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PROCEEDINGS

OF THE

INDIAN ACADEMY OF SCIENCES

VOL. XV

SECTION B

BANGALORE CITY

i'KINTED AT THE BANGALORE PRESS, MYSORE ROAD

1942

CONTENTS

SECTION B VOL. XV

No. 1 January 1942

PAGE
Studies on the Growth and Breeding of Certain Sedentary

Organisms in the Madras Harbour M. D. PAUL 1

The External Morphology of the Brain of Semnopithecus entellus

(A Comparative Study) ... A. ANANTHANAHAYNA AYER 43

The Egg-Capsule of the Millipede, Thyroglutus malayus Attems

(Syn. Thyropygus malayus Carl.) M. B. LAL 58

Cytogenetical Studies in Datura. I. Cytology of the Parents and of
the Fj Hybrid between Datura fastuosa and Datura sp.
T. S. BAGHAYAN AND A. E. SRINIVASAN 61

No. 2 February 1942

New Concepts of the Solid State SIR 0. V. BAMAN 75

A Contribution to the Life-History of Vahlia viscosa, Roxb., and

Vahlia oldenlandioides> Roxb

T. S..BAGHAVAN AND Y. K. SRINIYASAN 83

Trypsin-Kinase in Blood ........ 1ST. K. IYENGAR 106

Anti-Tryptic Components of Blood 1ST. K. IYENGAR 112

Prothrombin and Plasma Trypsin N. K. IYENGAR 123

No. 3 March 1942

The Grain Sorghums of the Durra Group

0. "N. EANGASWAMI AYYANGAR, M. A. SANKAEA AYYAR, T. E.
NARAYANAN AND A. KUNHI KOEAN E"AMBIAK 133

Age and Affinities of the Bagh Fauna . . . G. W. CHIPLONKER 148

On the Internal Bundles in the Stem of Rumex patientia L. . .

P. MAHESHWAHI AND BALWANT SINGH 153

The Embryo-Sac of Euphorbia heterophylla L. A Reinvestigation

, P, MAHBSHWARI 158

PAGE
No. 4 April 1942

A Leaf Spot Disease of Zingiber Officinale caused by Phyllosticta

zingiberi n.sp T. S. EAMAKRISHNAN 167

The Origin of Siphonostele in Three Species of Selaginella Spr. .

GIRTJA P. MAJUMDAR 172

The Genus Cephalogommus in India and Burma . G. D. BHALERAO 178
Bionomics and Control of Aeolesthes holosericea F. (Cerambycidae:

Coleoptera) . KHAN A. EAHMAN AND ABDUL WAHID KHAN 181

Phragmotelium mysorensis, A New Rust on Indian Raspberry . .

M. J. THIRUMALACHAR 186

Female Gametophyte and Embryogeny in Cymbidium bicolor Lindl.

B. G. L. SWAMY 194

A Study of the Life-History and Control of Batocera horsfieldi
Hope (Lamiidae: Coleoptera) A Borer Pest of Walnut Tree in
the Punjab . . KHAN A. EAHMAN AND ABDUL WAHID KHAN 202

Indian Water Moulds III ABDUL HAMID 206

Indian Water Moulds IV . H. CHAUDHURI AND M. L. BANERJEE 216

Indian Water Moulds V. A New Genus of the Saprolegniaceae:

Hamidia Gen. nov H. CHAUDHURI 225

No. 5 May 1942

Contributions to the Bionomics, Anatomy, Reproduction and
Development of the Indian House-Gecko, Hemidactylus flavi-
viridis Rtippel. Part III. The Heart and the Venous System
BENI CHARAN MAHENDRA 231

Cytological Studies in Indian Parasitic Plants. II. The Cytology

of Loranthus L. S. S. KUMAR AND A. ABRAHAM 253

On the Biology of Red Spider Mite (Tetranychus telarius Linn.)

in Baluchistan NAZEER AHMED JANJUA 256

No. 6 June 1942

Stages in the Spermatogenesis of Siphonops annulatus Mikan. and

Dermophis gregorii Blgr. (Amphibia: Apoda) B. E. SESHACHAR 263

Origin of Intralocular Oocytes in Male Apoda . B. E. SESHACHAR 278

Analysis of Raspuri and Badami Varieties of Mango (Mangifera

indica) Grown in Mysore . C. SBIXAKTIA AND N. L. KANTIENGAR 280

Enzymic Proteolysis. Part V. The Liberation of Cystine . . .

