its reduced secretory activity is a functional
change correlated with the relatively small changes in the appearance of the
epithelium described above.
Taking the above histological data at their face value, one may
summarise the relations between the physiological and histological findings
as follows. The cells of the midgut and caeca of newly emerged adult, with
almost negligible invertase and proteinase activity, lack a striated border,
show no peritrophic membrane, and contain relatively few cytoplasmic
inclusions. Within 24 hours, however, even in starved insects, there is an
increase in tissue enzymes accompanied by the formation of the border, the
delamination of peritrophic membranes and an increase in the cytoplasmic
inclusions. Continued starvation for three days tends to reverse all these
changes except that the striated border persists and there is an extrusion
of epithelial nuclei indicating cell degeneration. Renewed feeding, however, produces marked increase in invertase and some proteinase activity, new 37. delamination of peritrophic membranes end the return of e rich complement of cytoplasmic inclusions. But such en actively secreting epithelium does not show nuclear extrusion nor ere cytoplasmic (secretory) globules to be seen at the free margin of the cells (such as are visible after starvation for
1, 3 or 8 days).
The above observations are supported, so far as they can be, by the work of Day and Powning (1949) on Blattella. Before leaving the histological section, however, one must comment briefly on the limitations of the technique used. Baker (1960) points out that one of the defects of formalin as a fixative is its tendency to induce the formation of small blobs of cytoplasm at the free border of cells, and it could therefore be argued that the presence of formaldehyde in Bouinls fixative was responsible for this pheno— menon (which would then be an artefact) in the newly emerged and starved insects. Eut such extrusions were consistently absent in fed insects fixed in the same way and there is, in fact, no evidence that formaldehyde in the presence of the other constituents of Bouinis fixative behaves in the same way as when used alone. The extrusion of small amounts of cytoplasm (and, for that matter, of nuclei) seems therefore to be a genuine feature of the behaviour of the epithelium in starved insects.
It is not, of course, suggested that the very simple techniques of fixation and staining used here could reveal a fully adequate picture of the cytological changes which accompany secretion. The full investigation of such a relationship, however, is outside the scope of this work. 88.
OBSERVATIONS ON THE DIGESTIVE ENZYMES OF DYSDERCUS FASCIATUS.
(1) pH optima for amylase and invertase activity in the midgut extracts
of adult Dysdercus fasciatus.
The digestive enzymes of phytophagous Heteroptera were studied in
West African Cacao capsid bugs (Goodchild, 1952), Leptocorisa varicornis,
Dysdercus koenigii (Saxena 1954 and 1955) and Oxycarenus hyalinipennis
(Saxena et al. 1958). Both amylase and invertase were reported in these
insects but the observations on the pH optima of various enzymes were only
made in Oxycarenus hyalinipennis. It was, therefore, resolved to study this
problem for amylase and invertase of Dysdercus fasciatus midgut before
exploiting this species for further work.
In some preliminary tests both amylase and invertase were detected
in the midgut extracts of Dysdercus fasciatus. The invertas4 activity was
studied by applying the Sumner's method as used for the invertase activity
in Locusta migratoria migratoides. The amylase activity was also studied
on the same principle. These preliminary observations were made by using a phosphate buffer, pH 6.5.
The pH optimum for amylase activity in the midgut extracts was studied from a culture stock of adult Dysdercus fasciatus. The adults used for this experiment were kept on water for about 24 hours prior to dissection so that their midguts were cleared of the contents. For each treatment a concentrated extract was prepared in distilled water. The dissected midguts were homogenized with a pinch of Kieselguhr and mixed with a known quantity of distilled water and then centrifuged at 1,500 r.p.m. approximately for
15 minutes to get the clear supernatant. The selected pH range was from pH 6.0 to pH 8.0 with a gradient of 0.5 units. Therefore solutions of 89.
phosphate buffers for these pH values were prepared according to the table
given by Cole (1955). These solutions were further checked by Cambridge
pH meter and adjusted accordingly. The midgut extract was divided into
ten samples of 0.5 ml. each. Five of these samples were heated in boiling
water for ten minutes and then cooled down in ice cold water for half an
hour. These samples were the boiled controls for the respective pH values.
Therefore for each pH valueone sample was a boiled control and the other
one with normal enzyme extract. Both the samples received 1 ml. of one
buffer solution. All these samples received 2 ml. of 1.5% starch solution
with 0.01 MNacl solution, and subsequently they were incubated at 400C. for
3 hours. A control with 1.5 ml. distilled water and 2.0 ml. starch solution
was also incubated with other samples. Since this control had everything
except a buffer and enzyme therefore it was called 'reagent control'. The
pH value of the final solutions before incubation could neither be checked
nor adjusted due to unavailability of fine electrodes for this purpose. It
was assumed that the mixture of the extract and the respective buffer solut—
ions would show very little shift from the adjusted pH value in the buffer
solution because buffer solutions are not altered much when diluted with
water.
After the incubationall the samples including the reagent control
were given the usual treatments as described in the technique. The colour density of the diluted solutions of all samples was measured by comparing
those of samples with the reagent control and amylase activity was recorded 90.
in terms of colorimeter scale divisions. This experiment included a number of treatments with different stock extracts. The complete data is given in Table 22. Since the boiled controls of the respective treat— ments did not show any measurable activity therefore these ere omitted in the table.
The observations on the pH optimum for invertase activity in the midgut extract were carried out in the same way as described for amylase.
In this experiment the substrate was 2 ml. of 5% sucrose solution. A reagent control contained 1.5 ml. of distilled water and 2 ml. of 5% sucrose solution. These samples were incubated for one hour only which was quite enough to get appreciable readings. The data for this experiment is mentioned in Table 23. Here also the boiled controls gave very negligible activity.
The data on amylase activity shows that the maximum enzyme activity occurs at pH 7.0 the neutral point. It is also clear that the alkaline range gives higher reading than the acid pH values. On the contrary in the case of invertase activity the acid medium is more favourable than the alkaline side (Fig.13). The maximum invertase activity occurring at pH 6.0, does not lie very near to the neutral range. It is slightly in acid range.
In the more alkaline range invertase activity falls down very markedly. The pH optimum for amylase activity in Dysdercus differs from that observed in cockroaches (pH 6.0) by Wigglesworth (1927b) and later confirmed by Day and
Powning (1949). According to Shinoda (1930a) the optimal activity for amylase lies in the alkaline range in herbivorous and omnivorous insects. In 91.
Table 22. pH optimum for amylase activity in the midgut extract of Dysdercus fasciatus.
Treatment Extract Concentration Amylase activity in colorimeter units No. per ml. distilled water pH values 6.0 6.5 7.0 7.5 8.0 5 midguts, contents 12.0 15.0 22.0 18.0 16.0 mostly washed away. 5 midguts, contents 8.0 10.0 16.0 14.0 12.0 mostly washed away. 9.0 11.0 17.5 15.0 13.0 4 5 midguts, all adults 12.0 14.0 18.0 14.0 12.0 were males. 5 midguts, all adults 25.0 34.0 46.0 40.0 39.0 were females.
Table 23. pH optimum for invertase activity in the midgut extract of Dysdercus fasciatus. Treatment Extract Concentration Invertase activity in colorimeter units No. per ml. distilled water pH values 5.5 6.0 6.5 7.0 7.5 8.0 1 5 midguts. 52.0 74.0 64.0 48.0 22.0 10.0 2 5 midguts. 48.0 58.0 50.0 37.0 17.0 8.0 3 4 midguts. 30.0 38.0 34.0 25.0 16.0 8.0 4 4 midguts. 29.0 37.0 32.0 25.0 16.0 8.0 5 2 midguts. 9.0 12.0 10.0 8.0 5.0 3.0 F1G.13
CURVES SHOWING pH OPTIMA FOR AMYLASE AND INVERTASE IN DYSDERCUS
amylase activity inyerlase activity 130 _ tn 70.
CC 60 _ (2 O 50 . U
40
30.
NJ 20
10 -
5.5 6.0 6.5 7.0 7.5 8.o pH UNITS 92.
Oxycarenus (Saxena et al. 1958) and Trogoderma larvae (Krishna, 1958) the
optimum pH for amylase are 7.2 and 7.5 respectively which are almost neutral
range as observed for Dysdercus.
The optimal pH for the invertase activity is 6.0 which is similar to
that observed in cockroaches (Wigglesworth, 1927; Day and Powning, 1949),
in Trogoderma larvae (Krishna, 1958). In Oxycarenus hyalinipennis — a
Lygaeid bug (Saxena et al. 1958) — the pH optimum for invertase is 5.4 which
suggests an acidic range is suitable for the invertase activity in heteropterout
insects including Dysdercus fasciatus.
According to Wigglesworth (1927b) and Day and Powning (1949) since
the optimal pH values for amylase and invertase are approximately the same
and they have got the same working range, therefore these enzymes can work
at the same time and in the same region of the gut with this pH range avail—
able. But in Dysdercus fasciatus the optimal working conditions with regard
to pH values of amylase and invertase are different. It is very much possible
that in Dysdercus, one pH condition in a section of the gut may be suitable
for a normal working of one of these enzymes while the other one may have
slower activity. It is interesting to mention here that in Oncopeltus
fasciatus, with feeding habits similar to Dysdercus, amylase is secreted
from the salivary glands which digests starch mostly extra—intestinally
(Miles, 1959 and 1960) during the process of feeding. 93.
(ii) Distribution of invertase and amylase in the midgut tissue of adult
Dysdercus fasciatus.
Although invertase and amylase have been reported from the digest—
ive tract of some Heteroptera (Goodchild, 1952; Wigglesworth, 1953; Saxena,
1954 and Saxena et al., 1958) there is no information on the site of their
production in the alimentary canal of these insects. As the experiments on
the pH optima of these enzymes reveal that both invertase and amylase are
present in the midgut extracts it was thought to examine their distribution
in the tissue of different regions of midgut. From the anatomical point
of view, the midgut of Dysdercus fasciatus can be arbitrarily divided into
four sections (Fig.14). This division is only based upon the external
features of the various parts. The first and anteriormost ventriculus is
a bag—like structure which has been labelled as crop in certain Heteroptera.
This leads to the second ventriculus which is very long, coiled and narrow
tube of almost uniform width. This may or may not be one uniform histological section. Because no histological information is available on the digestive
tract of this species this region is considered here as one section of the midgut. The third ventriculus is a spherical structure connected to the second section through a short and very narrow tube. The fourth and the last ventriculus is a short and very narrow duct opening into the hindgut.
A group of fifteen adults of one sex was sorted out from a stock culture comprising the adults of approximately same age. The adults verc dissected in Ringer's solution and their digestive tracts were transected FIG.14
ALIMENTARY CANAL OF DYSDERCUS FASCIATUS
(male) (female)
2nd. ventriculus
3rd. ventriculus
4th. ventricuculus
hindgut 94. into the above mentioned regions. Then these portions were opened longitud— inally and the tissues were quickly washed and dried quickly on a filter paper. The tissues of each section of midgut of all the individuals were pooled and extracted in 5.0 ml. distilled water. The extract of each section was divided into eight portions of 0.5 ml. each. Two of these portions were heated in boiling water for ten minutes and, then cooled down in ice cold water for half an hour. One of thes4 samples was used as a boiled sample for invertase and the other one served as boiled sample for amylase. Out of the remaining six portions,three were used for the measure— ment of invertase activity and the rest for amylase activity. The samples
for invertase activity end its boiled control were mixed with 1.0 ml. phosphate buffer adjusted to pH 6.0 and 2 ml. of 5% sucrose solution;while the samples for amylase activity and its boiled control received 1.0 ml. phosphate buffer adjusted to pH 7.5 and 2 ml. of 1.5% soluble starch solution with 0.01MNacl solution. The entire set of these samples including boiled controls were incubated at 4000. for 3 hours. After incubation the reaction was stopped and the colours were developed as described before. The enzyme activity was measured by comparing the samples with their respective boiled controls.
This experiment was done four times in different sex groups. In each group the number of adults being constant the individuals were of the same sex.
The data is given in Table 24. It shows that the bulk of invert— ase is synthesized in the epithelium of first ventriculus and the second ventriculus provides much less quantilly. In contrast to invertase, amylase
is very poor but its production is also mainly confined to first ventriculus. Table 24. Distribution of invertase and amylase in the midgut tissue of Dysdercus fasciatus. L Treatmenti Sex I Invertase activity in colorimeter units Amylase activity in colorimeter units Number 1st Vent- 2nd Vent- 3rd Vent- 4th Vent- 1st Vent- 2nd Vent- 3rd Vent- 4th Vent- 1i riculus riculus riculus riculus riculus riculus riculus riculus 1 la 1 85.0 15.0 1.0 0.0 6.0 2.0 0.0 0.0 1 lb Females r 86.0 13.0 0.0 0.0 6.5 2.0 0.0 0.0 lc ! 85.5 15.0 0.0 0.0 6.0 1.0 0.0 0.0
Mean = 85.5 14.3 0.3 0.0 6.1 . 1.6 0.0 0.0 2a i 33.0 4.0 0.0 0.0 3.0 2.0 0.0 0.0 2b Males 30.0 3.5 1.0 0.0 4.0 1.0 0.0 0.0 01 2c 32.0 4.5 0.0 0.0 3,0 1.0 0.0 0.0
Mean = . 31.6 4.0 0.3 0.0 3.3 1.3 0.0 0.0 3a 70.0 8.0 0.0 0.0 4.0 2.0 0.0 0.0 3b Females 66.0 12.0 0.0 0.0 i 5.0 0.0 0.0 0.0
3c i 65.0 10.0 0.0 0.0 7.0 1.0 0.0 0.0 _____ Mean = i 67.0 10.0 0.0 0.0 5.3 1.0 0.0 0.0 4a 38.0 6.0 0.0 0.0 3.0 1.0 0.0 0.0- 4b Males i 35.0 5.0 0.0 0.0 3.0 0.0 0.0 0.0 4c____ i 37.0 6.0 0.0 0.0 2.0 1.0 0.0 0.0 Mean _ = ., 36.6 5.6 0.0 0.0 2.6 0.6 0.0 0.0 96.
The quantitative comparison of invertase activity between the corresponding
tissues of the two sexes shows higher concentration in females. This difference is not unexpected if the size of their digestive tracts is compared;
The females are much longer than males and the female digestive tracts are more massive than those of males. This experiment reveals that both invert- ase and amylase are secreted in the digestive tract of Dysdercus fasciatus
and most likely the first ventriculus only is secretory in function while other ventriculi are mainly absorptive areas of the midgut.
(iii) Distribution of invertase and amylase in the salivary gland of adult
Dysdercus fasciatus.
The secretion of salivary enzymes in the phytophagous Heteroptera was studied by some workers mainly in the last two decades. Baptist (1941) demonstrated either invertase or amylase, and in some cases both enzymes, in the salivary gland of a number of species belonging to different families of
Heteroptera. In Dysdercus howardii he reported an invertase only. Very strong amylolytic activity was observed by Goodchild (1952) in the salivary gland of West African Cacao capsid bugs. The invertase activity was not detected. Nuorteva (1954) recorded the presence of amylolytic activity in the saliva of some bugs injuring wheat kernels. This enzyme was found in all the species studied by him and the maximal activity was noted in the vicinity of pH 7.1 but he did not study the invertase activity. Strong amylolytic activity was found in the salivary gland of Leptocorisa varicornis by Saxena (1954) but no invertase could be detected. Both amylase and 97.
invertase were reported to be absent in Dysdercus koenigii (Saxena, 1955).
