VARIATION IN THE HAEMOGLOBIN CONTENT OF DAF’HNIA 625

Causes of variation in the haemoglobiri content of Daphnin (Crustacea : Cladocera) in nature.

BY A” CHANDLER,

Bedford College, University of London.

[Communicated by Professor H. MUNRO FOX, F.R.S.-Received 13th October 1953.1

(With 3 figures in the text.)

CONTENTS.

Introduction ........................................... Material and methods. .................................. Oxygen and haamoglobin .............................. Iron and haemoglobin ................................. Seasons and haemoglobin .............................. Species and hasmoglobin ............................... Acknowledgments ..................................... Summary ............................................. References ...........................................

Page 626 625 626 627 627 628 629 629 630

INTRODUCTION. In the laboratory the haemoglobin content of the blood of Daphnia has

been found to be inversely proportional to the amount of dissolved oxygen present in the water in which the animal lives (Fox, 1948). Other environ- mental factors which increase the amount of haemoglobin in Daphnia are abundant food, high temperature, dissolved iron compounds and duck faeces (Fox, Hardcastle & Dresel, 1949; Fox & Phear, 1953). I n nature the haemoglobin concentration in the blood of Daphnia varies from pond to pond, and from month to month in any one pond ; the present investigation was undertaken to study the causes of these variations under natural conditions.

MATERIAL AND METHODS.

The species investigated were Daphnia rnagna Straus, D. pulex (De Geer) and D. obtusa Kurz.

A group of ponds at Newdigate, Surrey, and another group a t Whipsnade Zoological Park, Bedfordshire, were visited periodically. Some other ponds were studied in England and also in Denmark. The investigation lasted two years.

Por the determination of dissolved oxygen in pond water, samples were taken from mid-depth so as to obtain a mean value in the event of there being a vertical oxygen gradient. Oxygen was determined by the Winkler method, the first two reagents being added to the sample in the field. To avoid a possible error due to oxidizing or reducing substances present in the water, which would increase or decrease the amount of iodine finally liberated, the modified method described by Ellis, Westfall & Ellis (1946) was used. The oxygen content of the ponds was measured two hours after sunrise and two hours before sunset, in order to approximate to the minimal and maximal oxygen concentrations, due respectively to nocturnal respiration and diurnal photosynthesis. Mid- depth temperature was recorded whenever sl water sample was taken for oxygen estimation.

Parthenogenetic females alone were studied.

626 ANN CHANDLER

100

90

80

X fO-• W

The iron content of the water was determined by dipyridyl after reduction (Hill, 1930). The colour developed in fifteen minutes was compared in a Duboscq colorimeter with that from a standard iron solution.

The haemoglobiii content of Dophnia was determined by the method of Fox (1948). The mean haemoglobin value for ten iiidividuals gives the “ haemoglobin index ” of the population. This index was corrected for the mean size of the animals. The length of each of the ten, from forehead to base of tail-spine, was measured with an eyepiece micrometer, and the mean length reckoned. On the assumption that the thickness of the animal, through which the haemoglobin concentration is measured, is proportional to the animal’s length, the haemoglobin index for each population was corrected to a mean length of 2 mm. Indices below the value of 20 cannot accurately be determined and are given the arbitrary value of 10.

OXYGEN AND HAEMOCLOBIN

Figure 1 shows the relation between haemoglobin index and oxygen content, of the water in thirty-seven determinations of each variable, made in twenty-six ponds. It is clear from the figure t,hat red Daphnia populations were generally

-

-

-*

-0

E 6 0 -

c .- - :so-

: 40-

Cn

01 m I 30

20

10-

*.

7

-

0

0

0 a

0

0 8

0

0

8 0

8

0 em0 a 0

01 I 4 I I I I ! 1

Mean Oxygen concentration in water: (mE./e). 0 1 2 3 4 5 6 7 8 9 1

Fig. 1.-Haemoglobin content of Daphlzia in ponds with different contents of dissolved Each of the 37 points gives the haemoglobin and oxygen determinations

Daphnia magm 0, oxygen. for I?+ certain pond on a certain day ; 26 ponds were studied. D. p l e x 0, D. obtusa .