- - - M. DAMODARAN AND T. K. KRTSHNASWAMY 285

STUDIES ON THE GROWTH AND BREEDING OF

CERTAIN SEDENTARY ORGANISMS IN THE

MADRAS HARBOUR

BY M. D. PAUL, M.A., M.Sc.

(From the University Zoological Research Laboratory, Madras)

Received October 14, 1941
(Communicated by Prof. R. Gopala Aiyar)

CONTENTS PAGE

1. INTRODUCTION . . . . ... . . . . 1

2. ENVIRONMENT . . . . . . . . . . - . . 2

3. MATERIAL AND METHODS . . . . . . . . 4

4. RATE OF GROWTH, AGE AT SEXUAL MATURITY AND PERIOD OF
BREEDING

(a) Hydrozoa . . . . . . . . . . 5

(6) Annelida . . . . . . . . . . . . 6

(d) Mollusca . . . . . . .. . . . . . 9

(e) Crustacea . . . . . . . . . . ...12

(/) Ascidiacea . . ... . . . . . .. . . 14

5. DISCUSSION

(a) Growth . . . . . . . . . . . . 16

(b) Breeding .. .. .. 21<

6. SUMMARY . . . . . . . . . . . . 28

7. ACKNOWLEDGEMENT . . . . . . . . . . 28

8. BIBLIOGRAPHY . . . . . . . . . . . . 35

9. EXPLANATION OF FIGURES . . . . . . . . . . 41

Introduction

IN recent years methodical work has been carried out on the growth and
breeding of marine animals in order to gain an understanding of life in the
sea and considerable information has been gathered on the biology of marine
organisms. On the Indian coast, however, excepting for a study of the
growth and breeding of the pearl oyster (Margaritifera vulgaris Schum.),
(Herdman, 1903 and Malpas, 1933) and of the edible backwater oyster,

1
Bl F

2 M. D. Paul

Ostrea madrasensis Preston* (Hornell, 1910) very little work has been done.
Sewell (1925) has recorded his observations on the growth of a few marine
forms in the Nicobar Islands. Winckworth (1931) has studied the rate of
growth and breeding of Paphia undulata (Veneridae) from specimens
dredged in the Madras Harbour and sent to him. Quite recently Srinivasa
Rao (1936, 1937, 1938) has studied the habits, rate of growth and breeding
of Trochus niloticus Linn, and Pyrazus palustris (Linne) in the Andaman
Seas with very interesting results. A preliminary survey has also been
made by Erlanson in 1936 on the growth and breeding of animals, chiefly
boring organisms, in Cochin Harbour.

For purposes of this study the following sedentary organisms that com-
monly settle down and grow on the concrete piles and other substrata in the
Madras Harbour were selected : (1) Laomedea (Obelia) spinulosa Bale var.
minor Leloup, (2) Hydroides norvegica (Gunnerus), (3) Crisia sp., (4) Mem-
branipora sp., (5) Ostrea madrasensis Preston, (6) Mytilus viridis L.,
(7) Patella (Cellana) cernica H. Adams, (8) Balanus amphitrite Darwin,
(9) Polycarpa sp. and (10) Diandrocarpa brackenhielmi Michaelsen.

Environment

This work was carried out in the Madras Harbour (Lat. 13 06' N., Long.
8018' E.) situated in a typical tropical coast. This Harbour (Text-Fig. 1)
is an artificial one, with an area of about 200 acres roughly rectangular in
shape enclosed by concrete breakwaters. These and the concrete piles offer
a good place for attachment to several sedentary organisms. The entrance
to the Harbour is in its north-eastern corner and it is here that any .roughness
in the sea is first felt. In the south-west portion of the Harbour there is
the c Boat Basin ' in communication with the main Harbour. . .This part is
farthest away from the entrance and consequently is much less subject to the
action of waves. From the Boat Basin a narrow channel leads into a pond
of calm sea- water, the ' Timber Pond '.