Bronskill et al. (1958) found both the enzymes concentrated in the lateral
lobe of the salivary gland of Oncopeltus fasciatus. In contrast to his
previous observations Saxena et al. (1958) reported both invertase and amylase
in the saliva of Oxycarenus hyalinipennis. Although no attempt, was made to
study the invertase activity, Miles (1959 and 1960) demonstrated the presence
of an amylase in the posterior lobe of the salivary gland of Oncopeltus
fasciatus. This report differed from Bronskill et al. with regard to the
concentration site in the gland.
These varied accounts reveal that both invertase and amylase were
detected only in very limited number of species, while in the majority of
species either one or the other enzyme was found and sometimes only one of
them was studied. These records were made only qualitatively and were mostly
based upon starch iodine technique for amylase and Fehling's solution for
invertase.
As observed in the last section, amylase activity was very poor
comparable to the invertase activity in the midgut tissue. It wasI thereforel
thought desirable to compare the concentrations of these two enzymes in the
salivary gland. To examine this possibility it was proposed to measure the
activity of these enzymes quantitatively and concurrently in the salivary
extract. The comparative measurement of these enzymes from the same extract
was based upon Sumner's method for reducing sugars. This method was hitherto unused by the previous workers. 98.
Each treatment was run from a concentrated extract of fifteen pairs of adult salivary glands from one sex. The salivary glands were removed by dissecting live adults in Ringer's solution. The head and appendages were removed and then thorax was longitudinally cut on the lateral sides to remove the thoracic terga. The salivary glands were floated in the dissecting medium and finally the main glandsl along with their accessory glandst were gently picked up by their ducts leading to the salivarium. In this way fifteen pairs of glands were homogenized and extracted in 2.5 ml. distilled water. From each extract four samples, containing 0.5 ml. each, were obtain— ed. Two of these samples were reserved for boiled controls for invertase and amylase respectively as already described. Out of the remaining two samples, one was used for invertase and the other for amylase activity.
Finally, these samples were treated in the same way as in the previous section.
The enzyme activity was measured colorimetrically by comparing the colour density of the final solutions of the samples with their respective boiled controls. This experiment included several treatments using the salivary extracts from the adults of either sex in various post—emergence
conditions. The data is given in Table 25.
Invertase activity was never found in any of these treatments.
Amylase activity was always present. There is no appreciable difference in amylase concentration between the sexes. However, it appears that starvation after emergence decreases the amylase production in both sexes. Also in the last three treatments starvation after feeding tends to reduce the amylolytic activity in the salivary extracts. Table 25. Distribution of invertase and amylase in the salivary gland of Dysdercus fasciatus. Treatment Sex Invertase activity Amylase activity Condition of adults at the time cf in colorimeter units in colorimeter units dissection. No.
1 Males 0.0 14.0 One day starved after emergence. 2 Females 0.0 -17.0 One day starved after emergence. 3 Males 0.0 10.0 Two days starved after emergence. 4 Males 0.0 8.0 Three days starved after emergence. 5 Females 0.0 6.0 Three days starved after emergence. 6 Females 0.0 19.0 One day fed after emergence.
7 Females 0.0 16.0 Two days fed after emergence.
8 Females 0.0 18.0 Three days fed after emergence. 9 Females 0.0 24.0 Four days fed after emergence.
10 Males 0.0 15.0 One day fed after emergence.
11 Males 0.0 11.0 3 days fed after emergence.
12 Males 0.0 15.0 24 hrs. starved from a stock culture.
13 Males 0.0 10.0 3 days starved from the above culture.
14 Males 0.0 8:0 3 days starved from the above culture. 100.
Thus to summarise, in Dysdercus fasciatus, the digestive tract
has demonstrated (in previous section) very strong invertase activity but
amylase activity is very weak. On the other hand invertase activity has
not been detected in a fairly concentrated salivary extract whereas amylase
activity is present considerably. This suggests that the entire demand
of invertase is probably supplied by the midgut tissue and the digestion of
sugar is confined solely in the digestive tract. But amylase is mainly
contributed from the salivary gland and it is utilized for the extra—intest—
inal digestion of starch. This view is supported from the experimentsl
evidence on Oncopeltus fasciatus, in which Miles (1959 and 1960) demonstrated
the secretion of amylase from the salivary gland on to a substrate. Then
the substrate was sucked back along with the saliva into the digestive tract
where starch grains were found completely digested.
(iv) Invertase activity in the serial dilutions of midgut extract of
Dysdercus fasciatus.
In the preceding experiments it was observed that invertase activity
was very high in the midgut tissue as well as in the whole midgut extract of
Dysdercus fasciatus. Therefore this enzyme was selected for detailed observ—
ation on its secretion in relation to feeding and non—feeding conditions.
But before starting to study the quantitative measurement of invertase activity
it was essential to work out the calibration of the technique whereby changes
in enzyme activity can be appreciated. Therefore separate dilution turves
were required for the invertase activity in the midgut extract of this species 101.
The method used for this observation was based on the same lines as for proteinase and invertase activity in Locusta migratoria migratoides.
The extracts were made from a stock adults fed on water for 24 hours before dissection was performed. Each stock extract was the contribution of one or more midguts per ml. of phosphate buffer at pH 6.0. Two samples of
0.5 ml. each were obtained from each stock extract and then one of them was serially diluted by the buffer solution in the same way as described earlier for Locusts. The weakest concentration was 1/32 of the strongest sample. The relative enzyme concentration in serial dilutions was represent— ed with respect to the weakest solution. A reagent control was also prepared
with only 0.5 ml. of phosphate buffer at pH 6.0. Then 2 ml. of 5% sucrose solution was added in each sample including the control, and these were incubated at 40°C. for 3 hours. Then the colour was developed in the usual
way.
The colour density of various dilutions was compared by adjusting
the reagent control at zero of the colorimeter scale and colorimeter units
were recorded for each sample. Similar treatments were performed from a number of extracts. The result of all treatments is given in Table 26.
From the data it is clear that within the range 1 to 48 colorimeter units the recorded units are almost doubled corresponding to the ratio of
the successive concentrations. But above 48 units this ratio is not maintained.
Fig.15 shows some curves drawn visually by plotting the colorimeter units against the serial dilutions from treatment Nos. 3, 5 and 6. In these 102.
Table 26. Invertase activity in serial dilutions of midgut extracts of
Aysdercus fasciatus.
Treatment Strength of stock Invertase activity in colorimeter units extract per ml. Relative enzyme concentration in serial dilutions .No, buffer solution. . 1 2 4 6 16 32
1 4 midguts 13.0 26.0 50.0 78.0 100 over 100 2 2 midguts 6.0 11.0 23.0 44.0 68.0 95.0
3 2 midguts 5.0 10.0 19.0 38.0 56.0 82.5 4 1 male midgut 1.0 1.0 3.0 5.0 10.0 22.0
5 1 female midgut 3.0 6.0 13.0 25.0 48.0 76.0 6 1 male midgut 0.0 1.0 2.5 5.0 11.0 23.0
curves the points lying between 1-48 units are almost on the straight part of the curves, As observed from treatment Nos. 4 and 6, very weak concen- trations also show corresponding differences between their colorimeter units e.g. as low as 2 units represent a significant change. Therefore various degrees of low enzyme activity can also be quantitatively differentiated in further experiments.
In future experiments the concentration of extracts has been, therefore,modified to work within 1 to 48 colorimeter units and these units are recorded as direct measure of the invertase activity in Dysdercus fascists no.15 INVERTASE DILUTION CURVES FOR DYSDERCUS
32 1 2 L 8 16 EXTRACT CONCENTRATION. 103.
(v) Variations in the invertase activity and weight of 1st ventriculus
tissue of adult Dysdercus fasciatus, unfed and fed following emergence.
As mentioned in the introduction Dysdercus fasciatus was selected as a contrast to Locusta migratoria migratoides on account of differences in their feeding behaviour. Since preliminary attempts showed weak proteinase and amylase but very strong invertase in the midgut extracts of Dysdercus fasciatus and also the 1st ventriculus tissue vas found to be the main site of invertase productionI therefore this region was used for the determination of invertase concentration in adults which were unfed and fed following emergence.
After emergence unfed adults especially females did not survive more than 4 days. The death rate was very high on 3rd and 4th days following emergence. The surviving adults were never observed to mate and when dissected, ovaries of the females showed absence of development in all age groups. The starvation resulted in shrunken abdomen mostly in 3 or
4 days old adults. The 1st ventriculus was always full of air but the other parts of the midgut contained some larval contents. The invertase activity was concurrently measured in 4 to 6 individuals, including males and females, from each unfed age group and also the newly emerged adults which were dissect- ed within an hour after emergence, The individual data is recorded in the experimental groups (in Experimental section) and the mean values of the respective groups are given in Table 27.
It is observed that soon after emergence the midgut tissue lacks enzyme but on the following day some invertase is produced in unfed adults of both sexes and females have higher concentration than the males. While 104.
Table 27. Mean values showing variations in weight and invertase activity in
the 1st ventriculus of adult Dysdercus fasciatus, unfed from
emergence.
Experimental Age (days) Sex Weight (mg.) Invertase units Category after emergence
148 Newly emerged Male 2.3 4. 0.4 0.1 0.3
Female 2.6 4. 0.2 0.1 - 0.2
149 1 Male 1.8 0.4 3.2 t. 1.5 Female 3.3 ±0.3 4.1 ± 0.8
150 2 Male 1.7 ±0.3 2.0 ± 0.9
Female 3.2 t. 0.5 6.7 - 2.1
151 3 Male 1.3 ± 0.4 1.0 t 0.7 Female 3.0 0.5 3.8 ± 1.7
152 4 Male 1.1 1. 0.3 . 0.5 10.7 Female 2.8 ± 0.6 1.5 4" 1.1
unfed males do not show further increase, maximal activity of tissue invertase occurs on second day in unfed females. When starvation is continued film
4 days after emergence, tissue invertase gradually decreases in both sexes after the maximal activity. These variations are similar to those observed for proteinase and invertase in the midgut and caeca tissue of adult Locusta, 105. unfed from emergence, or the tissue invertase in 5th instar Locusta hoppers, unfed following moult. Thus the initial enzyme build up in Dysdercus may
also be regarded as a pre-requisite for an actively feeding stage following
emergence.
The invertase activity was also determined in the tissue extracts
of 1st ventriculus of adults which were continuously provided with food after
emergence. Four to six adults from each age group were used at a time.
The individual data is given in the relevant experimental category and their
mean values are listed in Table 28.
In the presence of food, the tissue invertes e is double that of
the starved adults within 24 lee 48 hours after emergence and a peak of maxi-
mal concentration is observed on the 3rd post-emergent day in both sexes.
Thereafter, considerable fluctuations occur and another peak of higher
concentration is developed on the 8th post-emergent day. The invertase
activity and tissue weight are comparatively higher in females than males.
During the 3 days following emergence and in the continuous presence
of food, the 1st ventriculus is always full of food and this condition was
also accompanied with progressive development of eggs in the ovary. Later
on, 4th to 6th days, the variation in invertase activity coincided with
partially full or empty condition of 1st ventriculus in many females which
had their ovaries with more developed eggs than the females with completely
full gut. Thus increasing enzyme activity in the early stage of egg develop-
ment may be suggested due to initial nutritional requirements, It was
observed that the majority of females lay the first batch of eggs between 6 106.
Table 28. Mean values showing variation in weight and invertase activity
in the 1st ventriculus tissue of adult Dysdercus fasciatus,
provided with food following emergence.
Experimental Age (days) Sex Weight (mg.) Invertase units Category after emergence
•153 1 Male 2.5 ± 0.4 6.0 ± 1.3 Female 5.0 4. 0.8 10.2 ± 3.2 154 2 Male 2.5 ± 0.4 6.6 ± 1.2 Female 5.3 ± 0.7 19.3 + 2.1 155 3 Male 3.0 ± 0.2 9.0 ± 1.3 Female 6.1 ± 0.8 30.7 ± 5.4 156 4 Male 2.2 ± 0.4 6.8 ± 2.1 Female 5.6 ± 0.8 17.4 ± 6.0 157 5 Male 2.4 ± 0.4 7.1 ± 1.9 Female 5.2 ! 0.6 33.3 + 10.2 158 6 Male 2.6 ± 0.5 4.7 ± 1.2 Female 3.3 ± 0.5 14.8 ± 1.9 159 7 Male 2.2 ± 0.4 4.2 ± 1.1 Female 5.7 ± 1.0 37.3 + 8.0 160 8 Male 2.5 ± 0.5 7.5 ± 2.6 Female 6.2 ± 1.3 53.5 ± 11.8 161 9 Male 2.8 ± 0.5 5.2 ± 2.0 Female 4.5 ± 0.9 29.3 ± 12.6 107. to 8 post—emergent days and the indications for the development of next batch of eggs, were clearly observed from 7th day. These are supported by the data of Singh (1954) on this species. According to him most of the females
ley their first batch of eggs between 6 to 8 days after emergence at 27°C.
when food is supplied on alternate days, and that the second batch of eggs
is laid after a further 3 to 4 days. Therefore the second peak of invertase
activity i.e. on 8th day, is very likely related to the nutritional require— ment for the development of 2nd batch of eggs in females and for more fertile
condition in males.
The individual data for unfed experimental groups and the mean
values in Table 27, show that the tissue weight is very slightly increased
in females on the day following emergence and later the changes are very
little. On the other hand, in males, tissue weight declines following emergence without any increase. In males, fed continuously following emergence (Table 28), the increase in tissue weight is very little, but in
females the increase is more significant and its variation corresponds almost
to the enzyme activity (Fig.16). However, the individual tissue weight bear no constant relationship with the corresponding invertase activity.
(vi) Effect of feeding on the weight and invertase activity of 1st ventricul—
us tissue of adult Dysdercus fasciatus.
In the last section it was observed that if food is available, the
invertase activity is significantly increased within 24 hours following
emergence. In the present experiment it was proposed to examine the no. 16 WRIATION IN WEIGHT Ale INVERTASE ACTIVITY OF T. VENTR1CUUJS TISSUE OF DYSDERCUS FASCIATUS FOLLOWING EMERGENCE kworps• activity inmale unfed —••--e— • Image . -- --0-- „ male' ted -n-9- female'
9
TIAIE(DAYE) AFTER LNERGENCE 108. secretory response caused by food in adults following prolonged starvation for 3 days from emergence. It was thought that water, being a component of normal diet, might also influence the secretion. If that is so it may account for a mechanical or physical nature of stimulus es well.
For this experiment adults 3 days unfed following emergence word regarded as suitable because as observed before, such insects were unable to produce any more enzyme without food. These insects were either provided with distilled water or germinating cotton seeds. Invertase activity was concurrently measured for both the categories at 3, 6 and 24 hours after the provision of these substances. A control consisting of 3 days old end unfed adults was used for comparison. The individual results are listed in the relevant experimental groups and Table 111 includes the mean values.
It was observed that feeding response wes not immediate in all insects for any type of food. However, few insects were attracted to either diet within an hour of food provision but later the number increased further.
Mating also occurred in adults on normal diet within 3 hours following the provision of food and the 1st ventriculus of these insects was always full of pastey food at the time of dissection.
