Trypsin activity associated with reproductive development in the cockroach, Nauphoeta cinerea (Blattaria)

J. Ins. Phyiol.,

1965, Vol. 11, pp. 961 to 971. Pergamon Press Ltd.

Printed in Great Britain

TRYPSIN ACTIVITY ASSOCIATED WITH REPRODUCTIVE DEVELOPMENT IN THE COCKROACH, NAUPHOETA CINEREA (BLATTARIA) BALAKRISHNA

R. RAO*

and

FRANK

W. FISK

Department of Zoology and Entomology, The Ohio State University, Columbus, Ohio 43210 (Received

29 December

1964)

Abstract-The trypsin activity of female Nauphoeta ciwea cockroaches was related to ovarian development under varying circumstances. Because of the specificity of trypsin for the synthetic substrate, TAME (p-toluenesulfonyl-Larginine methyl ester), the trypsin assay employed TAME, its first reported use with insect material. Mated females on a normal diet were used as controls. Starved, protein-fed and carbohydrate-fed unmated females showed a general decrease in trypsin activity as opposed to an increase in trypsin in both mated and unmated females on a normal diet. In mated females the trypsin activity decreased after ovulation, while in the unmated females on the same diet there was a continued rise in trypsin activity even after ovulation. These data are in general agreement with the findings of others regarding the interaction of brain median neurosecretory cells and corpora allata secretions and their control of digestive protease, haemolymph protein concentration, and ovarian yolk deposition. INTRODUCTION

PROTEIN synthesis in the adult life of a female insect is mostly directed towards the production and development of the oocytes. The raw materials like amino acids, dipeptides, carbohydrates, etc. needed for such a synthesis are produced enzymatically from ingested food. Of the several enzymes taking part in the breakdown of the complex, native proteins of the ingested food, trypsin is considered to be the prime initiator, attacking the centrally located and the terminal peptide bonds. Hence it was decided to investigate the quantitative relationship between trypsin activity and oiicyte development in an insect. THOMSEN (1952) postulated on the influence of the median neurosecretory cells (m.n.c.) controlling protein synthesis and showed the relationship of m.n.c., corpora allata, and the corpus cardiacum to ovarian development in female Cdiphora erythrocephalu Meig. THOMSEN and MP~LLER (1959, 1963) found that the stimulation of the intestinal protease activity in Calliphora was also controlled by the m.n.c. HIGHNAM (1962) reported that a hormone from the m.n.c. regulated ovarian development in Schistocercu gregariu Forsk. Further, HIGHNAM and LUSIS (1962) showed that the blood protein concentration, which normally * Present address : Mergenthaler Laboratory for Biology, The Johns Hopkins University, Baltimore 18, Maryland. 61

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BALAKRISHNA R.RAo AND FRANKW. FISK

increased just before and during yolk deposition, decreased as the oijcytes reached their maximum length. HILL (1962) showed that the high haemolymph protein concentration correlated with the developing ovaries in the same species. ROTH and STAY (1962) showed a strong correlation between mating and oijcyte development in the false ovoviviparous cockroach, Nauphoeta cinerea (Olivier). An attempt was made to establish a correlation between the midgut trypsin activity and the oijcyte development in adult females of N. cinerea and the effect of the mated versus unmated condition on this relationship. With the unmated females the effect of high carbohydrate diet, high protein diet, or starvation on these parameters was also studied. MATERIALS