Jutting into the water from the west Quay is another Quay, the New
North Quay, about 600 feet in length. The concrete piles of this afford
excellent place of attachment for sedentary organisms and it is in the eastern
part of this, where there is considerable wave-action and probably abundant
food supply, and where the dirt of the Harbour does not accumulate, that
much of this work has been carried out. Projecting into the Harbour from
the south wharf are some steel jetties the frame-work of which also serves as
substratum for the attachment of these forms.

Ostrea virginica changed into Ostrea madrasensis Preston. See Preston, 1916.

,;,/,/ /,V,v,//// A r / G-

gansms

1 X

-, v
? i
! * *X If

! *\ -'

'* s ' '

i tf 1 1 K Madras Harbour lowing the places referred to in the text

lltr icinpcraturc of the surface water in the Harbour ranges throughout
>- ir !rn|1 5 ^ ' C in 33 (\ (Table XIII). According to the Harbour
iMa-r liver,* is an a\craiic temperature of about 27-6 ' C. Unfortunately
rcaihfifs ucrc tjot taken throughout the year but the salinity of the
\\d\-cr u!f the ei>ast of Madras according to ScwelFs charts (1929) varies
the iiiiicrciii parts of the year as follows:

Sepfemher ti> November
December to I ebruary
March to May

to AlHMiNt

> 30-00 to 32-00 pcrmille

, 33-25 to 33-50
, 33-50 to 33-50
34 "(X) to 34-50

The iiMiiil itiiiil rafi^c is only from 2 to 3 feet of water. During the rains
fiiciv is a jMv.it admixture of fresh \\ater in the Harbour, especially in those
portions taithest from the entrance.

4 M. D. Paul

Material and Methods

In studying the rate of growth of organisms suitable objects had to be
devised to serve as places of attachment for the sessile organims. Wooden
racks (PL-Figs. 1 and 2) in which ordinary glass slides 3 inches by 1 inch could
easily be inserted were the most useful and the most widely used. This rack
was made of two pieces of wood, 4 inches by 2 inches and 1 foot in length,
with grooves sawed on one of the broad sides of each piece at a distance of
about 1 inch apart. The two wooden pieces were fixed by bolts with their
grooved edges opposite each other at a distance of about 2J inches. Glass slides
could be conveniently inserted in these grooves and taken out. To prevent
them from being washed off two long but narrow strips of zinc were screwed
on to the apparatus at the top and at the bottom, thus keeping the slides in
position. By unscrewing one of these strips the glass slides could be easily
removed from the wooden rack. The larvas of the organims studied settled
down and grew normally on these slides (PL-Fig. 3). At desired intervals
the slides were brought to the laboratory in a jar of fresh sea-water and after
the early stages were studied under the microscope the slides were conve-
niently stored in formalin for future reference.

Other objects used were (1) wooden pieces scooped out on one side and
tied down below low water level and weighted down by short lengths of iron
rails, (2) cement blocks suspended in water at definite depths and (3) iron
pieces tied down below low water level. The last, however, did not generally
allow the larvae to settle down during the first few days because of rusting.
Because of the large size the wooden and iron pieces and the cement blocks
allowed enough space for forms like oysters and mussels to settle and grow
on them. Further, definite areas on the concrete walls of the Harbour were
scraped down thoroughly and the animals allowed to settle down. These
marked areas were frequently examined, the samples removed and measure-
ments taken. .

In addition, several of the sedentary organisms were collected from
boats and buoys. The respective dates on which these were launched or set,
after being thoroughly scraped down, were ascertained from the Harbour
authorities and from these the approximate age of the organisms collected
was calculated. These data were very helpful for verifying the results obtain-
ed by the other methods employed.

It was found after some experience that conditions in certain places in
the Harbour were more favourable for growth than in others and it is in the
former localities that more concentrated work was carried out. Of such
places mention may be made of the eastern side of the New North Quay big

Growth and Breeding of Certain Sedentary Organisms 5

buoys Nos. Ill and IV facing the main entrance to the Harbour and the North
and South Buoys in the Boat Basin. Regular observations, week to week,
were made from November 1935 to February 1937. Observations of a more
general nature, though not weekly, were continued even after the period men-
tioned. During the period of study visits were made to the Harbour at fre-
quent intervals so that direct information could be obtained of the settling
of young ones in various places.