On comparison from the control both individual data and the mean values at different intervals, after the food is provided, show that on the normal diet invertase activity is significantly increased within 3 hours and continues further in both sexes but the changes are always more pronounced in females (Fig.17). On the other hand, distilled water alone has a comparat— ively small effect which is significant after 6 hours but only in females. Table 29. Mean values showing effect of feeding on the weight and invertase activity of 1st
ventriculus tissue of adult Dysdercus fasciatus, unfed for 3 days following emergence. Control (unfed for 3 days following emergence). Experimental Category 162. Sex Weight (mg.) Invertase units Male 1.2±0.3 0.6 ± 0.6 Female 2.8 ± 0.7 4.4 ± 2.4 Time (Hours) Sex On distilled water On germinating cotton seeds. after provision of food. Expt. Weight (mg.) Invertase units Expt. Weight (mg.) Invertase units. Category Category + 3 Male 163 1.3 -1. 0.5 1.0 - 0.0 164 1.6 0.7 1.3 1.3 r? I ..oo Female 2.8 4. 0.4 2.3 ± 1.2 3.8 ± 0.7 8.0 ± 2.8 6 Male 165 1.7 + 0.3 1.7 + 1.5 166 1.8 ± 0.3 3.5 + 1.3 Female 2.8 ± 0.7 9.8 ± 2.0 3.8 ± 0.6 13.8 ± 2.4 24 Male 167 1.2 ± 0.5 3.8 ± 2.2 163 2.3 4. 0.4 9.0 ± 3.2 Female 2.8 + 0.4 10.8 ± 3.6 5.6 + 1.0 27.6 ± 9.2 V EFFECT OFFEEDING 1ST. VENTRICULUSOFDYSDERCUSFASCIATUSUNFEDFOR
IN ERTASE UNITS _ 3 DAYSFOLLOWINGEMERGENCE o 10 20 30_ _...._._invertase activityinmole,.fedonwater • •••• ••• • 3
...• TIME (HR5)AFTERPROVIDING FOOD woo 6 • •••••
amb •ammo41.••ma ••••• • ••••.••••••= ON ,, male,fedongerminatingcottonseeds /1 ,, female, . femal, FIG. 17 •••• ••• TISSUE INVERTASEACTIVITYOF 12
8 , „ • ••••• • •••••••••••••• • •mo
•••• • tam. • es..do.
• 4... ••••••.4=0 am.. • •••• •40 24 110.
Leteroo marked change is observed for a period of 24 hours.
The tissue wkight is significantly increased between 6 to 24 hours it both sexes on normal diet. Similar changes are not observed in adults provided with distilled water.
From the above data it can be concluded that in Dysdercus, the secretion of invertase is partially influenced by the ingestion of distilled water, a chemically inert substance, which may cause the stimulus by mechanical or physical means. But the presence of normal food is essential for higher production of invertase activity. DIPEPTIDASE ACTIVITY IN LOCUSTA MIGRATORIA MIGRATOIDES AND DYSDERCUS
FASCIATUS.
(a) Introduction.
The investigation, of various components of proteolytic enzymes in
the digestive juice of insects has received very little attention. A
dipeptidase capable of hydrolysing dipeptides and tripeptides unaffected by
trypsin, was partially separated from trypsin in the midgut contents of
Fe4oplaneta (Wigglesworth, 1928). However, Aminopolypeptidase, carboxypoly—
peptidase and dipeptidase have been demonstrated in Carabus, Dytiscus, Bombyx
mart, Orthoptera and some caterpillars (from the textbooks; Raeder, 1953
and Wigglesworth 1953). According to Roeder in carabids (Schlottke, 1937)
and Bombyx mori (Shinoda, 1930a) the dipeptidase is only an endoenzyme which
suggests that complete hydrolysis may not be necessary for absorption.
Wigglesworth also suggested the same idea from his conclusion that whereas
proteinases are active in the gut lumen, peptidases are much more abundant
in the cells. This interesting idea was examined in Locusta and Dysdercus
by investigating the distribution of dipeptidase in the tissue end contents
of midgut and salivary glands.
(b) Method for dipeptidase activity.
The dipeptidase activity was studied by paper chromatography. The
extracts of tissue as well as contents were made in 1 ml. phosphate buffer
adjusted to pH 8.0. A 5% solution of DL—alanyl•glycine (L.Light & Co.) was used as substrate and the mixture of the extract and substrate was incubated at 40°C. The samples were placed on a square sheet of filter paper (8" x
8" approximately) on about an inch spaced points which were previously treated
112.
with a few drops of 0.01M mercuric chloride solution to stop further enzyme
activity on the paper. One or more sheets were simultaneously used in a
frame (Datta et al., 1950) which was equilibrated for an hour in a Shandon
developing Chamber and then solvent was added to run the chromatogram in
ascending way. The solvent was a mixture of 9 : 1 (v/v) 2—Methoxy—Ethanol
(B.D.H.) and water (Bender, 1951). When solvent reached the top of the
papers, these were removed and dried at room temperature. It was sprayed
with 0.16 ninhydrin (Hopkins and Williams) solution in 9 t 1 (v/v) mixture of
n—fkitanol and water. After drying the paper at room temperature, colour of
the spots were developed by heating the paper at 1000C. for a minute.
Since dipeptidase splits a dipeptide into free amino acids, there—
for in a preliminary experiment solutions of DI, alanyl.glycine, alanine and
glycine were used as markers to know the positions of these spots on the
paper. It was observed that alanine travelled further than glycine while
alanyl—glycine formed a big spot between the positions of other two spots.
(c) Dipeptidase activity in Locusta migratoria migratoides.
In the first experiment the tissue and contents of midget and caeca
were separately extracted in 1 ml. buffer solution from 3 days old adult
female unfed from emergence. From each extract 0.4 ml. was mixed with
equal quantity of substrate solution and subsequently 0.01 ml. sample was
placed on the paper at 0# 1 # 3 and 24 hours after incubation. Similar
quantity of extract as well as substrate was used as control. It was
observed that substrate was hydrolysed within an hour in all the samples
except the control. 113.
The second experiment included the crop contents also and the extracts were obtained from a mature female which was kept on water overnight.
Each extract was divided into two portions of 0.4 ml. each. One of them was heated in boiling water and used as boiled control with substrate. The samples were placed on the paper at 0# 1 # 21- and 3 hours after incubation.
In these extracts dipeptidase activity was observed within 30 minutes.
Visually the intensity of spots of alanine and glycine was less in crop contents than those of caeca contents but equally developed as compared to midgut contents.
A pair of salivary glands was obtained from a mature female and freed from the adhering thoracic muscles and the fat body. Then washed in
Ringer's solution and extracted in 1 ml. buffer solution. Although the glands were cleared off the adjoining tissue as far as possible, an equivalent amount of washed muscles and fat body tissue were also extracted separately and used for comparison. Each extract had its boiled control. The substrate control was also included. Samples from incubating mixtures were withdrawn at 0# 1 and 2 hours after incubation. Both fat body and salivary gland extracts hydrolysed the substrate within 15 minutes but muscle extract took about an hour to do so. The controls were unaffected.
This suggests that the dipeptidase is present in the salivary glands of
Locusta dnd also that muscles and fat body have intracellular dipeptidase.
In a final experiment on female Locusts, starved overnight, dipep— tidese activity was again observed in caeca tissue, caeca contents, and midgut 114. contents within fifteen minutes of incubation but midgut tissue extract could hydrolyse the substrate after 2 hours of incubation, suggesting that dipep— tidase was probably weaker in this tissue.
(d) Dipeptidase activity in Dysdercus fasciatus.
In a preliminary experiment 4 midguts were extracted in 1 ml. buffer solution. This was divided in two portions of 0.4 ml. each and one
of them was used as a boiled control. Each portion was given 0.4 ml. of
5% Alanyl glycine as substrate. From each mixture 0.01 ml. sample was withdrawn at zero, 1, 3 and 24 hours after incubation, and placed on the paper. A substrate control sample was also included. It was observed that experimental samples hydrolysed the substrate within an hour. Thus it was concluded that a dipeptidase was present in the total gut extract of Dysdercus fasciatus. It waso thereforelproposed to investigate the distribution of dipeptidase in the tissue and the contents of different regions of midgut and the salivary gland extract. This was carried out in two sets of experiments, one for the tissue and the salivary gland and the other for the midgut contents,
For the determination of dipeptidase activity in the tissue, ten midguts were dissected from females only, and transected into 4 regions as described in Fig.13. These were slit open longitudinally to wash the gut contents by Ringer's solution and were quickly dried on a filter paper.
Finally, tissues of each region of all the guts were collectively extracted in 1 ml. buffer solution. The salivary gland extract was the contribution of 5 pairs of glands including their ducts from females in 1 ml. buffer 115.
solution. All the extracts were divided into en experimental and a boiled
control sample as in the preliminary experiment. Separate sheet was used
for each extract and 0.01 ml. of extract—substrate mixture was obtained at
zero, 1, 3, 6 and 24 hours after incubation to be applied on the paper.
Similar quantity of a mixture of respective boiled control and substrate as
well as substrate only, was also applied on each sheet.
In the second series, the contents from first sections of the mid—
gut of five females were washed in 1 ml. buffer solution. The rest of the
procedure was the same as for tissue dipeptidase.
The comparison of the spots showed that dipeptidase activity occurred
in all tissue extracts in an hour following incubation. Similar result was
found for the salivary gland extract.
As regards the midgut contents strong dipeptidase activity occurred
within an hour in the contents of 1st ventriculus but 2nd ventriculus demonstrated the hydrolysis of substrate in 6 hours. The third ventriculus contents did not show any dipeptidase activity within 24 hours of incubation.
These observations suggest that dipeptidase is not localized to any particular region of the digestive tract of Dysdercus fasciatus. On the other hand, the lumen of 1st ventriculus contains more dipeptidase than the lumen of second ventriculus. Therefore 1st ventriculus may be regarded as main site for final hydrolysis of protein diet in this species.
(e) Discussion.
The foregoing observations on Locusts and Dysdercus are based on semi-..quantitative method and thus a quantitative distribution of dipeptidase in different parts of digestive organs can not be assessed. But, however, 116. in Locusta, a dipeptidase capable of hydrolysing alanyl-glycine, is fairly distributed in the midgut and caeca tissue; in the midgut, caeca and crop
contents, and the salivary gland. In Dysdercus, also, a dipeptidase is not
localized in only one region of the digestive tract. Thus this dipeptidase
is not the so-called Pendoenzyme" (a term wrongly used for an enzyme present in epithelium by some authors) in Locusts and Dysdercus which would mean that alanine and glycine are set free in the lumen of the gut before absorption of digested food.
Schlottke (1937a and b), in carabids and Periplaneta, although observing a dipeptidase active everywhere in the gut yet suggested a ferment chain in the activity of proteolytic enzyme by observing more powerful dipeptidase activity posterior to the site of proteinase activity (in crop).
Similar suggestion is available from the text book of physiology by Roeder
(1953) that the proteolytic enzymes may be arranged serially in the gut, those capable of hydrolysing higher molecules may be located more anteriorly.
This idea is not supported by the presence of a dipeptidase in the salivary glands of tocusta and Dysdercus. The salivary dipeptidase probably supple- ments the quantity in the regurgitated digestive juice of the crop following feeding in Locusta. In carabids, Schlottke (1937a) observed an increase in dipeptidase in the crop following feeding, which was accounted for the dipeptidase contained in the food. In Dysdercus, a dipeptidase in the saliva may be either involved in the extra-intestinal digestion of protein food or it may be transported to the midgut with the intake of food in a manner similar to that observed in Oncopeltus fasciatus (Miles, 1959). However,a watery 117. saliva is secreted by Dysdercus also in the same way as in Oncopeltus.
Whether this is involved in the digestion of protein extra—intestinally has not been demonstrated.
The presence of a dipeptidase in the tissue of thoracic muscles and the fat body of Locusts may be concerned with the metabolic functions in those parts. 118.
DISCUSSION.
Some results have already been discussed in the relevant sections.
It is therefore proposed to discuss only the remaining observations in the present section.
The gut contents of newly emerged Locusta migratoria migratoides have abundant proteinase which has been carried over from the last nymphal s stage. The presence of considerable protease activity in the newly moulted
Tenebrio larva also demonstrates the carry over of enzyme from the previous
stage (Dadd, 1954). But the newly emerged adult Tenebrio has almost negligible protease activity in the whole midgut extracts. This suggests
that the intermediation of a pupal stage during the metamorphosis does not benefit the imago by providing the larval stock of digestive juice, and as
seen in Tenebrio (Dadd, 1954) the newly emerged adult has to synthesize some
enzyme before the resumption of normal feeding (5 or 6 days following emergence). In Locusta feeding is normally resumed at twelve hours following emergence (Chen, 1954), enzymes being readily available to start digestion.
Thus feeding is approximated to a more continuous process in a hemimetabolous
insect such as Locusta than a holometabelous insect like Tenebrio.
According to Babkin (1944) secretion of a digestive enzyme involves two fundamental processes, the elaboration of organic substances in the glandular cells and the subsequent discharge of this material from the cells.
He named these processes as "ecbolic" and hydrelatic respectively. In mammals, the secretion of digestive enzymes may be spontaneous, continuous or intermittent. Babkin describes "spontaneous" secretion as a result of 119.
inherent ability of certain secretory cells to elaborate and discharge the digestive juice continuously without being initiated by a neural or humoral mechanism, although such mechanisms can increase or decrease secretion.
According to him "continuous secretion" is the property of such secretory
cells which cannot secrete spontaneously and their secretory ability depends upon external stimuli. Theftintermittent" secretion occurs only under the
influeneeof adequate stimuli.
It is difficult to make a clear distinction between "spontaneous"
and "continuous" secretion in the midgut epithelium of insects because the
existence of stimuli similar to those in mammals (such as neural or hormonal) has not been definitely established. The fact that some enzyme is produced
in unfed Locusta and also Dysdercus following emergence may represent a
"spontaneous" secretion. But a stimulus caused by continuous feeding is required in order to raise enzyme production to the normal level. Therefore,
in these insects continuous secretion is in fact the normal process of digestion. Proteinase activity in the tissue of midgut and caeca of Locusts,
fed or unfed following emergence has been recorded as very low in comparison
to the proteinase activity of the whole gut, and since the midgut contents do not appear to have an activator for tissue proteinase we may conclude that
the ecbolic and hydrelatic processes occur simultaneously and the digestive juice continuously accumulates in the gut lumen. The continuous secretion
of digestive enzyme in Locusts is in accordance with its continuous feeding
behaviour. Whenever feeding is stopped for a temporary period such as
before moulting, the process of secretion is slowed down but not stopped
completely. This is supported by the very low invertase activity in the 120. midgut and caeca tissue of 5th instar Locusta hoppers in the pre—moult stage:
Further evidence of continued secretion in the pre—moult stage is available from the higher concentration of proteinase in the gut of newly emerged Locusta than in that of 5th instar hoppers in the early pre—moult condition
(approximately 12 hours after the last meal).
The continuous secretion of proteinase involving simultaneous uccbolie and "hydrelatic" processes is also concluded from the following result. When adult Locusts, starved 3 days after emergence, starts feeding continuously, the marked increase in the proteinase of total gut is parallel with similar change in tissue proteinase and also that tissue proteinase is very little as compared to its concentration in the total gut which means the storage of digestive juice in the gut lumen.
The low level of enzyme concentration recorded in Tettigonia,
Stenobothrus (Schlottke, 1937b) and Periplaneta (Schlottke, 1937c); Blattella and Periplaneta (Day and Pouning, 1949), after starvation, also suggests a slow rate of continuous secretion which is stimulated by intake of food.
In Tenebrio unfed following emergence, some protease activity is always detected in the midgut tissue as well as in the contents (Dadd, 1954) though the latter shows much higher concentration than the tissue. It was therefore suggested that synthesis and discharge of enzyme occur together and it is stored in the gut lumen. In Tenebrio larvae protease activity is markedly decreased following prolonged starvation. This has been suggested to be a result of slow secretion rather than complete cessation of enzyme production. On the basis of these observations Dadd suggested a "continuous', secretion in Tenebrio. 121.