AND METHODS

The cockroaches were reared in polystyrene containers, fed on Austin dog biscuits (Sunshine Biscuits, Inc., Long Island, N.Y.) and watered. Advanced immature stages were separated from the stock cultures to obtain freshly moulted adults. The adults, as they emerged, were placed in separate dishes and identified by number, date of emergence, sex, and proposed treatment. All were watered and fed according to the prescribed treatment, namely, normal diet (dog biscuits), high protein (Menhaden fishmeal), carbohydrate (80 per cent starch, 10 per cent sucrose, 10 per cent cellulose), or starved (water only). Mating was accomplished on the seventh day after adult emergence since copulation was always effected at this age. Unmated females were kept isolated throughout their adult lives. The cockroaches were dissected in cold saline, exposing the alimentary canal, from which the midgut was severed, removed, and frozen. The oijcytes in the ovaries (or the ova in the ootheca) were removed and their lengths were measured by means of an ocular micrometer. The midguts were homogenized individually (three cockroaches for each day’s observation) in tris-citrate buffer of pH 7.8 and the supernatant, after centrifuging and filtering through glass wool, was made up to 1 ml. The homogenate was stored in the deep freezer until needed. Total protein was measured by the Folin-Ciocalteau phenol method (LOWRY et al., 1951). The procedure was further modified as follows. A 50 ~1 portion of the homogenate to be assayed was diluted to 1 ml with water. One ml of water served as the blank. The alkaline copper-tartrate reagent was made up to 50 ml, then diluted to 250 ml with water. Five ml of this diluted solution was added to each 1 ml sample and stirred. Crystalline bovine albumin (Armour Laboratories, Chicago, Illinois) was used as a protein standard with each run and the results were represented graphically in terms of pg/ml of this standard protein. TAME, p-toluenesulfonyl-L-arginine methyl ester HCl (Nutritional Biochemicals Corp., Cleveland, Ohio), has been established as a most suitable and specific substrate for trypsin by SCHWERT and TAKENAKA (1955), HUMMEL (1959), and others. The procedure used was adapted from SIEGELMAN et al. (1962) and measured the methanol released by the trypsin hydrolysis of TAME. A 50 ~1 portion of homogenate was used for each trypsin test. It was incubated with 0.2 ml of 0.25 M TAME at 37°C for an hour along with suitable blanks. One ml

TRYPSIN

ACTIVITY

ASSOCIATED

WITH

REPRODUCTIVE

DEVELOPMENT

IN COCKROACH

963

of the supernatant obtained from the subsequent precipitation by 10% trichlorowas oxidized with potassium permanganate acetic acid and centrifugation, and treated with chromotropic reagent. The resulting purple colour was read in the Klett-Summerson photoelectric calorimeter at 600 mp. Crystalline, 2x , salt-free trypsin (Nutritional Biochemicals Corp., Cleveland), prepared in 0.05 M calcium chloride buffered at pH 2.5 with HCl, was used as a standard for trypsin activity. RESULTS

Data on trypsin activity, as related to age since emergence, were analysed by the method of least squares to seek a linear relationship. Since a linear function could not be fitted with the above results, they were further analysed by computer for the polynomial curve fitting. Accordingly, theoretical values of y were plotted

Age,

days

FIG. 1. Trypsin on normal brood sac.

diet.

activity and oiicyte development in mated female cockroaches After day 32 ‘olicyte length’ refers to the length of the ova in the

against corresponding x values. Secondand third-degree polynomial curves were drawn by joining these points. The mean values of the average oocyte lengths were also plotted in the same graph. Linear regression lines were fitted by computer analysis for total protein values against age by means of the method of least squares. In the case of mated females on normal diet the trypsin activity increased with the development of the oijcytes until ovulation, and then started decreasing gradually as shown in Fig. 1. This increase coincided with the period of active deposition of yolk in the oocytes. The oticytes shortened in length and increased in width during the ovulation period. Following oviposition the ova continued to grow in the uterus due to the absorption of water (ROTH and WILLIS, 1955).

964

BALAKRISHNA R. RAO ANDFRANKW. FISK

Hence there was a slight dip in the oijcyte growth curve as it was based on the Another feature of the mated condition was that there length measurements. was a rapid growth of oijcytes following mating, confirming the findings of ROTH and STAY (1962) and ROTH (1964a).

5

0

IO

15

20

25

30

35

FIG.