VARIATION I N THE HAEMOGLOBIN CONTENT O F DAPHNIA A27

This found in poorly aerated waters, and pale ones in well-aerated waters. shows that the laboratory results apply also to natural populations.

IRON AND HAEMOGLOBIN.

Haemoglobin index plotted against dissolved iron content of the waters is shown in fig. 2. There was clearly no correlation between these two variables. In spite of the fact that additional iron in laboratory experiments increases the haemoglobin content of Daphnia when the oxygen content of the water is low (Fox & Phear, 1953), in nature iron is not a determining factor in the redness of the animals. No doubt oxygen deficiency is the overruling cause.

i O O r

90

80

x 70- Q) 7J C -60- c z 5 0 - - rs,

0 -

- 0

,ao

i 1

10 *.. i ..

e

I

0

0

0

0

* .. 0

0 0.2 0.4 0.6 0.8 1.0 12 14 16 1.8

I I I I I I I I I

ison concentration in water ( m g m Fig. 2.-Haemoglobin content of Daphnk in ponds with different contents of kon. Each

of the 41 points gives the haemoglobin and iron determinations for a certain pond on a certain day; 21 ponds were studied. Duphnia magm 0, D. pulex 0, D. obtusa .

A few- exceptionally high iron contents of very shallow reddish-brown waters cannot be shown in fig. 2 . In one of these the iron content was 51 mg./l., but despite a low oxygen concentration in the water, namely 0.1 ml./l., the haemoglobin index of the Daphnia obtusa present was only 45.

SEASONS AND HAEMOGLOBIN.

It is difficult to foretell how the different seasons of the year might affect t'he hnemoglobin of Daphnia in ponds, largely influenced as this is hy the amount

628 ANN CHANDLER

of dissolved oxygen present. In summer, photosynthesis will increase oxygen and so diminish haemoglobin, but, on the other hand, a high temperature (Fox & Phear, 1953) and abuntlzlnt algal food (Fox, Hardcastle & Dresel, 1!)4H) tend to increase haemoglobin. In winter, these jnfluerlces are reversed. Figure 3 shows the haemoglobin indices of Daphnia obtum populations in six

80y

70 -

60 - x W 0 c - 50- t 40- -

c3, 0

a, (D

E 30-

* 20-

0 “ I 1 ! I l l 1 ! I 1 1 1 1 1 1

J F M A M J J A S O N D J F M A M J 1951 M 0 N T H S ’952

Fig. 3.-Haemoglobin content of Daphnia obtusa in 6 ponds at different times of year. The initial letters of the months are given.

ponds studied during different months of the year. In all these cases there was more haemoglobin in winter. This bears out two preliminary series of determinations by Fox (1948, p. 199). It is not, however, an invariable rule, for in a settling tank at the Hampton Water Works, Middlesex, in two successive summers (1952 and 1953) a crowded population of Daphnia m g n a was very red, while in the intervening winter the sparse population was pale (personal communication from Mr. J. Green).

SPECIES AND HAEMOGLOBIN.

It is known that the haemoglobin indices of two species of Daphnia living in the same pond may be different from one another (Fox, 1948). Although present in the same pond, however, i t is possible that when one species is redder than another, the two do not really live together, but the redder one may frequent regions in which the water is poorer in dissolved oxygen. It was therefore thought desirable to test a mixed population in the laboratory, keeping both species together in the same water. This was done with D. pulex and D. obtusa, cultured as by Fox, Gilchrist & Phear (1951). In each experiment forty pale individuals (twenty of each of the two species) wer0 put into a conical flask of water containing Chlorelln as food, and kept in the dark.

VARIATION IN T H E HAEMOGLOBIN CONTENT O F DAPHNIA 629

The oxygen content of the water remained low, since oxygen is consumed by Daphnia and Chlorella, and little enters through the small air surface. Sclditional iron, in the form of ferrous ammonium snlphate, was added to increase the amount of haernoglobin syii thesized in response to a low oxygen content of the water (Fox & Phear, 1953). After eight days the animals, initially pale, had become red, having synthesized haemo- globin, and the haemoglobin index and mean body length of the animals were then determined. which i t is seen that in nine of the ten experiments Daphnia pulex synthesized more haemoglobin than D. obtuua.