In contast to the "continuous" secretion of almost continuous feeding insect such as mentioned before) midguts of female mosquitoes Aedes aegypti have a very low residual protease activity which increases twenty—fold' following a blood'meal (Fisk and Shambough, 1952). This demonstrates an
"intermittent" secretion influenced by a food stimulus which is also ingested intermittently. In another intermittent feeder, Dytiscus marginalis, Dadd
(1954) explains a different type of secretory condition in which "ecbolic" activity in the midgut tissue is spontaneous but hydrelatic process is dis— continuous and depends upon the feeding stimulus.
The anterior and posterior parts of the midgut of Tenebrio, both larva and adult, differ in their histological appearance (Dadd, 1954). After
3 days starvation following emergence the anterior region has almost uniform epithelial lining with very few cytoplasmic extrusions, whereas the posterior region shows irregularities in the appearance of the striated border and the presence of large numbers of cytoplasmic extrusions. In mature adult es well as larva the anterior midgut has very uniform epithelium without any indication of cytoplasmic extrusions but in the posterior midgut such extrusions are more pronounced than those of the starved insects. The observed increase in protease activity was ascribed to the increased number of secretory cells but this was not supported by the histological appearance of the midgut. In Locusts, although midgut and caeca differ anatomically, there appears to be no contrast between them like the histological appearance of the anterior and posterior midgut of the Tenebrio.
Although the present histological investigations are based on simple 122. methods only, some results are worth consideration. Extrusion of nuclei
and cytoplasmic material is seen from the epithelia of midgut and caeca of
3 days old adult Locusts, unfed following emergence. Subsequent feeding
for 24 hours is marked with a rich compliment of cytoplasmic contents, uniform striated border and absence of cytoplasmic extrusions. These
changes are accompanied with increasing concentration of proteinase and
invertase in the midgut and caeca cells.
Since in Locusts it is very possible that continuous secretion
involves continuous tlecbolicv and "hydrelatic" processes, it can be inferred
that in this species normally hydrelatic process occurs through histologically uniform epithelium rather than by way of cytoplasmic extrusions or cell
breakdown as observed in the midgut of Dytiscus or the posterior midgut of
Tenebrio (Dadd, 1954). The cytoplasmic extrusions or the discharge of
epithelial nuclei from the midgut and caeca of starved Locusts, may be regarded only as indications of cell breakdown due to starvation. A similar process occurs in the midgut of starved Blattella, but when fed the greatest enzyme secretion is correlated with cytOlogically uniform epithel—
ium (Day and Powning, 1949).
The histological preparations of midgut and caeca of Locusts (kept on food for 24 hours after 3 days starvation following emergence) did not show mitoses correlated with increasing enzyme activity in the tissue.
Moreover, whereas the number of regenerative nuclei, as well as the number of mitoses, appear to be greater in the caeca of pre—moult hoppers (24 hours after the last meal) than in those of the actively feeding 5th instar hoppers, 123.
the reverse is true of the invertase activity, which is very low in the
former in comparison to the latter. These observations suggest that enzyme
secretion and especially its synthesis (i.e. ecbolic process) is not related
to mitoses as suggested by Day and Powning (1949). Dadd (1954) supported
the hypothesis of Day and Powning on the basis of a correlation between the weight and protease activity of midgut tissue of adult Tenebrio. Although he has not examined the relationship between the mitoses and the tissue enzyme, increased protease activity has been suggested as a result of increase in the number of secretory units (i.e. epithelial cells).
Brachet (1957) summarises many investigations which conclude that protein synthesis in various tissues depends upon the concentration of ribonucleic acid (RNA). Since all enzymes are proteins therefore their synthesis also depends upon RNA concentration in the cells of digestive glands. It has also been established that glandular organs (like pancreas, salivary glands, gastric and intestinal mucosae) are rich in RNA. Somewhat similar conditions occur in organs where mitoses are frequent. Thisso in the light of these biochemical data and the present histological observations it seems reasonable to suggest that in the midgut epithelial cells of insects, enzyme synthesis occurs in a manner similar to the digestive glands of vertebrates in which mitosis is out of the question.
The effect of feeding in previously starved Locusta adult is marked by en increase in the tissue weight which is much greater for caeca than for the midgut while invertase production IS comparatively high in the midgut tissue. Similar changes occur in 5th instar Locusta hoppers. These data suggest that changes in the enzyme concentration of the tissue are not the 124. main cause for marked variations in the tissue weight. Caeca of Orthopteroid
insects are more actively absorptive in function than the midgut. This has
been demonstrated quantitatively in cockroaches (Treherne, 1957) and
Schistocerce gregerie (Treherne, 1958a) in which a•solution of 14c glucose,
injected into the gut lumen, mainly passes through the wall of caeca to the heemocoel and it is rapidly converted to trehalose. In Schistocerca,
Treherne (1958a) also observed that some 14c becomes incorporated with the
glycogen in the caecal cells and therefore suggested that some glucose is utilized by the cells of caeca for metabolism. It is,therefore,very reason— able to conclude that the great increase in the bulk of caeca tissue of
Locusta, following feeding, is a result of that tissue having higher metabolic activity than midgut, and that cell multiplication by mitoses is not involved
to change the tissue weight.
In some insects, including Locusta, the peritrophic membrane is deleminated from the surface of the midgut epithelium (Mercer and Day, 1952).
In recently emerged unfed adult Locusta, the formation of peritrophic membrane runs parallel with the increased secretion of the digestive enzymes. But if starvation is continued for 3 days, both digestive enzyme activity in the tissue and further production of peritrophic membrane is adversely affected.
Subsequent feeding enhances tissue enzyme activity and again there is a parallel change in the membranes which are now more numerous. Dadd (1954) found similar changes in the larva and adult Tenebrio, and supported the view that feeding increases the rate of production of peritrophic membrane in certain insects (Waterhouse, 1954). This correlation is also obtained from the histological observations on Locusts as mentioned above. This is further 125.
confirmed by the complete absence of peritrophic membranes in the empty
midgut of non—feeding 5th instar Locusta hoppers in the pre—moult stage.
The formation of peritrophic membranes in the adult and larval Tenebrio as
well es adult Locusts following emergence also indicates a continuous secret—
ion on the part of epithelial cells in starved condition..
In 5th instar Locusts hopperslprovided with food continuously
following moult, invertese concentration increases almost regularly in the
midgut and caeca tissue for 4 days and declines subsequently to a very small
tissue enzyme level in the pre—moult hoppers. These changes suggest that
enzyme production is perhaps regulated by certain factors involved to control
the growth during the intermoult period. Wigglesworth (1959) discussed that
when a resting Bhodnius larva is fed, rapid growth occurs in various tissues
(such as fat body, muscles, and epidermis) accompanied by enlargement of
nucleolus and increase in the ribonucleo protein in the cytoplasm. Similar
changes occur by injecting ecdysone (moulting hormone). On these observatiall
Wigglesworth suggests that when growth beginseprotein synthesis is increased
and among the most important of such proteins will be enzymes which will
further catalyse growth. Thus it is very possible that in Locusta hoppers
the increase in invertase production following moult also represents the
initial growth under the influence of moulting hormone.
Davy (1954) has recorded the changes in the food consumption by
the hoppers of Schistocerca gregaria. According to him in 3rd, 4th and
5th instar hoppers the quantity of food consumed increases after the moult and shows a maximal quantity during the inter—moult period which subsequently decreases until the next moult. Similar changes occur in the concentration 126.
of ribonucleic acid (RNA) of the entire body in 1st and 2nd instar nymphs of
Gryllus bimaculatus (Krishnakumeran, 1961). Since protein synthesis depends upon the concentration of RNA (Brachet, 1957) therefore protein synthesis
starts after the moult and becomes maximal during the intermoult period. Thus according to.Wigglesworthis suggestion (1959) that growth is a manifestation of protein synthesis in Rhodnius it can be suggested that growth of hoppers in Grvllus and Locusta occurs rapidly and progressively after the moult and maximal growth of the organs is.perheps attained in the middle of intermoult period. .Somewhat similar growth curve is found in Tenebrio larva in which there is a progressive increase in total body weight following moult but there is an abrupt decrease prior to the next moult (Murray, unpublished observations). The growth curve of Tenebrio larva runs parallel with that of protease activity of the midgut (Dadd, 1954).
As from the data of the present investigations, resumption of continuous feeding following the emergence of Locusta, leads to increasing concentration of proteinase as well as invertase during the period of ten post—emergence days. This can be regarded as requirement for the rapid utilization of food essential for the initial growth'and metabolism of various tissues. According to the hypothesis of Wigglesworth (as discussed before), enzyme secretion (a form of protein synthesis) may be required to catalyse further growth. This is supported by the observation of Chet. (1952) who maintains that in Locusta migratoria migrdhides the process of build up of reserve substances is very closely related with the feeding activity of the insect. Soon after emergence adultsstart to build up fat body which 127.
reaches its magimal concentration in about two weeks and declines afterwards.
Similar changes follow in the total body weight.
Further, relationship between nutrition, metabolism, growth and
development is afforded from the present observations on Dysdercus fascintus.
When the adults are continuously provided with food following emergence,
invertase production is very rapid and increases progressively for first few
days. This has been observed as parallel with the fast developing eggs in
females. Such processes do not occur in adults unfed following emergence.
It is therefore very possible that enzyme synthesis is related to the nutrit—
ional requirement for the development of the eggs. It is well known that
egg production is profoundly influenced by the food supply (Wigglesworth,
1953). The quantity of food has been observed to influence the number of
eggs in Ciinex by Cragg (1923), Titschack (1930), Johnson (1941) and Khalifa
(1952); and also in Phymata pennsylvanica by Balduf (1941b). The second peak of invertase activity in Dysdercus prior to the production of 2nd batch of eggs is probably related with further nutritional requirements.
In Oncopeltus (Johansson, 1958) which lays eggs continuously, an initial feeding after emergence, is not sufficient to continue egg laying at normal rate, and in insects which lay their eggs in batches a further meal is essential for further development of eggs after one batch is laid. This has been demonstrated in Aedes aegypti (Roy, 1936), Anopheles hyrcanus (Detinova and Butenko, 1955), Haematotopa pluvialis (Cameron, 1934) and Lucille( sericata
(Hobson, 1938). 128.
In male insects fertility is comparatively little affected by nutrition (Wigglesworth, 1953). However, starvation effects the fertility of male Oncopeltus (Johansson, 1958) by marked reduction in the size of testes and seminal vesicles. Similar effect is found in Cimex (Cragg, 1923;
Titschack 1930) and Rhodnius (Buxton, 1930). It is therefore very likely that a small increase in the invertase activity of the midgut of Dysdercus males, corresponding to the 2nd peek of invertase activity in females, is also a nutritional effect.
In Rhodnius, rapid digestion of intestinal contents occurs when egg development is stimulated by corpora allata (Wigglesworth, 1936). But it is not clear whether the increase in digestion is a direct influence of endocrine secretion or it is an indirect effect resulting from egg develop— ment. However, in Dysdercus invertase production and also the ovarial development are stimulated by the ingestion of food following emergence.
Since such changes are not produced in the unfed adults, it is reasonable to believe that the intake of food initiates the mechanism of growth and development which are manifestations of protein synthesis.
Day and Powning (1949) analysed the possible factors involved in stimulating the secretion of digestive enzymes in Periplaneta and Blattella based on the analogy of the possible mechanisms (neural, hormonal or secretogogne) in mammals. A neural mechanism was considered doubtful becaust the techniques used aid not show the innervation of midgut epithelium. This was also supported by the lag of time between the feeding and the increase of 129.
enzyme secretion. In generals feeding of normal diet elways resulted in
increased secretion of digestive enzymes in Periplaneta and Blattella which
confirmed the observations of Schlottke (1937a, b and c). But water alone
did not stimulate the secretion in cockroaches. This made Dey and Powning
consider Ilsecretogogue mechanism" as a probable factor. On the other hand,
in adult Tenebrio, apart from the feeding of normal diet (flour) protease
secretion was also stimulated by providing water or cellulose powder (Dadd,
1954). Thus he suggested that a mechanical rather than chemical nature of
food is involved to stimulate secretion of digestive enzymes. Murray (1960)
observes that Tenebrio larvae are highly specific to the food material and
they do not ingest powdered cellulose even in starved condition. Therefore
it is very possible that if any small quantity of cellulose powder is ingested
by the adult Tenebrio, it will not be sufficient to cause a mechanical effect
in the midgut similar to that of normal food which keeps the midgut completely distended. Similarly„ingested water will also cause a partial mechanical effect in the midgut. Steeeesect. Locusta adults ingested insufficient moistened cellulose powder to fill the midgut completely. Those provided with distilled water only had some drinks and showed more stretched abdomen
than the controls (starved adults). Eut neither cellulose powder nor water could invoke increased secretion of invertase in the tissue of midgut and caeca. Similarly, ingestion of water did not produce any effect on the secretion of proteinase.
Howevc5 in Dysdercus fasciatus,ingestion of distilled water causes marked increase in invertase production. Althoueh water alone may produce physical effect on the midgut epithelium, its importance as a component part 130. of the normal diet cannot be ignored in sap sucking Heteroptera. In Oncopeltus, which normally live on milkweed seeds in nature, the longevity of adults is very short and almost equal when they are either provided with dried milkweed seeds or starved completely from emergence Potaansson, 1958). But adults kept on water only survive for a considerable long time.
Evidences, in support of a secretogogne mechanism for the secretion of protease in the midgut of female mosquito Ades aegypti, has been repeat— edly shown by Fisk and Shambaugh (1952 and 1954). They concluded that production of both invertase and protease is stimulated by a factor present in the normal diet (blood).
Day and Powning (1949) considered that caeca of Periplaneta and
Blattella were ill—fitted for a rapid diffusion of food material from the midgut lumen and also that their epithelium was not innervated. Therefore they did not support the observations of Schlottke (1937c) that in Periolaneta caeca are stimulated first to secrete proteinase and amylase following feeding.
In Schistocerca gregaria Treherne (1959) demonstrated that when a solution of amino—acids is injected into the gut lumen, it is rapidly absorbed by the caeca. But after the intake of normal food this process may not be very quick because the process of digestion may take some time to convert food substances into assimilable solutions• In Locusta adult, within half an hour following the commencement of feeding, food enters the midgut lumen which must receive secretory stimulus before the caeca. This is evident from the invertase activity of midgut and caeca tissue of 5th instar hoppers and adults (starved 3 days following emergence) following the commencement of 131.
feeding. In both cases midgut tissue is stimulated earlier then the caeca
tissue. The lag of time between the commencement of feeding and the appear—
ance of detectable increase in invertese activity suggests the absence of a neural mechanism but supports a secretogogue mechanism.
But the production of proteinase is stimulated later than the
invertase and also the stimulus is observed first in the caeca tissue which provides the bulk of proteinase in starved as well as fed adults. The
time relationship between the commencement of feeding and the response for
the secretion of invertase and proteinase suggests a "preferential" secretion
wfiich may be accounted for by the higher concentration of sugars than the protein in the normal diet.
Day and Powning (1949) were the first to suggest a factor in
the haemolymph of normally fed insects controlling the secretion of digestive
enzymes. They found that increased secretion of enzyme ran parallel with
increased mitoses in the nidi of midgut epithelium. In Tenebrio, injection
of blood into starved adults from fed donors resulted in increased number
of mitoses. But they did not demonstrate a chemically determined enzyme
increase following blood injection. However, Dadd (1954) demonstrated by
chemical determinations that a factor present in the haemolymph of adult
Tenebrio fed on normal diet or cellulose powder stimulates protease secretion
in unfed recipients. Fisk and Shembaugh (1952) did not observe a detectable
increase in protease secretion following blood injection experiments. An
attempt to investigate a blood factor in the 5th inster Locusta hoppers by the
blood injection technique gave a negative result.