45

40

50

60

55

’

days

Age,

2. Trypsin activity and oBcyte development in unmated females on normal diet.

The oiicyte development in unmated females on normal diet was slow and gradual as seen in Fig. 2. There was no high initial rate of growth as seen in the mated ones. Trypsin activity increased as the oijcytes developed until the commencement of ovulation. But thereafter, unlike the mated condition, the activity further increased until it reached a peak and then dropped off rapidly.

I

0

/

/

5

IO

I

I5

Age, FIG.

3.

Trypsin

1

25

20

30

35

days

activity and oiicyte development in starved unmated females.

The growth rate of the oijcytes of starved unmated females was still lower and the oijcytes never reached their full length before ovulation as shown in Fig. 3. The trypsin activity of these starved females dropped off rapidly the first 2 weeks, remained about the same for a week, then dropped off to near disappearance

TRYPSIN

ACTIVITY

ASSOCIATED

WITH

REPRODUCTIVE

DEVELOPMENT

IN COCKROACH

965

about the thirtieth day. Carbohydrate-fed unmated cockroaches (Fig. 4) also showed an initial lowering of trypsin activity like that of the starved ones. But after about 20 days it started rising gradually. The oiicytes grew gradually and

0

I

5

I

IO

I

15 Age,

FIG. 4.

Trypsin

I

20

I 25

f 30

I 35

-0

days

activity and oijcyte development in carbohydrate-fed females.

unmated

o 00

Age.

FIG. 5. Trypsin activity and okyte

doys

development in protein-fed unmated females.

reached their maximum length before ovulation. In the protein-fed unmated females (Fig. 5) there was a pronounced high initial trypsin activity which decreased to its lowest value by about the twenty-fifth day and then started rising. However,

BALAKRISHNA R. RAO AND FRANK W. FISK

966

the oijcytes reached full length before ovulation. In all the above categories of unmated females the oikytes started degenerating after they reached the uterus ; they turned yellow and translucent and the remnants were finally aborted. AS for the trypsin activity in all but the starved females there was a general trend of increase even before the oiicytes reached maturity.

IO 5

0

10

15

20

25

30

35

Age,

FIG.

Total

6.

3-

homogenate

protein

and trypsin normal diet.

50

55

60

activity

in unmated

females

on

0

0

+ &2

0

i

0

- 200

‘\ ‘\

‘\

0

0

0

0

0 -100

..______r--------_ . . .\

’

.c z f +

‘l

0

I 5

I IO

I 15 Age,

Total

homogenate

protein

I 25

I 20

4

< h i

0

z5.

FIG. 7.

44

- 300

0

I I / I

.s ::

40

days

\ I 30

I 35

5 ‘0 c

0

days

and trypsin

activity

in starved

unmated

females.

The total protein content of the midgut homogenate of the mated females remained nearly the same throughout the period of oiicyte maturation and gestation though the trypsin activity showed a rise and fall. In the unmated females there was a slight rise in the total protein content (Fig. 6), whereas in the starved ones there was a gradual fall (Fig. 7). In all the above cases the initial amount of

TRYPSIN

ACTIVITY

ASSOCIATED

WITH

REPRODUCTIVE

DEVELOPMENT

IN

COCKROACH

967

protein was almost the same. But in the carbohydrate-fed and the protein-fed unmated females there was a high initial content of total protein which decreased in a steep slope (Figs. 8 and 9).

0

o

Cl

,c--.

0

‘\

‘1 \

,I

I

I

1

I

I

I

I

23

5

IO

I5

20

25

30

35

Age,

FIG. 8.

Total

homogenate

protein

days

and trypsin females.

activity

in carbohydrate-fed

unmated

0

4-

0

0

-400

-200

4 2 a

-100

c 2 0 c =

-300

0

1

11

23

I 5

I

IO

I

I

I!3

20 Age,

FIG. 9. Total homogenate

1

25

I

30

I

35

a

JO

days

protein and trypsin activity in protein-fed

unmated

females.

