PHOSPHORUS DISTRIBUTION IN T H E GRASSHOPPER EMBRYO

VINCENT THOMPSON AND JOSEPH HALL BODINE Zoologiaal Laboratory, State University of Iowa, Iowa City

FOUR FIGURES

INTRODUCTION

It has been rather clearly demonstrated that various phosphorus compounds are of importance in many phases of vital processes (Kay, '34). Needham and Needham ('30) state that "the estimation of the various forms of phosphorus in organic and inorganic combination is one of the best methods we possess for gaining an insight into the metabolic changes taking place within living organisms. "

The eggs and embryos of the grasshopper, Nelanoplus differentialis, have been employed in this laboratory as a means of attacking funda- mental problems of cellular physiology. Considerable information is at hand regarding the developmental metabolism of this animal (Needham, '35 ; Bodine and Boell, '38). Such experimental material is also of especial interest since, during development, periods of intense cellular activity are separated by one of considerable duration in which the cells are relatively inactive (diapause) (Bodine, '32). The present paper is a report of a study of the phosphorus content, its distribution and changes in its partition during the embryonic development of this form.

MATERIALS AND METHODS

Grasshopper eggs of the species Melanoplus differentialis were used. Methods of collecting and care of eggs have been previously pointed out (Bodine and Boell, '38). Five whole eggs or from 10 to 100 embryos (depending on the age of the egg) were employed for each determination of total phosphorus, while 100 to 500 eggs and 1000 embryos were necessary for phosphorus partition analyses.

Phosphorus was determined by the colorimetric method of Fiske and Subbarow ('25). For total phosphorus the materials were wet- ashed with sulfuric acid and superosol for oxidation of organic sub- stances (Stearns and Warweg, '33).

l Aided by a graiit from the Rockefeller Foundatioii for research in cellular physiology.

24 7

JOUBNAL OF CSLLULAR AND COMPARATIVD PHYSIOLOGY, VOL. 12, NO 2

248 V. THOMPSON AND J. H. BODINE

Analysis of the following fractions of phosphorus was attempted : Inorganic (ortho-) phosphate, pyrophosphate, lipoid phosphorus and protein phosphorus. The procedure was adapted from that of Eggleton and Eggleton ('29) and Raldwin and Needliam ( '34). The material was ground at 0°C. in 5% trichloroacetic acid. After 30 minutes the acid-insoluble fraction (B) was separated by centrifugation and filtra- tion. The acid-soluble fraction (A) was neutralized with powdered haryta until neutral to phenolphthalein, and the barium insoluble frac- tion (A,) removed by centrifugation.2

The precipitated barium phosphates were dissolved in 0.1 N HC1, and barium removed by addition of sufficient saturated sodium sulfate. -4fter centrifuging to get rid of the barium sulfate, aliquots of the supernatant liquid were used to determine 1) inorganic phosphate, 2 ) pyropliospha-te, and 3) total phosphorus in fraction A,. Inorganic ( ortho-) phosphate was determined by adding the Fiske-Subbarow reagents to the sample at once. The increase in ortho-phosphate (i.e.' the condition in which the phosphorus must be present fo r the Fiske- Subbarow technique) after 7 minutes hydrolysis at 100°C. in normal HC1 mas taken as pyropliosphate phosphorus (Lohmann, '28). Total pliosphorus was determined in the usual manner.

Fraction B contained 1) protein and 2) fat soluble phosphorus. The latter was dissolved in Bloor's ( '18) mixture. Two hours of extraction were found sufficient to remove completely the soluble materials. After centrifuging off the undissolved material, alcohol and ctlier were boiled away and total phosphorus determined. This frac- tion lias been designated as lipoid phosphorus. The remaining substance was analyzed fo r total phosphorus. This non-lipoid portion of fraction B x-as called protein phosphorus.

RESULTS

1. Tota l phosphorus (fig. 1) The total phosphorus contained within a grasshopper egg was found

to be approximately 14 y,'egg, which amount was constant through- out development. The total embryonic phosphorus increased with growth of the embryo. As the yolk was engulfed by the embryo (5 to 12 days post-diapause) the embryonic phosphorus became nearly equal to that of the total egg. No attempt was made to remove engulfed yolk from the embryo since it is then actually a part of the embryo.

a The barium soluble fraction (9,) includes some hexose phosphate and phosphageii. This fractioii was not investigated, except to demonstrate (by means of the difference in lability of t h e two esters, Needhain et al., '32) that the pliosphaqeii mas argiiiiiie and not creatiiie phosphate.