The experiments of ligaturing the adult Locusta following emergence, provides an evidence for a hormonal factor secreted in the head to control the 132. production of invertase in unfed adults after emergence. The possibility of a similar factor has been suggested to stimulate the initial production of protease in the midgut of adult Tenebrio, unfed following emergence (Dadd,
1954). Thomsen and !Miler (1959) demonstrated experimentally that if recent— ly emerged blowfly Calliphora is deprived of medial neuro—secretory cells of the brain, the secretion of intestinal proteinase is stopped. Earlier
Thomsen (1952) showed that in the absence of medial neurosecretory cells of the brain the ovaries of female Calliphora are developed to a limited extent and the flies are unable to utilize the ingested meat. From these observ— ations it was concluded that in the absence of medial neurosecretory cells protein is neither synthesized nor utilized by the blowflies (Thomsen and
M5ller, 1959). The transport of neurosecretory material was also demonstrat— ed in the nervi oesophagii via the corpus cardiacum and ganglion hypocerebrele
(Thomsen, 1954). But whether the effect of this neuro—hormone on the prod— uction of intestinal proteinase is a direct or indirect process in Calliphora is not known. Wigglesworth (1959) suggests that growth and moulting hormone
(as discussed before) is likely to produce the same effect as observed by
Thomsen and Miler (1958) on the synthesis of intestinal proteinase.
Thus it it very likely that the initial enzyme production in the unfed insects following moult (as observed in adult Locusta, 5th instar hopper and adult Dysdercus) is initiated by a neurohormone suggested by Thomsen and
Mailer (1959) or by the growth and moulting hormone suggested by Wigglesworth
(1959). But this hormone alone is unable to catalyse further growth because feeding following moult stimulates enzyme synthesis tremendously. Thus in— take of food is an important factor. Further investigations are essential 133.
to explain whether feeding causes a direct stimulus for the production of digestive enzymes or secretion is mediated by a hormonal factor influenced by feeding. 134.
SUMMARY.
(a) Proteinase activity in adult Locusts migratoria migratoides.
1. In the newly emerged adult Locusta proteinase is abundantly present in the total gut extracts (except hindgut). On starvation from emergence proteinase activity is decreased in the beginning, then rises for a short time and finally decreases until death occurs.
2. Adults unfed from emergence show a rise in total gut (except hAndgut) weight on the following day which is followed by a gradual decrease.
3. While caeca provides the bulk of proteinase, the midgut has only a small quantity. Some proteinase is also regurgitated into the crop.
4. Some proteinase is lost from the gut contents of 5th instar hoppers in the beginning of pre—moult condition but before the emergence of adult this loss is compensated by further secretion.
5. Newly emerged adults have almost negligible proteinase activity in the midgut and caeca tissue. Later, on continued starvation some enzyme is built up in the tissues, which show maximal activity on 2nd day after emergence, and then it continues to decrease.
6. When adults are continuously fed after emergence, tissue proteinase is higher than that of unfed adults.
7. Although caeca tissue has always higher enzyme value than that of midgut, tissue enzyme in either fed or unfed adults is very little in compar— ison to its concentration in the lumen of caeca, midgut or the whole gut.
8. In unfed adults, the variation in tissue weight runs parallel with the tissue enzyme activity. In insects feeding continuously following emergence, tissue weight increases progressively for 3 to 4 post—emergent 135.
days but later considerable fluctuations occur. The increase in caeca
tissue is proportionately higher than that of midgut tissue.
9. The tissue of adults starved 3 days following emergence is not
stimulated to secrete proteinase immediately after the resumption of feeding.
However, in about 72 hours after the first intake of food both tissue and
total gut proteinasc is significantly increased. But tissue proteinase is
little in comparison to that of total gut, showing that enzyme is mainly
stored in the gut lumen.
(b) Invertase activity in Locusts migratoria migratoides.
10. Newly moulted 5th instar Locusta hoppers have negligible invertase
in the midgut and caeca tissue. When they are unfed, someiinvertase is produced on the following day and later further production is stopped.
11. Hoppers provided with food continuously after moult show progressive increase in invertase activity both in midgut and caeda tissue for about 4 post- smelt day and then subsequently tissue enzyme decreases regularly to almost very small activity in the pre-moult stage which is accompanied by empty gut.
12. The variation of weight of midgut and caeca tissue of unfed hoppers following moult is similar to that of respective tissue invertase.
13. In hoppers fed continuously from the moult, there is a small increase in the weight of midgut tissue on the day following moult. Subsequent changes are not marked. But the changes in the weight of caeca tissue run parallel to that of invertase activity.
14. Resumption of feeding following 3 days starvation from moult results in stimulating the invertase production in midgut tissue within 3 hours. Caeca tissue is stimulated later. 136.
15. Like newly moulted hopper, newly emerged adults also have negligible invertase in the midgut and caeca tissue. Similar to proteinase some invertase is also produced endogenously in the tissue of unfed adults after emergence.
16. In adults fed continuously after emergence invertase concentration in midgut and caeca tissue is higher than the unfed adults. Invertase activity increases rapidly for about two days and then the rate of increase is slowed down. Within a week both midgut and caeca have almost equal enzyme concentration.
17. There is no constant relationship between individual tissue weight and the corresponding invertase activity.
18. Invertase activity is very varieble in the range of high tissue weight,especially in caeca.
19. After 3 days starvation from emergence the intake of a single meal for 30 minutes is not enough to stimulate the secretion of invertase either in midgut or caeca tissue.
20. Viten above mentioned adults were kept on food continuously, similar to hoppers invertase production was first stimulated in midgut tissue (in about 6 hours after the 1st intake of food) caeca tissue was stimulated later
(in about 12 hours).
21. Ingestion of cellulose powder or water did not cause increased secretion of invertase in the midgut and caeca tissue of newly emerged adults.
22. Ligaturing the head of adults immediately after emergence, and of those starved for one day following emergence demonstrated lower production of tissue invertase than unligatured controls.
23. Injection of blood from the actively feeding hoppers to 3 day old 137. end unfed hoppers did not demonstrate a blood factor to stimulate the secret— ion of digestive enzyme.
(c) Histological observations on Locusts.
24. Secretion of digestive enzymes from the midgut epithelium may be evidenced by the appearance of a number of peritrophic membranes in the midgut lumen of one day old but unfed adult.
25. Continued starvation for 3 days following emergence results in the extrusion of epithelial nuclei and loss of cytoplasmic contents from the eJithelie of both midgut and caeca.
26. Resumption of feeding by the above adults for 24 hours results in the production of rich complement of cytoplasmic inclusions in the epithelia of both midgut end caeca, and new delemination of peritrophic lamellae from the midgut epithelium. These changes are correlated with increased enzyme production.
27. A replacement of nymphal epithelia of midgut end caeca does not occur either before or after the moult to adult.
(d) Observations on invertase and amylase in Dysdercus fasciatus.
28. pH optimum for amylase lies in the vicinity of neutral point whereas an optimum pH for invertese is 6.0.
29. The bulk of invertase is produced in the tissue of 1st ventriculus.
30. Amylase is very weak but it is also mainly confined in the tissue of 1st ventriculus.
31. Both invertase and amylase are much higher in concentration in females than the males.
32. In salivary gland amylase is present considerably but invertase has not been detected. 138.
.~J• Like Locusta, newly emerged Dysdercus adults have also negligible invertase in the 1st ventriculus tissue. Later, in unfed adults some invertase is synthesized endogenously, more significantly in females than males. But further production does not occur on continued starvation.
34. Starvation following emergence results in decrease of tissue weight of 1st ventriculus in both sexes.
35. In the presence of food, following emergence, adults show a marked increase in invertase activity which is significantly higher in females than in males.
36. In females continuously fed following emergence, the tissue weight follows almost a parallel course with invertase activity but in males such correlation is not apparent.
37. Adults starved for 3 days following emergence, when fed on normal diet showed increased production of invertase within 3 hours from the provision of food. Invertase secretion was also stimulated by the intake of water.
(e) Dipeptidase activity in Locusta and Dysdercus.
38. A dipeptidase, capable of splitting alanyl-glycine is distributed in the midgut and caeca tissue; midgut, caeca and crop contents; and the salivary gland of Locusta.
39. In Dysdercus similar dipeptidase is present in the tissue of all the regions of the midgut but the lumen of 1st ventriculus has higher concen— tration of dipeptidase then any other region. The salivary glands also passes this enzyme.
40. In Locusta, thoracic muscles and fat body also demonstrate a dip— eptidase which may not be a digestive enzyme. 139.
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Partially translated.
* Cited from other references. 1.
Appendix.
( Operimental data; )
ii
Variation in Proteinase activity and eight of whol= Gut of ktclul# migratoria migraSoides, unfedfollowing emergence.
71seaimental cateLory !(recently emerged(adults) Weight in D1gs. !Proteinase unitk. 78 60 87 57 81 50 75 37 81 50 83 47 58 33 77 47 '59 43 TOI 56 75 33, 9.6 62 • BirrimenAal Category 2 (one day old) 9/ 26 2 TO5 19 9 134 29 9 117 55 8 66 ;i8 f 127 8 f 116 :6. d' 76 31 109 40 V 83 27 dr 91 23 ce 76 20 9 III 47 d' 105 50 106 41 6d'76 ..)...)Tr , cr 76 61 Experimental category 3(2 days old) 9 106 20 di 79 3 9 112 17 9 104 17 6- 83 24 d 91 14 9. 113 26 e 77 12 122 32 IOI 19
iii T 113 14 ? 78 10 e 63 25 9. dr 119 23 d: 66 15 d: 75 34 6' 94 30 62 22 Experimatfal Ott tort 41 three days old adiAlts1 9 4 8 9. 106 20 72 15 ce 72 16 9 91 24 9. 104 17 d; 7 1 /3 6 76 27 61 63 7 9 80 22 di 79 /7 , 108 25 dr 73 16 e 65 /9 d 77 33 78 27 d 72 4„)elr Apkerinent41 category 5 1 four days old aduAs ) 9 9 L5 9 74 I? 9 103 28 0.1. 85 28 dr 67 18 9 85 17 67 54 cr" 72 26 e 69 18 dr' 82 50 9 93 23 ? 87 33 d' 54 36 de 68 35 d 60 38 59 19 ? 100 62 e 67. 32 Z 4Derimpta4 category 6 ( five days old adults ) e 60 34 dr 90 24.5 9 III 41 83 50 105 19.5 9 61 14 5 56 19 59 di 15 ol, 52 19 dr 71 32 75 46 iv. cf 51 12 57 23 u 50 .:6. e 44 LB di 52 41 61 47 18 4xoeriMsntal qgtegorY 7 (six days old adults ) _ ‘? 08 55 77 20 i 94 40 oA 64 LI 9 76 9 °A 57 5 47 9 9 7D 55 9 62 & L 3:.. oA 64 24 os 55 42 e 50 27 0,CI 49 21 60 33 Elkorimenta4 qategory 8 ( ueven day,s old adults ) i 60 31 q 89 46 9 70 26 ? 84 33 9 66 i' 70 30 e 54. 27.5 crl 60 20.5 9 U6 40 c? 41 8 20 9 80 31 Experimentalt category 9 ( eiOlt days cld acluit2, ) dr' 53 5 o'l 54 13 V 90 d' 39 ce 60
? Zk 3.4' ? 87 15.5 ce 52 10.5 el 55 11.0 ? 64 '49 51 12 Experimental category 10 C nine day. old adults ) c? 57 6 dI 53 10 o4 62 13.5 9 50 14 i 73 15 ? 60 16 9 60 19 q e 7.-P. lig 2I 41
V.
Variation in weight and i-yoteinaze il6tvit; in Crop, Mickutjaud Caeca of 14du1t Locutz th. in., emergence. creD Midut Wt. Pf:ot,7inse- t. p 3. t. n ui L. ilerimental datogorT II ( dr.rgrd . T I§. I . ;30 Ic-,- to, ' 7 . 0 IC 21 7 4 35 7 ii ili .:.E.i 6 6 56 16 ar `,.::.:: ::: 2 dr 75 Experim-,nta1 c4:1togory T !,• ouo day old adult:; ) .5 • L 0 I 6:1 10 15 ' 0.0 41 ....a • IU 1.6 19 1 13 I '.i.i. 17 :17 7 I: CP 12 1Z' 7 1.7 44.:::) • St II Jaen catez ory 7.2.1 ( two day s old (iv1t)s e 1.5 13 1.5 :,)I 12 13 03 16 1 57 19.5 o 12 2 16 0.5 54 14.5 10 1.5 20 0 , 'L''- 39 7.5 9 II '24 6.0 04 14 I 17 1.5 31 3.0 ce L.', 0.5 14 0.5 46 14 e • I9 1.5 17 I . 57 13.5 0a 16 2 IS 0.5 52 16.5 O 26 2,5 16 1.0 75 20 23 7 IC 2.0 53 14 d 16 1.5 Ti:' I •d' 19 6 I2 Z-.5 3b 9 t7 6.5 49 16 CP :5 0.0 IZ) 0.5 40 8. dr 15 , . 26 6 40 20.5 9 23 r 27 5 51 lb Experimental category 74 ( threg days old adults ) & 13 19 1.5 II 5 46 17 26 14 2.5 6.5 141 17 Sd IU.b e 21 12 0.5 IS..) 22 8 22 11 55 11.5 ol 23 8.5 11 4.5 52 17 7 59 15
vi. 22 6.5 :7 720 .5 e ,15 2 - 19 1.5 31 G.5 el 16 I 34 Li..5 le 13 16 - I• ..:.,...- ' II er 14 4.5 17 L:. b *,:? 1445 e 15 4 1, 5 12.5 25 9. 20 3.5 1'6 0,.• L,4 7.5 i'.zporimeatal catelory 15 ( our i7.ays orc ac,.ults j +. I2 1.5 ;;.','; o.b :,,f-c5 4 ii 28 0.5 I'.: ,t, 61 ::.;.5 • I€ 1.5 46 r.,,b d/ 17 c.,, 12 -, ci, :2 5 IL , 41 14.5 7 e I'J ti 17 34 14 o'7 15 5 IT 4.5 .:1 21 I 27 d' 1.t:: 2 0.;5 . •,,:', E.::?) 5 Ci.) 50 '.!xoerimmtal cateorY_ 16 (•five .clays old .,(:::ults ) 20 •3.5 17 0.0 ,-'8 T',: Ili 3 1.-) - 1 • 153 U. d, Il.i I. 13 5.; d' 19 ,6.5 I::: ,:..:-.) .I - 24 . 5 13 :,,.5 ',:.i.:- I:.:.5 21 5 16 6 ;:~:7 . II
Variation in weight aria !'roteinase activity in the Midgut and Caeca tissue of adult Locw,ta m. m., unf,?ti anti /fed following emergence
Midgut Lls;.Ue caca tissue Sex Weight Proteinase 'ieiht ProL.:.ini-se (mgs) units. "Irs9') units. 'verilliental catggoa 20 C newly emerged adults ) 9 IC) 0.0 21 0.0 d/ 6 0.0 13 0.0 64 6 0.0 I0 0.0 15 1 d4 7 0.0 12 0.0 q t-1 0.0 .*1 3 I 6 0.0 IC 0.5 6 0.0 I 0.0 d4 7 0.0 14 0.0 0/ 6 0.0 L: 0.0 g 7 0.0 15 0.0
vii.