The haemoglobin indices were, as usual, zdjustcd to a body length of 2 mm. A comparison between the two species after this correction has been made assumes, however, that in each species the ratio of thickness of the body to its length is the same. This assumption was tested by measuring the thickness and the length of twenty-two individuals of each of the two species. The ratio of length to thickness was found not to Ile identical ; the mean ratio (with RE.) was 3.81 &On03 in D. ~ I / Z P X but 3.54+044 in D. obtusa. The consequence is that the haemoglobin indices for D. pulex should be multiplied 1)s SH1/364=1*08. This has been ( h i e in table 1, the result being that in nine experiments D. pulex synthesized more haemoglobin, while in the tenth case the amounts formed were virtually equal.

Tell experiments were made.

The results are given in table 1, from

TABLE 1. Comparison of haemoglobin synthesis in water of low oxygen content by Dupi~nia pz6fer and D . obtirsu living together. All unimels were initially pale, with e haemoglobin index

below 20 for enrh speries.

The different capacities of Buphnin p d e x and I) . obtusa to synthesize haernoglobin furnish another distiiiction between these two species, recently separated on morphological grounds (Scourfield, 1948) and by the absorption spectrum of oxyhaemoglobin (Fox. 1945).

ACKNUWLE I)GMHNTS.

I wish to thank Professor H. Muilro Pox a id members of the Zoology Department a t Bedford College for much help and advice. I am most grateful to the Zoological Society of London for hospitality a t Whipsnade, to Professor Kaj Berg for hospitality a t his laboratory at Hillerod in Denmark, and to Mr. T. B. Rothwell of L. Haig and Co., Newdigate, for allowing me to investigate his ponds.

SUMMARY. 1 . The haemoglobin content of Daphnia in the ponds studied was found to

vary inversely with the oxygen concentration in the water, but not to be proportional to the iron content of the water.

2 . In six ponds Daphnia was found to have a greater haemoglobin concen- tration in winter than in summer, whereas in one body of water the haemoglobin concentration was greater in summer than in winter.

3. Haemoglobin may be more concentrated in the blood of one pond-dwelling species of Daphnia than of another species in the same water.

PROP. ZOOL. SOC. LoNI).-VOI,. 124. -1.3

630 V-4RIATION IN THE HAEMOGLOBIN CONTENT OF DAPHNIA

REFERENCES.

ELLIS, M. M., WESTPALL, B. A. & ELLIS, M. D. (1946).

Fox, H. M. (1945). Fox, H. M. (1948). Fox, H. M., GILCHRIST, B. M. & PHEAR, E. A. (1951).

Fox, H. M., HARDCASTLE, S. M. & DRESEL, E. I. B. (1949).

Fox, H. M. & PREAR, E. A. (1953).

HILL, R. (1930).

SCOURFIELD, D. J. (1942).

Determination of water quality. Res. Rep. U.S. Fkh & Wildlife Serv. No. 9.

Haemoglobin in blood-sucking parasites. The haemoglobin of Daphnia.

Nature, Lond. 156, 475.

Functions of haemogoblin in

Fluctuations in the haemoglo-

Factors influencing haemoglobin synthesis by Duphnia.

Dipyridyl method for the estimation of iron in biological material.

The Pulex forms of Duphnia and their separation into two Ann.

Proc. Roy. SOC. (B) 135, 195.

Daphnia.

bin content of Daphnia.

Proc. Roy. SOC. (B) 141, 179.

Proc. Roy. SOC. (B) 107, 205.

distinct series, represented by D. pulez (De Geer) and D . obtusu Kurz. Nag. nat. Hist. (11) 9, 202.

Proc. Roy. SOC. (B) 138, 514.

Proc. Roy. SOC. (B) 136, 388.