PHOSPHOIlUS I N EMBRYOS 249

Slightly over 1% of the total phosphorus (about 0.2 y/egg) was present in the egg membranes.

2. Distribution of phosphrus i"r2 whole eggs Examination of fignre 2

indicates that there are two periods of rapid inorganic phosphate rise. One of these occurs in pre-diapause development, while the second is evident during the whole of post-diapause growth. These two periods are separated by a 'plateau' of little or no change, corresponding to the entrance into and maintenance of diapause.

a,. Inorganic ( o r t h o - ) phosphate (fig. 2).

Fig. 1 Total phosphorus. Abscissa : days of dcvelopmcnt, pre-diapause, diapause and post-diapause at 25°C. Ordinate: y phosphorus/egg.

These periods of rapid free phosphate increase correspond with those of high water absorption (Bodine, '29). This osmotically active form of phosphorus map aid in maintenance of a constant osmotic medium for the embryo contained in a greater volume of embryonic fluids.

b. Pgroyhosphate (fig. 2). Pyrophosphate phosphorus was fonnd to exhibit most striking changes. This fraction was high during the first few days of development, and then rapidly dropped to a level, reached about the seventh day, which was then constant throughout development. The significance of these changes will be discussed later.

250 T. THOMPSON AND J. H. BODINE

1.5 -

c. Ligoid phosphorus (fig. 3 ) . Lipoid phosphorus (i.e.' the phos- phorus in a form soluble in alcohol and ether) was high at first, but gradually dropped to a fairly constant level. This phosphorus com- bination is related (Needham and Needham, '30) 1) t o the utilization

1.5 - FIGURE 2

- I NORCANIC PHOSPHATE PIROPHOSPHATE --

. .

I FIGURE 2

- I NORCANIC PHOSPHATE PIROPHOSPHATE --

. .

0

c i! 0.5 . 8 P

I --- 5 10 IS 20 40 5 10 15 I

PRE-DIAPAWE DIAPAUSE POST - DIAPAUSE

D A Y S

Fig. 2 Inorganic phosphate and pyrophosphate. Abscissa and ordinate as in Inorganic (ortho-) phosphate and pyrophosphate phosphorus content of whole indicated ages.

I-

5.0 - F I G U R E 3 - PROTEIN PHOSPHORUS LlPOlD PHOSPHORUS ---

4.0- a ."\

0

w '. \

;\, > 3

2 3.0- 0 P

P

2 .--_ >--< _ _ _ - - - 2 c 8

-_- 2.0- 5 10 I5 I 20 40 5 H) I5

PRE- DIAPAUSE DIAPAUSE POST- DIAPAUSE

D A Y S

figure 1. eggs as

Fig. 3 Lipoid and protein phosphorus. Axes as in figure 1. Lipoid soluble phosphorus (solid line) and protein phosphorus (broken line) of whole eggs at indicated ages.

PHOSPHOIZUS I N EMBRYOS 251

of fat (yolk), representing the transformation of lipoid phosphorus into other types (as inorganic and protein), and 2) to that phosphorus in lipoid soluble combination which is deposited in the organized, perma- nent integrity of the organism. During early development the first process is dominant, while later the two may become balanced as indicated by a more or less constant lipoid content.

I r

FIGURE 4a.

EGGS

I D H

0.1 I

F I G U R E 46

EMBRYOS

Fig. 4 Summary of phosphorus distributioii i n eggs and embryos.