Elmerimentel eatecory 2I ( One day old adults, unted ) 13 0.0 23 0.0 9 I0 0.0 20 2 1 9 0.0 25 I ? 8 0.0 20 5 9;9 0.0 21 I 7 I 20 2 a 0.0 18 0.0 oe 10 1 20 I on 7 4 13 0.0 g 10 I 21 I ad 8 3 15 I e 9 0.0 II 0.0 oa 7 0.0 12 0.0 o4 8 0.0 II 0.0 61' 8 0.0 17 0.0 o4 9 0.0 15 0.0 o4 II 0.0 20 0.0 Bxperiment4 category 2? (Avg day* old adults, fed ) 9 0.0 30 3 9 0.0 23 5 f 13 0.0 27 3 d' I0 2,0 IS 2 e 8 0.0 22 5 o4 II 2 20 1.5 cr" 9 2 18 2 e II 2 17 3 e 10 3.5 19 2,5 o* II 1.5 15 2 ce °0 2.5 20 2 M 7 0.0 17 0.0 o4 8 I 23 1.5 et 6 0.0 18 0.0 12 0.0 25 0.5 o'Y 9 0.0 17 0.5 12 0.0 30 0.0 jarlrimentAl cltegory p ( 'se days old agults wifed 3 II II 28 5 6 13 6 24 5 re 9 7 25 I0 f II 0.0 20 I II 0.0 40 2 II 0.0 25 I coq- I0 4 25 I0 ce 9 3 22 9 es' 8 I 12 0.0 e 6 I 15 I * I0 0.5 22 1.5 e 8 1.5 6 I f- 16 4 33 15 9 4,r, 23 0.0 te 9 6 22 8 f- I0 1.5 24 0.0 ",.,": "j .. ,~ "J ~~D ~ 18 4- ~::,~ :) ;::1 ~ piu. t.*eYltA. fuu , Lj.: ... 17 •. ~ -I ?:.7 .".... .J ;~D- XU ;~7 ~.5 ~~'2 0,0 ,'I":' ,I:' I I~l ...... ~: ~'" I,ll ;, 17 tv ::.~;. :') ~~~·z '";~~'. j " 1°~, ..~j :.:7 1·1 ·14 e .. ' ~ ;~ sr: .:..~:" ... "~ ~r; r~ ';', '\ I'i J;" .... ;'~~b l'~ E7 IU f:{:: ~.o Ll /.• .0 titr9t-~ g'n" 8 o~rJ uq,lJ, til" . oofeg. : , II I 16 ::...:.> I~j ~.5 17 I It) 1.5 17 2.0 "t ~~I ~ :' " ~., I .. , .~. I lb ....': ~4 a.\) U.i 1 H; C.O ~.:~~ 1.:} It I 10 1 ;::~. I t}~l'§ua~ b? 019 /.;;.9uk~§!, 17 ~ ;:::1:',~ 7 ix.,
~ 13 2 42 3.5 ~ 16 1.5 36 3 A 9 ...,7 26 1.5 a 14 I 30 2.5 I) IS' 9 2 29 G· ! 10 I 30 I fl 12 I.5 55 I .:::1,- ~ 13 0.5 .3I I ~ 14 I 37 1.5 f 16 I 53· j ~ 10 I 26 2 Experimental pateGorY 27 ( four daYS~dults. unfed) 13 3 20 2 ~ 8 I 15 2 f! 9 I 17 4 I 9 0.5 17 2 /-.tf 7 I 13 4 8 I 12 2 ~' 12 0.5 20 0.0 7 0.5 9 I ~ 6 0.5 10 0.5 "f 9. 0.5 18 2 10 I 13 0.5 ~ 8 0.5 II 0.0 ~' II I 18 2 ~ 9 I 17 I ,?, 14 0.0 20 0.0 ~ 10 2 14 0.5 9 I 15 :2 ~ 9 I 13 2 EJper1m§ntal cat,&orx 28 ( 'foyr daYS old ra dults, feg j I 9 4 29 10 G /, 8 4 27 13 10 ti ~3 8 12 8 ~1 12 ".t 10 6 :29 8 /, 10 10 35 16 f' 10 I 41 2 q' 12 0.0 47 1.5 14 4 39 2 ~ 9 I 19 I ~ II 0.0 37 I f' 10 I 42 4 t II 2 45 4 16 is 48 5 ~ 8 0.0 25 ;::.... 6' II I 20 I ~ 16 4 55 6 x.
~eX:lm9D:ta~ gi~e.&o.r;z ~9 ( r1!~ 9il'.:§ 212 j!S!lJatil.l YDfSlg ) e .:-~ I~ 4 ! a ,;;,<. S 4 ~ 7 3 I7 4 10 7 16 7 ~ 6 ~.5 I~ 4.0 if 5 1~5 14 0.0 r! 6 0.0 I7 0.0 t II 1.5 7 I ~ 7 I r.;I 4 10 I 19 0.5 ~ 5 I 16 2.5 I G m,,5 8 0.0 13 I 19 I- ~ f) 0.0 12 O~b ! 7 0.0 ;';'0 2 if 10 2.5 17 O.b I 6 0.0 14 0.5 ~.J?er!rofl) tal categorz30 ,fiv! da.E:§ .'p1d adlll t§ I ,grid) ,I' 9 9 23 9 0 5· 29 5 I $ 14 4 2,9 7 ~-I 10 5,., :c,G 9 $ 9 u 3~ 6 ! 6 3 26 :> 9 1£ 2 36 3 ~ lil 3 30 4.5 ~ II 2 29 3 12 3 :~::.~ 2 r, $ 14 r,., 4""-' b.5 t 10 10 ~'O 7.5 21 ~ 56 4 ~ II 2 53 4 S 8 ~ 25 2 ~ 10 0.0 0"u 2.5 '"\ ~ ~ 0.0 31 4 ~ 14 6 52 a ~! limegtS!l· 2ategorl: 31 ( §!~ d xi. Experimental category 32 ( six days old adults,fed ) 9 It 0.5 38 10.0 ? 15 2 43 7 9 12 1.5 46 14 8 I 40 IG 9 14 I 37 8 e 12 0.0 35 3 12 0.0 35 3 9 12 I 40 6 16 2 50 3 11 I 38 2 er 12 0.0 38 5 9 15 1.5 44 4 9 I? 1.0 45 5 e 9 2 35 3 e II 2 28 2 di 8 1.5 30 2 e 7 1 32 I Experimental category 33 ( seven_ days .old adults, unfed ) dr' 06 0.0 8 0.0 dr 7 0.0 9 0.0 9 8 0.0 II 0.0 9 6 1 12 0.0 9. 7 0.0 16 0.0 a- 5 0.0 9 0.5 1 9 0.0 12 0.0 9 9 I 14 3 e 4 I .8 0,0 9 6 I IS 4 9 7 2 14 5 1 8 I 20 4 9 8 0.0 14 4 9 7 1 Ib 2 6 0.0 12 '3 as 6 I 9 0.0 e 7 0.0 8 0.0 dr 6 0. 0 8 0.0 -10x1grimental category 34 C seven days old adults, fed ) , 9 14 .0 51 4 I 39 5 9IL 2 38 5 12 I 42 ,-,c. dr IO 0.5 39 2 9 16 0.0 40 2 1 12 2 27 8 9 8 I 21 4 9 14 8 24 9 12 5 32 1 11 3.5 29 :6. 04 11 I 31 6.5 9 14 0.0 40 5 16 2 36 IO 1 IO I A 6 dr 8 0.0 30 4 dr 8 I 30 4 a II I 32 3 xii. Experimental catei.iorx 3§ ( eilbt dayfi old aciAtts, unfed 1 t 7 2 8 2 64 3 I II 2 3,4 8 3 I6 2 e 6 4 14 2 9 6 0.0 II 0.0 de 5 2.5 8 1.5 6' 5 0.0 8 I e 5 0.0 II 2 9 8 0.0 I0 I 9 6 0.0 8 2 o 7 I II 2 9 6 0.0 I0 I t 5 1.5 9 1 9 8 0.0 8 0.0 e 6 0.0 II 0,5 e 7 0.0 7 0.0 Experimental capNory 36 ( eight days 014 a4ultsa fed ) 9 I2 6 36 6 12 5 26 7 14 4 36 6 q 3 .(A q 10 0., 6 9 II 6 39 7 * 12 3,5 48 II 9 Ii 9 35 7 9 II 7 38 II e 15 3.5 50 12 Q 10 2 27 4 eil II 2 22 7 a 8 3 30 4 9 I 39 70 ell 12 0.0 30 9 e a 2 25 7 6' 9 I 24 3 xoerimental category 37 ( nine days 91d adults, unfed ) q / 2 12 3 e 6 2 6 2 6 I 16 2 al 6 2 9 2 5 1,5 8 2 e 5 0.0 10 0.0 Q 7 0.0 13 I 9 7 I 14 0,5 9 6 0.5 I0 I ei 5 0,0 ICI 2 on 6 0.0 8 0.0 64 4 0.0 7 0.0 jxperimental category 38 ( nine uay$ old adultat fed, ) tri 8 I 22 7 * I0 2.5 3I 5 t 15 1.5 45 4 ell 9 4 34 14 01 16 4 28 25 qL I0 30 5 Cr 8 I 24 6 vve. 44z -141. csitve 4° 1°144 %14;r.--:j % %+0 40+0+0 +0 C:VC' -14440.03 %cy 1-4 1--4 /-4 1-1 I-I 1-4 I-I 1-4 1--I 1.4 rm. c Cr3 tt• 4-4 Crt C: C-10 Cg 1.4 CS 1.4 0 • it+ kb. • a • • • • • 17)r C jc.1 fr." 1-s• • P. . zvs 7%1 c,,' h t7; • cC - C C. ' it* ,.:;"! Cr- 1/:::f fp` i. .--; -,2 i;s ED 1-4 C 1-4 1-t -...1 it, t.:7'. 7.C. t.I.' Cl 7.‘:' Cr% -471 1.::: r.-':: pt). 0 t;',,, !..-.7. t:-.+. t:_.-, -1 t-t ?.7. • 4::-.. r0 ..7':-. • ..,, . (2 e-) 1.,..,. • • • • a Cs • • a r,z. • ...., ..,, o... CDC c, C-- C.:•- r.- c:i •xiv. ChiJANs ia ig1it;:41(7, PvoLciaao Lot.Lvity of Mir:4;;ut an0 Caocc, t.i.Jut of:..c11..dt.Licu;:t ( thric u.Lyi; 1:cliaJlag 01).or6ence ) tr ( [cr Mie211.41. t1t.12u0 (.,:ca i'..1ue • ex :14.:htt i:Jcti;,1, ,T oight i:'.votciaa ( ..ii;:.)- Lialt, tfili::n.) ,11.11it. catc,lv ,,I ( (cati'cl.- thrc.7 _ay-3 ol6 aiAlic;::,, u.Iftf.i from (..., e.1,Lunc,.:, ) , 1 ....: ,. „• 7 e •1 I.',., 9 10 ..'. L.1. 17 ( 1::::1-ditc,iy Lf or th,,- j:idi,LLi.,) cf :17 ...-, e 0 0,„C 17 .1 9 ii .,,,,i,,' ,.._, -7 7 11 - M.; 4. 9 ::.,7 9 7 a' L.. •.:., :,:l 004 6 ',. , 9 ;. .5 9 10 ,Lx1tl /...;,'_,:toy .4. ( 1 j, :Lftor i:.:,ecinr timo)., • ce• 11 - v "- 7., T 04 II ...... ELO 7 e 1:: 0.:.) 6',1 .1 9 1.-:, • if ... • o4 7 al 7 ,1....)' a4 6 ; 1 1 r d U .. ., 4?. IL; - ,'..)$: i.' 0 1 !i,...- rit;.2.1 c.ii...: t. , . ,- r ''' " ' '':i."1 : L .1.. C t er .C ,L..-c.1:1_,Lti- ..,.. ) e .i. , t.., „L.Til ...1, 16 .:„.6 :...]: '1 .1'..: ,..5.- q 6 ,-.i.'O., '.:-1 ICIV. Z4i'efiLe4tal, catc!IIPAT 45 ,- 4,,,e • , (.., hrli, ;:ftor feeCia, ) 9 1 .1 13 1.5 60 i 10 1 es L.)-- ,„.. 15 . 4 el ;,.3- - 2 ;,'.:. • 0.0 ecrit442, catec;ory 4L ( IL tr3; &I:tor .Ck.0(1.1.1.k-• Licv ) se 7 ,.:,. 14 5 ? 10 , ' , 10 , . 17 C.. porimentLi gatogory..47 %.( ....;' ,1 lvellz,_ u rter -feciiiiit. t::, ) aq ..,, 0.0 I. Lac I Is I,();: .5 C oe v:t.-'. , I 8 t:o 15 .....0 dr 4. L.0 ...... ••••••••••••.•-• C1.a.:...eL5 in wc.ALlit, zliic.;i, 1::,•%..teirta:.5e Letivity cf uhelc .u'r. s except hinogut) of Locu-AL ru. D. ( unL'ef-J. Zor throe .followinc er.x.rgence ) ;.1.tei-- L •11,1Qz41 for L:b Eilnuk:.0:jr. ox licioht Preteinaz:K! ;,J(.111,;11t • FrottrdlikLze ....11.t. ii,...xerimeati..1,1 catejory 411i aperizicatca cate6orJ- 51 t;olitiol— - oi,40 olc, c..6.Lit, ,( 12 11:,-z„ after feec.:ia;:, ti.silq ) p4feci frolf, cr-. " T ce 68 IL et ...,, 04 ce • •.A.i:i . :;.'..:;.: • a-7 i'.. ;.:4 cfi . 70 10 ...!:,,,::=, f2ri'..:,eatc.1 c:....,t.:,..,6c,3%;.• oi. q II4 f.,,,..- I 16 hr:i. ,:A't.,....'... ,:f.:(34x4; ti; 9 :::;.....:::: q 100 ,1.; x, can:xi-L.1 et. -tei;ory ilizcLia-tely aftov fe,:.sci.LA.L; LAA:o ) , Lu ...i.,..;:'../...11,za ..t:::,„or b;: cfi ;L:Z2 ',f;..., r L :..::`,. Ix AL ;-.i ,,'•` tc.!..,L..y.,,,:,os, in:,,.., t4....i.Li e L... ,L., 10 . • . cr ILL. f,...1 e - Cl FOXf.' erifin en tf;:i 1 CZ.: t.:e. (_0 hr. after 1'oeCiac_t_Le ) e Y • '., di q- l',.,,r/ ;..:.• XVi. Changes in weight and Proteinase activity of whole gut (except hindgut ) of Locusta in. m. (unfed for three days following emergence ) after the prevision of food for continuous feeding. Sex Weight Proteinase Sex eight .Proteinase (mgs.) units .units (Control-3 days ola adults, Experimental c,ategory 56 unfed from emergence ) • (48 hrs, after the commencement Experimental category 54 of feeding) e u8 16 i 356 16 e 19 39 e 246 16 dz ' 73 42 64 249 33 e 58 23 i 373 28 oe 70 19 C' 274 25 9 114 42 4? 326 30 9 96. 35.5 Experimental category 57 i I00 42 ( 72 hrs. after the comL.:ncement f 131 • 22 of feeding ) ce 94 27 9 382 47 a' 83 13 e 207 35 ICIEL 22 489 z5 e 86 18 e 362 32 Experimental category 55 2 418 35 (24 hrs. after the commence - ? 165 3‘z: ment of feeding ) f 383 49 411 • 12 e 236 ,Z7 444 220 • 26 e 205 35 q 366 24 e 207 36 e 232 20 Experimental category 58 9 342 29 (96 hrs. after the commencement q 279 27 of feeding) 9 327 29 9 303 43 ? 382 20 Q. 341 41 e 266 23 287 52 de 262 21 er 162 e 263 20 409 39 Q 288 43 4 269 44 309 55 205 20 oa 19 189 33 Variation in weight and Proteinase activity in the Midgut and Caeca tissue of 5th. imstar hoppers of Locusta, unfed from moult. . Midgut tissue Ca ca tissue Sex Weight Pavafuldase ';eight- Invertase nit m ,s Experimental category 74 (newly moulted ho ers 4 0.5 16 0.0 ce 5 I II I e 4 0.0 II 0.0 di 3 0.5 9 0.5 ? 