968

BALAKRISHNA R. RAo

AND

FRANKW. FISK

DISCUSSION From the pattern of trypsin activity found in the mated females it becomes evident that the enzyme titre in the midgut was higher when there was a demand for yolk protein in the ovaries. During the period of egg development and yolk deposition the proteolytic enzymes showed a higher activity, supplying the metabolites for the protein synthesis. In N. cinerea, as with other false ovoviviparous cockroaches, increase in the size of the uterine eggs was accounted for by their absorption of water only, without the need for further protein synthesis (ROTH and WILLIS, 1955). Several workers observed that when females were kept with males the eggs matured rapidly. In N. cinerea mating stimulates oiicyte development so that the preoviposition period is about 17 days shorter than in virgin females (ROTH and STAY, 1962). NORRIS (1954) and HIGHNAM and LUSIS (1962) reported that when female desert locusts were kept in the presence of mature males their oiicytes matured faster and fewer were resorbed, so that more viable eggs resulted. HIGHNAM (1962), working with the same species, has shown that the hormone from the median neurosecretory cells (m.n.c.) is released into the haemolymph shortly before ovarian development begins and continues throughout the major At the conclusion of yolk deposition, just before part of ovarian development. oviposition, the release of the material ceases abruptly, resulting in its accumulation in the m.n.c. and corpora cardiaca. HILL (1962) showed that protein concentrations in the haemolymph of the desert locust correlated exactly with the release of m.n.c. hormone. Hill postulated that the increased haemolymph protein during ovarian development represented a neurosecretory control of protein synthesis. This concept is further strengthened and extended by the present study in which the midgut trypsin concentration of mated N. cinerea females increases during ovarian development and is drastically reduced when the oijcytes pass down the oviducts and are retained in the uterus. Our results may be interpreted to confirm the findings of THOMSEN (1952) and THOMSEN and MILLER (1959, 1963) with the fly, Calliphora erythrocephala, that the m.n.c. hormone regulates protease activity of the midgut as well as ovarian development. THOMSEN and MP~LLER (1963) state: ‘There is some evidence that the m.n.c. hormone might be involved in the regulation of protein synthesis in general.’ The influence of the corpora allata secretion in reproductive activity and ovarian development should not be overlooked. It has been investigated in Culliphora by THOMSEN and MBLLER (1963) and THOMSEN (1964), in the desert locust, Schistocerca gregaria, by HIGHNAM et al. (1963), in the ovoviviparous cockroach, Leucophaea maderae (Fabr.), by SCHARRER (1946) and ENGELMANN (1960, 1964), in the viviparous cockroach, Diploptera punctata (Esch.), by ENGELMANN ( 1960) and ROTH and STAY (1961), and in other cockroach species by ROTH and STAY (1959, 1962). In general, the corpora allata show increased activity and their secretion of ‘gonadotropic’ hormone is released during periods of active ovarian development. In the two cockroach species the glands were relatively inactive during the period of uterine development of the oocytes. In