Pr 47 %

4 a. Phosphorus distribu- tion in whole eggs a t three stages of development: 1, 1 day old; D, diapause; H, hatching. 4 b. Phosphorus distribution in embryos at diapause (D) and shortly a f te r diapause (post). L, lipoid phosphorus; Py, pyrophosphate phosphorus ; A,, barium soluble, acid soluble fraction ; Pr, protein phosphorus; I, inorganic phosphate ; A,, barium insoluble, acid soluble fraction less pyro- and ortho-phosphates. Note that each unit in 4 a represents l y phosphorus, while in 4 b eaeh unit indicates but 0.1 y phosphorus.

d. Protein phosphorus (fig. 3) . Protein phosphorus (i.e., that phos- phorus insoluble in acid or lipoid solvents) undergoes a rapid increase, broken only by a diapause ‘plateau.’ A part of this fraction represents nucleoproteins which are being elaborated as more and more cells are formed. I n fact, this fraction is thought to be roughly indicative of the relative number of cells and cell nuclei present.

252 V. TEOMPSON AND J. H. BODINE

3. Distribzrtiovz of phosphorus in tlae enibryo (fig. 4 b)

Embryos in diapause are morphologically similar to those just out of this dormant stage, while physiologically they are much unlike (Bodine and Boell, '38). Three lots of 1000 embryos each were used for the distribution determinations. The diapause embryos were selected from eggs which were 30 to 50 days of age. Post-diapause embryos were from eggs in which blastokinesis (Slifer, '32 b) had barely commenced.

The total phosphorus for these diapause and post-diapausc embryos was 1.5 and 2.2 y/embryo, respectively. The distribution of phosphorus, when expressed in terms of per cent of total phosphorus, does not show any outstanding difference in the various fractions between the two types of embryos.

DISCUSSION

Inasmuch as the grasshopper egg is a closed system the total phos- phorus content as would be expected remains constant (Needham, '31, for hen's egg). It is the transition of phosphorus from one combina- tion to another within this closed system which is of interest. Such changes are summarized in figure 4 a , in which the phosphorus frac- tions have been represented as per cent of total phosphorus content.

This is high at first, gridaally dropping to a level (fig. 2) which is constant until hatching. According to Lohmann ( '28) this pyrophosphate indicates the amount of adenylpyrophosphate, a co-enzyme concerned in one of the steps of Carbohydrate metabolism. Pyrophosphate determina- tions have thus been employed as indirect measurements of degree of carbohydrate utilization (Reller, '36, for example). Boell ( '35) has shown that the R.Q. for the grasshopper egg is unity at the beginning of development, indicating a carbohydrate metabolism. This figure drops to the fat level, 0.7, by the seventh day, where it remains until hatching. The change in pyrophosphate content parallels closely this decrease in R.Q.3 Needham's ( ' 33 ) sequence of energy sources (first carbohydrates and then fats) is, therefore, admirably exemplified in the chemical embryology of the grasshopper egg.

Of most significance is the change in pyrophosphate.

Heller ( '36), using butterflies, found low pyrophosphatc content during the pupal (dormant) eoiiditioii aiid high aniouiits in active nymphs. He coiisiders that this, with other evidence, iiidieates variation in carbohydrate metabolism. No change iii pyrophosphate was found in diapause aiid post-diapause grasshopper eggs (fig. 2 ) . This was expected, since there is no evidence for carbohydrate metabolism after the seveiith day of development. It should be stressed that there is no point of analogy, except for the relatively illactive conrlitioii between pupal and diapause states.

PHOSPHORUS I N EMBRYOS 253

Despite the physiological difference between diapause and early post-diapause embryos, no striking variation in phosphorus distribu- tion was found (fig. 4 b). I f all phosphorus combinations could be determined some of them would undoubtedly exhibit differences. How- ever, pyrophosphate is the only fraction determined which can be linked with a metabolic process, and that process (i,e., carbohydrate nietabo- lism) is negligible, as indicated by R.Q. values of 0.7, for both diapause and post-diapause eggs.

SUMMARY

1. Total phosphorus content of grasshopper eggs remains constant

2. The following variations in phosphorus fractions of the .whole eggs were found : a) Inorganic phosphate increases during active development. b) Pyrophosphate is high during the first few days of pre-diapause growth. c ) Lipoid phosphorus decreases during early embryonic life, but remains fairly constant during the late post-diapause period. d ) Protein phosphorus increases rapidly in the active egg.

3. The possible relation between high pyrophosphate content in early ontogeny and carbohydrate metabolism is discussed,

- throughout development.

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