5 I 16 0.5 e 4 1 IC 0.0 de 4 0.0 8 0.0 50 I 12 0.5 q 6 0.0 13 0.0 e 4 I I0 0.0 e 3 0.5 12 0.0 6 I 13 0.0 4 0.0 I/1 0.0 Ex;:erimental c-tegorY 75 ( ore day old hoppers ) ? 7 4 16 I e 6 3 16 I e 5 6 13 I 6 3 13 I 6 8 14 5 ? 4 4. 15 I e 4. 4 II 2 8 • 3 13 2 a" 5 3 13 0.5 al 5 2 9 I 4 I q 6 II r, I? 7 . 5 17 ‘, Experimental category 76 ( two dais old hoppers ) d' 5 2 10 0.0 cry 4 2.5 II I 4-) 9 6 5 21 0-, ? 4 5 8 I 04 5 .6.5 15 . I 7 3.5 . 17 2 e 4 2 II 2 04 . 5 7 12 - 3 6 6 19 2 $5 3 12 I .e 4 5 13 I ox 4 3 IC • 2 ExtperimeAtal category 77 ( three days old hoppers ) 4 3 9 2 a 3 I0 0.5 5 4 15 I 8 -.4 4 7.5 ,., 3 2 8 7 6 ' 14 2 xviii. V 4 3 II_ 2 di 4 5 9 I os 5 3 8 0.5 V 6 8 12 2 q 4 4 12 0.5 Experiment4_categorY 78 (Igur dayl old_hoppers ) T 5 3 8 0.0 cle 3 4 10 1.5 6 3 I0 1' e✓ 6 5 10 0.5 V 6 2 14 0.5 V 6 7 I0 2 d 4 2.5 6 I ce 3 4 I0 2 dt 3 3 8 0.0 ce 4 3 8 2 5 6 12 2 ? 4 4 I0 I Experimental_ category 79 L five days old hopes ) cry 3 0.0 8 I.5 ce 3 2 8 2 f 5 5 10 I 9 4 4 II 0.0 9 5 6 9 2 4 4 3 10 1.5 e 4 2 4 0.0 el 3 4 8 I q 6 6 9 2 f 4 2 II I . d 4 0.5 7 0.0 gaverimental category 80 ( six clays ola hoppers ) e 3 I 5 0.0 cry 3 2 3 0.0 cei 3 3 5 I re 2 I 5 0.0 ? 5 2 7 0.5 4 2 10 2 ce 3 I 5 0.0 q 4. 4 4 1 Variation in weight and Invertase activity in the Midgut and caeca tissue of 5th. instar hoppers of Locusta, fed from moult. APerimental category 81 Gone day old hoppers ) cry 10 20 .,K.cn 7 9 9 1E 2:5 0 V 12 16 27 7 ce 8 14 20 5 V 9 18 34 8 V 8 I0 42 14 e 6 II 23 6 q 12 13 30 6 9 12 18 29 8 9 I0 15 26 7 e 6 18 15 4 e 6 14 23 8 In i---1.,,.0 0 c0 :.0 H L•-• to H r..0 iil p--/ 03 0 al k;) Z- 0 X) CD to ::.: r- :-.\-/ l;) c0 0 0.1 .7.4 0-) 0) 4--1 t•- Z:- c() ..0- I:- 0 0 0 W.---.L0 LO in c) c2 ‘:.3- H 1-4 H I-I 04 H H:: H C4 r--, 1-4 1-4 0.2 1-4 C,N2 H H 1-1 1-1 al 4-I c.1 zO 4.4:: H I-I 4-1 U) Cq 1-I k.-,,I i-4 1-1 H 1-4 1-4 C‘2 1-4 :;%2 ,-1 i--4 1-1 e) ;-1 7-1 al C) a) s-, o .0 p 0 •-0 0 0 O H 0 O c0 C12cot V V 0 02 r- C;) c0 0 V :0 1-1 0 0 0 02 V tf) Vl D- tr.) H io t0 to V Ott) O'r.)OcOtDCQO :S") H c.t4 :o -77! 02 1.0 H :11 tO CQ dl G12 j 1-1 c0 1-1 CC ca di '44 16Q C,2 cr3 Cc) 10 in V 1(3 rr3 et) CA2 V :s) t0 LO 10 02 odat4 ciO V LO cQ c4 03 rci a) rc:4 to a) 0 O 4-) 4-3 ti-i 4-▪ . U) to CO co O d30 c0 t0 ki) 1-4 02 cr) H C 0‘ 0V Ca 02 G) ci) CQ CCi v-4 LC) C\2 4-1 H co 0) 3):.0 4400 H ‘`.1 I-1 i---IC‘IH H H C11 HHHH H 14 CQ H 02 02 0.2 CQ H Fi 1-4 I-1 H H C.\1 H H I-1 C2 `;',2• H 0 0 a) a) 4-) ad to C) 0 C.) 0 I-4 H .-1 6-1 rl 41 cd tb CI 02 co 0 Si r- c7) (c) 0 0 t- +-.4 cO L.- a) at) c) t- a) :.0 L.- o ck) 03 10 r- to 40 t- 01-4Hcr)010401-4001a)003;Oc0C--0cDC0 ,21-40';)zOL9 _ 4 H H H `-' H H a 1-4 1-4 1-4 H a H 1--1 1-1 1-I El H 1-4 I-I -4 -1-1 -4 s-1 1.4 C1) 0+ 04-‘) S3 eki• S3 04-% 064-00 04'04'04-1%0 (14- 01- 01-153 01, 0v-N060 04Nr.) o4- o- 04-% c1A-N3 01- 0%-50 0-4-Vt 04- 04- 5:1 04- ok,-VIcereo e 7 17 25 8 q I0 18 38 22 Experimental category 87 ( seven days olddhoppers ) Cr 9 8 22 I0 II 8 21 17 cr 12 15 30 20 q I0 17 26 17 ce, II 13 26 12 q 9 3.5 I0 I 9 5 31 12 i II 2.5 13 I Experimental category 86 ( eight days old hoppers ) Q I0 4 2'.; 4.5 ce I0 I 16 I 9 1 20 4 8 8 9 3 c„2 lb .-)g, 9 7 I II 0,0 $' 10 3.5 23 6 d' I0 16.5 24 6 8 5 18 4.5 Experimental category 69 ( nine days olu hoppers ) 10 2 23 I 64 6 2 Ib I ? 10 .I 16 I 8 2 20 I '? II I 18 1.5 e 6 0.0 15 0.5 e 7 1 16 I e 8 0.0 14 0.5 dkanges in Invertase activity and weight of the Midgut and Caeca tissue of 5th instar hoppers of Locusta ( unfed for 3 days following moult ),after the provision of food. 2cperimental category 9 ( control— 3 days old hoppers, unfed followinA moult ) or 4 3 is I e 6 3 14 1.5 i 6 4 II 0.0 cr• 7 9 14 3 e 5 6 1.5 ? 4 2 6 2 T 7 4.5 9 2.5 d' ,-,4, 7 1.5 & 3 I 6 2 q 4 3 10 3 9 4 4 8 2 9 5 2 Ib ti (34 5 4 9 6"5 3.5 12 i.5 ?s 5 7 8 0.5 I- 7 2.5 6 xxi. Experimental category 91 ( 3 his. after the provision off:004J 5 14 13 ,2 9 6 5 16 3 e 5 • 5 13 1.5 8 2:', 3 7 6 e 5 3.5 10 4 e . 6 'II 14 4 e 5 6 6 I 4 ,0 1 ,c 3 , ce 5 7 ,:, e 4 7 12 2 7 12 I 7 2 7 7 5 5.5 3 d' 4 4 I0 3 9 5 2.5 9 6.5 ? 4 5 12 3 ,Experimental category 92 ( 6 hrs, after the 2rovision of food e 6 7 12 1.5 e 6 8 17 3 e 10 12 I ..3 cs4 4 2 8 1.5 d4 8 2 10 I 5 7 11 2 q 6 . 2 14 3 7 8 14 20 4 9 7 14 20 04 5 5 15 4 7 6 8 IS 2 re 6 • . 6 16 5 ExreriAntal cateory 93 (12hrs2 after the provision of food) 04 8 4 10 2.5 T 8 8 16 7 7 12 18 4 e 6 8 8 3 t 8 12 17 6 a* 5 5 18 2.5 04 5 8 I0 3 t 8 4 10 1.5 t 6 6 16 4,5 7 6 II 19 5 t 6 6.5 15 3 e 6 6 /3 2.5 CP 4 8 18 2 c? 5 4 9 3 Experimental category 94 (24 hrs, after the provision of foe(/' 9 6 15 10 7 V 5 8 15 8 cer 5 13 20 8 0-7 5 8 24 13 8 12 32 16 t 7 11 25 a/ 6 9 20. 1. e 7 ,0 L3 IG 7 II 34 17 64 7. 20 28 15 XXii . e 7 . 13 31 12 9 6 17 30 15 Experimental category 95 (48 hrs, after the provisthn of food) i 9 IO 28 7 0* 7 .16 25 13 e 6 23 ti 107 q 9 9 24 I0 15 37 14 e 7 IC 26 - 9 te 8 8 18 8 7 . 5 31 II Experimental category 26 (72 hrss after the provision of food) al 8 5 25 '3 d' I0 7 32 10 / 12 16 40 22 II 1.0 30 . Ii I. 12 14 45 20 0/ 8 6 29 7 t 14 19 50 21 9 II 28 8.5 Variation in Invertase activity and weight in the id_dgut and Caeca tissue of adult Locusta migratoria iLigratoi(As, unfod following emergence. Experimental category 97 ( newly emerged adults ) e 7 0.0 9 0.0 9 I 20 0.5 8 0.5 20 I I0 0.5 18 0.0 cly 6 0.0 9 0.0 e 7 0.0 13 0.0 e 8 0.5 16 0.0 q 9 0.0 21 I 0/ 7 I I0 0.5 00 6 040 IL' 0.0 Experimental catcyory 98 ( one day old adults / 10 3.5 26 8 10 7 19 2 01 8 8 16 3 T II I2 28 7 e 8 4 . II 3 e IC I'. 16 2 ow II 12 18 3 8 a 18 5 e 7 9 II 2 e 6 II 16 6 C 6 7 13 4 i-- 9 9 20 5 Experimental category 9 tor() days old adults ) e 6 9 16 2.5 c?T U 7 lu 1 II 7 28 .5 i ii a 8 1.5 16 0.5 di a 16 1.5 e 9 24 15 6 20 q 26 2 • II 7.5 o 12 10 38 5 (? I0 8 16 3 e 8 5 18 2 T 12 .10 • 20 2 ir . to •-- 4 , • • _ Expezim 2 •2 e 8 9 7.5 18 2.5 q e 7 • 10 2 9 6.5 ;2 i.' 3 g 7 14 I - 67 Iel 1.5 le 7 4.5 2.5 II 5.5 24 e IQ- cr 7 7 .:.ri 0 - 9 7 5 ‹, 2. 6 I 10 14 ,-, e 5 2 12 2 . 7 2 . ,:i ,1 5 e 7 6 10 1.5 7.0 10 13 2.5 ExPerimental category ICU ( four days old adults ) 7 5 14 0.0 6 3.5 IC I a 7 4 . 20 5 g 10 7.5 17 5 f9 5.5 24 6 , r e 7 $:; .0 14 1.5 I0 7 20 1 6* .I0 7.5 15 2 .2 ,-,, 74 7 d' .1 9 I 6 3.5 16 3 4 8 5 12 4 Experimental category 102( five (I:ss old adults ) f 8 6 ...2 3.5 e 4 1.5 8 0.0 cP 5 3.5 a 1.5 ? 6 3 rJ I e 5 4 IS 0 crl 7 3 II 1.5 2 7 6 15 3 2. 6 4. 15 , a' 6 3.5 14 .-, 2 6 4 17. 4 • cir 6 5 12 3 5 7 12 1.5 E)u,)erimental category 103 ( six days did adult:, 7 b 15 ,•, 6 2 6 0.5 ce 5 3-.s. II 1.5 9 8 ' 5 12 I e 6 IC 1.5 9 6 2 .6 I 9. 7 5 IC 1 9 81.5 o4 6 2 12 2 10 4 17 I of 7 7 10 I 9 8 • 2.5. 13 1.5 experimental cateory 104 ( seven days old at.Adt i 9 4 1.5 2.5 6 I 8 0.5 04 5 2 7 0.0 9 7 4.5 12 1.5 9 6 4 14 2 I;') 1.5 9 7 3 II I 6 • 2 11 I 9 9 • 4 7 12 1,5 ? 3 II IS e 6 2cp I0 1.5 cfY 6 4 6 I 7 4 II 1.5 e 4 1.5 II 2 9 8 :-.) 12 I e 6 4 14 Experimental category 105 eiLLt days old adults 1_ ..••••••••••••••• 9 8 5 ,, I o4 6 k. 7 0.5 GP 6 3 7 I.5 , 9 5 ID 2 io 1.5 9 7 4 5 1 ? 6 1.5 6 0.5 o4 4 0.0 7 0.0 e 0 6 1 9 6 2.5 • 7 0,5 .,0 5 12 2.5 ExperiTental category 106 ( nine days old 7 Ldutts 1 9 u I e 4 2 8 ,, . , f 5 2 7 I 4 6 I ? • a 4 II I 9 6 2.5 8 I al 5 I 7 I 0 4 2 6 0,5 9 . d 4 9 1.5 9 4 I 5 1 cl 6 4 6 I Experimental category 107 ( ten days adults ) 4 2 5 1.5 q 9 3.5 II I cer . 4 1.5 ' - 6 0.5 m q 3 I.S 6 0.6 9 5 I - 8 0.5 ce 5 2 7 I 9 5 I 9 I 9 10 .4.5 10 1.5 9 4 3 9 I dr 3 •1 4 0.0 9 0 2 8 I S 4 2 8 1 ExNerizontalxcatugory17:kxxyxi_xxxxxy.xxx:y. Variation in Invertase activity and weight in the Midgut and Caeca tissue of adult Locusts mi&ratoria wigratoides, feu after emergence Experimental catqgory 148 ( one day old adults ) 9 12 I9 29 7 22, 8 T 13 14 20 6 9 12 13 18 3 cr 8 18 23 Da d' 8 12 13 3 9 12 24 28 II g 14 12 $2 4 9 15 20 22 9 d' II 14 22 . 0 ce 8 15 19 5 'Experimental _category 109 ( to daald_ardlat_s_).. • d 11 29 27 27 T 17 27 43 17 9 15 II3 42 25 d II 27 26 II 9 12 18 29 8 13 14 45 20 cp9 I2 18 24 10 c? 9 9 16 3 9 10 25 30 15 9 10, 19 37 2I 10 21 24 12 9 12 16 33 21 e 10 II 29 IO 04 IC 18 24 7 T 10 17 37 17 Experimental categsry_ITO( three c: rid -dultc= ) — crl II 18 38 14 di 8 13 23 9 14 17 .56 16 9 13 26 3 17 re 13 25 31 Ii ce 13 28 23 Ii 14 22 13 9 17 26 43 25 d' 12 22 31 15 C 1.3 '21 28 9 =Vie c? 7 . d' lb L7 IL • a* II 17 ..!,(1 10 ',.:0 -')c.;er4aLijit4-4 c:.; t,e11.1.--(---L144.:16,--1 - — -a 4.1.ti• a du1Isj ef . II - 1.1 • g lb :.'t...-, 41 ',,A 1 IL ILI ID. __ii., :...0 IY 1I e e 11 1.1 .:i.ti II a' . ID .1 Z.:, Id, 3 • T • • 14 kl e 8 I% .vti 7 or' - • IC. ...;. ,,....),• .. - ,.:..1) II 0: II: ;:A:, ' :::,4:.', . IC, 4) 1tt74:1gata1SiAiSitglit II`.?. ( f ivq L“:j. o ..,, ,:.i .tal,..1,4_,I, i • z) / 4',.e..) ,:k' ce C Il '1 15 di d' •9 L.i 12 17 ir.414„.i e ic is cfi IC Ib 2:::•1 . .1:2,. , e ,,J 16 ,...A.., ,.: I., e ... 17 2,....dori:..,ant41 , cat.e4,;ic r:i iE .i. .;-- ;.;i:% 6:..::',11::: i...1,ct 1 17 ;../::-...... !...! 