TRYPSINACTIVI’IYASSOCIATED WITHREPRODUCTIVE DEVELOPMENT IN COCKROACH969 Calliphora and Schistocerca the investigators concerned are convinced that although the corpora allata and m.n.c. have a complex interaction and coincident periods of activity, it is the m.n.c. that exert a general stimulation of protein mobilization and synthesis which makes oocyte development possible. Our results with the virgin females on regular diet differed from those with the mated cockroaches both in reduced oijcyte development followed by resorption and in increased and extended trypsin activity. The maximum trypsin activity of the virgin females occurred about 45 days after emergence and was approximately double that of the mated females with a maximum activity on about the thirtieth day. It is obvious that the factors responsible for the abrupt cessation of m.n.c. secretions in mated desert locusts following oviposition (HIGHNAM et al., 1963) or for the inhibition of the corpora allata in mated ovoviviparous cockroaches following ovulation (i.e. beginning of pregnancy) (ENGELMANN, 1960; ROTH and STAY, 1962; ROTH, 1964a, b) were not operating in virgin N. cinerea cockroaches. These factors, which have been listed by HIGHNAM et al. (1963), are briefly as follows: (1) Terminal oijcytes of full size produce a factor which inhibits the secretion of the corpora allata. (2) The corpora allata may stop secreting because the haemolymph protein concentration begins to fall at this time. (3) The activity of the neurosecretory system, which controls protein synthesis, might be affected by the non-utilization of the protein produced. To these suggestions may be added the ‘trypsin inhibitor’ factor of RAO (1964) which inhibits trypsin activity in the midgut and, presumably, yolk protein synthesis. Rao’s postulated trypsin inhibitor may be identical with the inhibitor factor mentioned in statement (1). A humoral corpora allata inhibitor ‘agent’ is proposed by ENGELMANN(1964) for L. maderae and other ovoviviparous cockroaches. This agent is said to be ‘released by the egg case or the brood sac’ and to affect the central nervous system (ventral nerve cord and brain) which in turn inhibits the secretion of the corpora allata. An alternative hypothesis with considerable evidence to support it has been put forward ROTH and STAY (1959, 1961, 1962) and ROTH (1964a, b). They suggest that oviposition and the passage of the oijtheca to the uterus, followed by further increases in oijtheca size, stimulates stretch receptors in the uterus or elsewhere, resulting in a direct nervous connexion to the brain with resultant inhibition of corpora allata secretion. This inhibition, which lasts during most of pregnancy, not only inhibits oijcyte development but renders the female unreceptive to the male. Experimentally, the oothecae of pregnant females could be removed and replaced by similar-shaped glass beads which continued the pregnancy symptoms so long as the ventral nerve cord remained intact. Our results with unmated females indicate that often partial oothecae were formed and passed to the uterus, but degeneration and shrinkage followed so that inhibitory impulses to the brain would presumably not result. In the case of the starved unmated females there were at least two factors which could account for the observed lower trypsin activity and reduced oijcyte development, namely, the unmated condition and the lack of substrate. ROTH and STAY (1962) a1so reported reduced oijcyte development in starved virgin

970

BALAKRISHNA R. RAO AND FRANK W. FISK

cinerea, but not in starved mated females of the same species. KHAN (1963) starved from the day of adult emergence, noted that in Locusta migratoria L., ‘proteinase activity disappeared and was only restored after continuous feeding’. In the protein-fed and carbohydrate-fed unmated females the trypsin activity showed a similar decline. The protein-fed females showed a higher initial trypsin concentration (i.e. in the first 5 days after adult emergence) which decreased abruptly until about the twenty-fifth day, whereas, in the carbohydrate-fed females there was gradual decrease in trypsin activity. In both types a rise in trypsin activity was noted beginning about the twenty-fifth day after emergence, which coincided with the decline in trypsin activity of mated females. This period also coincided approximately with the time of oiicyte maturation in mated females. Thus it appears, by analogy with the desert locust, that the abrupt cessation of m.n.c. hormone noted in normal mated females at the completion of ovarian development does not appear at this time period in the unmated condition and hence the increased trypsin activity is accounted for. Because of a drastic change of diet at adult emergence (all larvae were on the standard dog-biscuit diet) there was an observed decrease in food consumption in the case of protein-fed and Thus a partially starved or nutritionally inadequate carbohydrate-fed females. condition was induced. The period of decreased trypsin activity as well as the decreasing midgut protein concentration could be attributed to this condition. The total midgut protein of mated females on normal diet remained the same during the period of study, while that of unmated females on the same diet increased gradually. Considering the high level of trypsin activity and the ample supply of food available for the virgin females an increase in midgut protein could be expected. In the starved unmated females total midgut protein showed a gradual reduction which paralleled general body-weight decreases as the stored reserves of the starved adults were utilized. Similarly, the reduction in midgut protein of the protein-fed and carbohydrate-fed virgin females was due in part to the lowered consumption of poorly balanced food. In conclusion, the observed pattern of trypsin activity in female N. cinerea cockroaches seems to be influenced primarily by whether or not they are mated, and secondarily by the nature of food ingested. With normally mated females our data strengthen the emerging complex pattern of m.n.c. and corpora allata secretions, elevated haemolymph protein levels, and oijcyte development. When oocyte development is complete and gestation begins there is a cessation of m.n.c. and corpora allata secretions and a lowering of trypsin activity and haemolymph protein levels. The primary cause for this curtailed activity seems to be incompletely understood, though several hypotheses, including stretch receptor impulses or a trypsin inhibitor substance, have been advanced by previous workers. N.

Acknozcledgements-We thank Dr. L. M. ROTH and Dr. G. F. SHAMBAUGH for critically reading the manuscript and offering valuable suggestions. Dr. R. L. SKAVARIL, The Ohio State University, using the IBM 7094 computer, and Dr. C. R. WEAVER, Ohio Agricultural Experiment Station, using the IBM 1620 computer, handled the statistical analyses.

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gregaria.

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HICHNAM K. C. and LUSIS 0. (1962) The influence of mature males on the neurosecretory control of the ovarian development in the desert locust. Quart. J. micr. Sci. 103, 73-83. HICHNAM K. C., LUSIS O., and HILL L. (1963) The role of the corpora allata during oijcyte growth in the desert locust, Schistocerca gregaria Forsk. J. Ins. Physiol. 9, 587-596. HILL L. (1962) Neurosecretory control of haemolymph protein concentration during the ovarian development in the desert locust. J. Ins. Physiol. 8, 609-619. HUMMEL B. C. W. (1959) A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Canad.J. Biochem. Physiol. 37, 1393-1399. KHAN M. A. (1963) Studies on the secretion of digestive enzymes in Locusta migratoriu L. I. Proteinase activity. Ent. exp. up~l. 6, 181-193. LOWRY 0. H., ROSENBAUM N. J., FARR A. L., and RANDALL R. J. (1951) Protein measurement with the Folin-phenol reagent. J. biol. Chem. 193, 265-268. NORRIS M. J. (1954) Sexual maturation in the desert locust (Schistocerca gregaria) with special reference to the effects of groupings. Anti-locust Bull. 18, l-44. RAO B. R. (1964) Trypsin activity associated with reproductive development in the female Tampa cockroach, Nauphoetu cinerea (Olivier). Ohio State Univ. Ph.D. Diss., 61 pp. ROTH L. M. (1964a) Control of reproduction in female cockroaches with special reference to Nauphoeta cinerea. I. First pre-oviposition period. J. Ins. Physiol. 10, 915-945. ROTH L. M. (1964b) Control of reproduction in female cockroaches with special reference to Nauphoeta cinerea. II. Gestation and postparturition. Psyche, Camb., Muss. In press. ROTH L. M. and STAY B. (1959) Control of oiicyte development in cockroaches. Science 130, 271-272. ROTH L. M. and STAY B. (1961) Oiicyte development in Diploptera punctata (Eschscholtz) (Blattaria). J. Ins. Physiol. 7, 186-202. ROTH L. M. and STAY B. (1962) A comparative study of oijcyte development in false ovoviviparous cockroaches. Psyche, Camb., Muss. 69, 165-208. ROTH L. M. and WILLIS E. R. (1955) Water content of cockroach eggs during embryogenesis in relation to oviposition behavior. J. exp. 2001. 128,489-510. SCHARRER B. (1946) The relationship between corpora allata and reproductive organs in adult Leucophaea maderae (Orthoptera). Endocrinology 38,46-Z. SCHWERT G. W. and TAKENAKA Y. (1955) Spectrophotometric determination of trypsin and chymotrypsin. Biochim. biophys. Acta 16, 570-575. SIEGELMAN A. M., CARLSON A. S., and ROBERTSON T. (1962) Investigation of serum trypsin and related substances. I. Quantitative demonstration of trypsinlike activity in human blood serum by a micromethod. Arch. Biochem. Biophys. 97, 150-163. THOMSEN E. (1952) Functional significance of the neurosecretory brain cells and the corpus cardiacum in the female blow-fly, Calliphora erythrocephala. J. exp. Biol. 29,

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