1.:,0 e :1 Q 1,7.., ;.:1,, „.0.., 17 e LL ce.' IC Ltd q .4,...: 14 ;-..:;...., ...•,.; •e ,,..: I', ix .L.A.,,c-,•,.,..41:1,:;:oii,.,.Le.A,,.:;:;ory i14.L....:(11,urt •ol lc: eft .11:0 .:;.'.:. LC) 17 er ie dl I . xxvii. 14 26 44 25 17 32 30 26 8 14 17 cf I0 '2 27 I0 9 14 27 32 16 9 15 21 41 25 Experimental category 115 Eight days old adults di 8 kg 15 IR 30 I8 cm 8 IU 19 13 9 I0 17 36 42 9 ' 15- • 36 36 45 0 7 19 31 26 g 12 • 40 48 42 9 12 24 24 d 43 - 29 37 20 d' 8 20 26 15 9 16 35 40 40 9 IC a 32 61 . . .8 . - • 18 35 18 Experimental category 116 bline. dayFt old a ciul ta ) o4 9 14 21 om 9 24 23 15 13 25 50 39 q ,... 29 17 28 15 4a I :0 6 19 33 17 d • 7 I0 13 9 12 23 40 9 12 16 59 31 9 13 32 V2 18 c? II 24 39 23 °4 II 23 30 2,8 V II 37 20 co II 18 41 25 c?. 14 35 30 17 c 9 32 51 Experimentaleategory II? C ten days old adults e 13 25 47 51 8 IL 24 II g 14 :,5- 39 15 it 13 26 36 14 03 6 20 14 10 ? 10 26 46 21.5 V_. 10 3o__ 38 30 or II 30 41 29 9 14 45 131, 48 9 IC 26 34 22 xxviii. CHanges in weight and Invertase activity of Midgut and Caeca tissue of -adult Locustajp.m. (unfed for three dc.ys following emergence ) ::fter a meal of 30 minutes, Experimental category 118 ( control— 3 days old adults, unfed from emergence ) . _..... i 8 5 17 3 7 9 13 2 8 . ,:::•; 15 3 e • 7 6 12 I.5 ,9 7 2.0 10 ,0. 18 4.5 e 8 5 14 d4 6 14 6.5 9 .,,0 e I4 3.5 3. 7 I 13 ,.) d' • ' 5 8 13 4 Experimental category 119 ( immediately after the feeding tirAe )- di 9 7.5 I0 9.5 14 I ce . 0 5 21 1.5 i 12 11.5 23 1.5 12 6 17 I u 7 17 2.5 ? II 6 Cr 8 7 18 2 Expenimontal category - ILO (1 hr. after feeding time ) 4 6 12.5 18 2 e 7 b II 0.5 04 7 I, 16 1.5 I0 8 21 1s5 e IC U .16 I ? 40 .10 21 2 LI 5 •20 2 T • 10 5 23 3 ;:xg±c.riental cate&pry 121 ( 4 hrs. after feediw timo 4 7 7.5 10 0.5 10 9 24 5 7 7 12 ._,-, cl4 e 8 IZ.5 Il e 7 9 15 3 4 (3- 5 20 4 I0 6 --t" 24 1 II 13 :2: 5 I. 10 IO - ;,:.:: 4 i 6 6 23 3.5 II 0 23 5 1 Experimental_gatei-Iety_12.2._(_6__hils„,.fter feeding time ) clw 7 9 18 5 g IQ 19 21 6 e I0 14 2 8 ,cr 6 13 14 5 q IC II 16 3 II 6 25 4 ill 9 5 I? 2 xxix . e 7 6 15 3 d' 5 7 12 4 or' 7 - 6 11 .. Z 9 II 'II 21 , 4 IC 10 17. 5 ::xperimental cateory IZ6 ( 9 brz, after feediiv iDcricci) 9 II 10 16 J. e 7 I lb q 17 II 23 2 v 10 IL 24 4 al 6 . • 9 14 ce 5 7 9 7 • • . 6 15 .. 2 Ev.erimental catel4ory I24_,LI2• hrs_t after fedini; period ) 7 •II 27 e 7 (3 10 x:f' •q 8 II 17 2.5 q 7 •• LS 1% •0.5 02 5 15 Z? e 6 U 14 5 9 IO 9 24 o o 9 21 ,:.n Invertase •Changes in weight and Rxateinasx activity of Midgut and Caeca tissue of adult Locgataax a. ( unfcd for & Cays following emergence ) after the provision of. food for continuous feeding) Experimental categgry 125 ( control— 3 days old aduis unfed from eorgenee ) d' 6 3 13 3 T 71 T1 25 4 9 13 12 ?!) 10 e 7 4 18 4 9 10 6 17 c:, 9 .10 II 21 • 4 de 8 14 1 e 6 2,:, 17 I 9 9 ..U',- . 2 4 - C, 02 f i 9 U I 8 Ce 7 1 ., 7 7 14 Z F) b r 7 7 19 I 9 9 9 18 2 q 10 iC 20 11 9„ 9 10 15 4, 6- 6 5 10 2,3 e 12 16 32 ;) e, 9 7 30 6 le II 18 27 I0 e I0 _8 34' 7 cr7 12 13 23 11 •am 8 15 -29 6 2 9 18 25 13 q 13 13 37 8 di 9 15 22 . 7 ? ..17 20 28 II 13 17 2_,4 15 i ,-: d 8 9 13 , 9 9 14 44. 17 ce 10 18 23 9 e 10 15 25 9 13 15 20 17 Experimental category 130 ( 48 hrs. after the commencement of feeding ) ci" II 15 33 It, 9 16 21 53 d' I 13 50 41 12 16 30 18 e II 17 26 16 9. 12 26 31 1:3 e 10 21 28 .,0T'-f e 8 10 9 14 13 23 ,,;,, II ,o c 9 19 19 13 20 . 39 • 14, 12 . II 4P 17 hxperimcntal,q category LA ( 72 hrs, after the commencement of feeding ) ••••••••••••••111••••••*.• d' ' 10 17 49 30 9 II II 40 18 o4 I0 I0 20 cr 8 6 22 16 a' 9 14 36 5 7 4 a 9 ..,0 ::4 14 10 37 14 a 10 12 • 30 • 15 q 15 27 49 20 9 II 13 42 12 61 12 15 22 2:6 experimental category 132 (96:.01) . after the commeneemmt of feeding ) 61 10 24 9 12 19 50 30 II . 14 37 16 ..,o - 6 7 17 12 xxxii. Effect of ligaturing on the invertase activity and tissue weight of adult Locust* E xperimental category 137 ( unligatured adults* 24 hrs. old ) ON 8 16 22 4 ? 14 12 19 3 04 II 8 21 3 9 13 21 23 5 1 I0 7 19 4 e 10 14 22 9 ell 9 II 20 5 e II 7 19 2 ? 8 8 19 2 e I0 13 23 2 al 10 9 20 4 Experimental category 138 ( 24 hrs. old adults* ligatured soon after emeEgence ) e II 4 27 3 9 I0 7 18 4 64 8 2 13 I0 9 14 II 25 4 ce 9 8 12 2 e 6 3 15 I e 8 5 IC 1.5 e I() 1.5 15 I t 12 9 16 2.5 e 7 8 13 1.5 e 7 I 13 2 ON 6 7 13 2 7 9 4 20 2 Experimental category 139 ( 48 hrs. 91d adults*, unlii,atured ) 4 I0 I0 18 4 9 8 15 2 T II 9 23 2.5 t cs4 8 8 I0 1.5 e II 10.5 18 4 f 14 12.5 27 5 e 9 8 17 3 e 7 6 9 te, 8 6 12 1.5 7 10 8 21 2 Experimental category 140 ( 48 hrs. old adults ligatured at 24 hrs. after emergence ) a 8 4 12 2 e 10 6 14 1.5 e 5 3 8 I 2 8 3 II I 9 9 7. 20 3 10 8 19 1.5 .!* 6 2.5 II 1.5 on 7.5 3.5 I0 0.5 8 5.5 I7 2 7.5 3.5' 13 1.5 xxxiti. Experimental catexow-I4r-f 72 Wise old adults, uraigatured ) ? / 8 18 3 8 6 21 3.6 d* 6 5 11 4 e 6 4 I5 I e 7 9 19 3 9 7 8 20 4 v 10 II 24 4 e 4. 3 14 2 9 8 6 14 1 Experimental category 142 ( 72 hrs. old adults, ligatured at 48 hrs. after emerginqe ) 9 8 9 15 4 9 8 8 16 2 e 9 I0 20 5 a 9 10 15 4 e 6 3 14 4 e 5 a 9 ,-q c? 7 6 II I 9 8 9 16 3.5 9 8 5 13 I 7 4 18 3 e 6 4 14 3 Variation ,in wweignv and invertase activitin the tissue of let. ventrieulns of istaxIsxxlyqiergusfasciatus, unfed after emergence. Exiotrimental category 148 ( pswlY emerged adults ) Sex weight Invertase . Sex weight Invertase (mgs.) units, (mgs.) units. e 2.5 8,0 Experimental category 149 2.0 0.0 ( one day old adults j 3 0.0 9 4 3 9 3 I 9 4 4 9 2 0.0 3 4 9 6 2 3 0.0 ? 4 9 3 0.0 f 3 2 9 2 0.8 f 3 6 9 3 0,0 9 3 4 di 2 0.0 9 3 4 a 3 0.5 9 3 4 tr 2 0.0 ay I 6 Experimental category 150 e 2 3 ( two data old adults ) o4 2 3 9 3 5 a/ 2 2 9 4 9 04 2 2 9 3 4 Experimental category 151 9 3 8 (Dare* days old adults ) 9 3 I0 9 2 8 1 2 2 9 2 0.0 9 4 10 9 3 2 9 4 8 9 3 4 e 2 I 9 3 4 cP 1 2 9 3 4 di 2 2 9 4 6 e 2 4 9 4 3 . ce a' 2 2 e 1 I dm 2 4 e I 0.0 ay I.5 2 d/ I 2 a/ 0.0 2 I Ce't 2 I e 2 2 Experimental category 152 e I 0.0 ( four days oleo Oults ) 9 3 2 9 3 0 9 4 3 2 I 3 3 g 2 0 om I 0 . 0 cr 2 2 0"101 I 1 0 e I 0 .5 61 I 0.0 xxxv,. Variation in weight and invertase activity in the tissue of 1st, ventriculus of Dys4ercus fas4atus, fed after emergence. Experimental category 153 Experimental category 154 ( one day old adults ) ( two days old 'adults ) 9 5 II 2 8 18 9 4 16 9 6 26 4 5 14 9 6 16 9 2 2 9. 5 20 9 5 6 9 • 4 .1') 2 4 10 9 4 22 9 4 11 2 5 20 os 3 8 2 • 6 24 ce 2 6 9 5 14 04 2 • 4 9 6 18 ce 2.5 6 4 4 16 d 3 • 6 - . d .c..,,. ..),_. 6 Experimental category 155 e 2 . 6 j... z.:, days old adults 1 cr 3 6 9 8 30 d' 3 8 ? 6 27 or 2 5 9 6 33 Experimental category 156 2 5 28 ( four days old adults ) 4 5 23 9 7 I0 6 40 9 4 7 9 5 24 9 7 23 9 •8 47 9 6 12 2 6 25 9 4 7 . d' 3 10 9 5 27 ce 3 8 9 6 15 di 3 IC i to 28 e 3 8 5 28 9 p e 2.5 7 e 2 3.5 II d' 2 2 Experitaental category 150 O 2 7 ( five days old adults ) cry' 4 oF 3 8 2 20 01 3 I0 4 6 50 e 2 a 6 43 Experimental category 158 6 54 ( six days old adults ) 4 20 9 3 14 4 15 4 17 5 30 3 12 6 35 3 15 4 e 9 4 18 2 5 5 13 d 3 9 9 cr 4? 3 . 6 3 II e . 3 . 5 al 2.5 8 dr di 2.5 2 6 cr 2 xxxvi Experimental category 159 Experimental category 160 (seven days old adults 1 ( eight days old adults ) 9 5 .21 9 5 61 5 38 i7 65. 9 4- 2S 3 20 9 7 • . 41 8 34 7 50 ? 7 50 2 ? - r 9 8 56 6 00 i 5 35 7.5 60 0 5 39 cf 2 5 2 5. . ci! •3 II cf 2 3 e 2 6 di 2 3 Ge 3 8 r, 4 3 6 Experimental category 161 ( nine days old adults ) 5.5 54 4 36 6 36 5 12 3 20 4 18 2 2 2.5 4 6 5 6 d' 3.5 8 xxxxii.. Effect of feeding on teigjit and invertase activity of Ist. vcntriculus of pysdercus fasciatus, after three days starvatiofl from emergence. Sex weight Invertase Sex weijit Invertase. (m41. ) units. Crags, 1 units. Experimental category 162 Experimental eategorylb5 ( control— 3days old adults ( adults kept on water for unfed from emergence ) 6 hrs. ) 4 6 1.5 I 9 e • ,,; e 1.5 I 3 ,, 4? 2g„ I 0" 1.5 0.0 e 2 I aq I. 0.0 9 3 14 g 4 I0 2 9 6 9 g 6 e 2.5 I0 1iL7 2.5 6 e I 2 07 1,5 4 0* g I iAcperimental cateLgry .166 f 2.5 2 ( adults kept on gerLiAating e I 0.0 cottonseeds for 6 hrs. ) 9 ,2 2; 9 4 15 e I I f . 5 13 Experimental category 16$ I 4 16 ( adults kept on germinating 9 3 8 cotton seeds for 3 hrs. 1 e 2 2K, g 4 I3 g 4 16 6- 4 9 c. 15 fQ 4 12 40 2 5 g 3 4 e ,.,,-, 3 e 1 0.0 e Ik5 4 e 2 ,.2 Experimental cateory 167 1 6 ,„,) - ( adults kept on water $ 4 9 for 24 hrs. ) 4 . 6 I 6 I0 $ 6 12 e 1 2 if 2 2. 9 4 21 ExperimentA. category 161 e I.5 a ( adults kept on water for 2.5 17 3 hrs. ) e I 3 9 2 I f 2 • I0 g 3 I a I 4 9 2.5 2 f z) 6 3 6 9 2 9 3 9 3 9 ,, I0 9 4 2 1 6 6 9 2 I g 3 IS 4 e 1.5 2 $f I3 I e 1.5 I xxxviii. Experiental c,AeLory 168 ( adults cept on germinating cotton seeds for 24 hrs. ) 5 28 de 3 16 ? 7 15 q 6 47 e 2 11 T 8 43 0 3 7 f 6 17 e 2 4 e a 10 T 4 26 5 22 ,, 7 2 9