For purposes of growth-study only the best grown individual of each
species attached to slides or blocks during a particular period of immersion
was selected. To illustrate, from Hydroides norvegica, measuring 5-1, 4-7,
3-8, 2-1, and 1-0 mm. in length, obtained from slides kept immersed in
water from December 26 to December 30, 1935, the best developed was taken
to represent the growth for the four days. After their measurements were
taken, the specimens had the condition of their gonads also determined in
the living state. Out of a large number of size measurments thus obtained,
only those representing the best growth for a particular period have been
selected and given in the following tables. It should be remembered that all
measurements recorded throughout this paper are actual measurements of
the maximum size of individuals and not averages unless otherwise stated
and the age in all cases denotes the length of life of the animal after
attachment.

Rate of Growth, Age at Sexual Maturity and Period of Breeding
Hydrozoa.

Laomedea (Obelid) spinulosa Bale var. minor Leloup. Of the Hydrozoan
colonies getting attached, Laomedea (Obelid) spinulosa Bale var. minor Leloup
was chosen. The planula of this form gets attached to and spreads on the
surface and from the basal stolon erect individual colonies grow. The
maximum height of the individual from the base to the tip is taken to
represent the growth. The number of polyps in each stolon is also counted.
Leloup (1932) mentions that the individuals of this species do not attain a
size of more than one cm. in the tropical seas. A maximum length of about
16 mm. is attained here (Table I).

Slides immersed on December 26, 1935, and taken out on January 3,
1936, contained several specimens and some were carrying well-developed
gonosomes. Thus, this species attains sexual maturity in 8 days. More
often, gonosomes were noticed after 9 or 10 days. The calculation assumes
that the larvse attach themselves to the slides on the day of their immersion.
If the attachment takes place actually a day or two after immersion, then the

6 M. D. Paul

attainment of sexual maturity must be considered even more raj
Small Nudibranchs (Amphorina ?) were seen browsing on the colonies ;
to lay their spawn. As Orton (1914, 1929) and others record, these NT
branchs live at the expense of the colonies, rush through their growth-sts
and deposit their spawn within a few days. Caprellids were found cliti^
to the colonies mostly during January and February. Table I gives
rate of growth of this species.

Breeding. A large number of Laomedea (Obelia) spinulosa was fo
attached from October to February and these carried a considerable m
ber of gonosomes, thus indicating that they breed during this period.

Annelida.

Hydroides norvegica (Gunnerus). Of the many sedentary polychs
in the Harbour, Hydroides norvegica (Gunnerus) attaches itself in very l
numbers, after metamorphosis, to all experimental materials. Atransluc
tube which later becomes calcareous is secreted. The tube grows vigoroi
adhering to the object of attachment but after a fortnight chiefly owin|
want of space it grows vertically. Soon after, the vertical part of the tub
usually broken off by the action of waves, etc., and it was found imposs
to pursue its growth after about two months. In the laboratory tanks wl
artificially fertilized eggs were kept, the worms underwent all the stages
normal development and attained a very large size (105 mm.) (Table II

In recording the growth, not only the maximum length of the tube
also the number of segments of the worm have been taken into considerat:
In every case the maximum length of the longest tube was accura
measured and then the animal was extracted and the number of segiru
and the nature of the gonads were made out under the microscope.

It was repeatedly found that this worm attained sexual maturity i
days after attachment. Glass slides let down into water on February 8 ;
taken out on February 17, 1936, just 9 days later 9 carried individuals v
ripe eggs and sperms. Adult worms removed from slides and put in dis
of fresh sea-water, extruded in the case of the male, a milky fl
containing millions of minute sperms and in the case of the fen
rose coloured eggs. Fertilization took place and gave rise to nor;
developmental stages.

Table II and Text-Fig. 2 give an idea of the rate of growth of Hydro\
norvegica from one day to about two months in the Harbour and to ab
nine months in the laboratory tanks. Text-Fig. 3 and Table XI give
growth of the form during all the months of 1936,

Growth and Breeding of Certain Sedentary Organisms

105
100

12O 16O

Age in Days

24O

28O

TEXT-FIG. 2. Graph showing the rate of growth of Hydroides norvegica (Table IT)

53
50

S
S

-c:

J FMAMJJASOND

TEXT-FIG. 3. Graph showing the growth of Hydroides norvegica
during all the months of 1936 (Table XI)

Breeding. It was found that this species was attaching itself to all
experimental materials throughout the year. Further, artificial fertilization
was successful every month of the year. Mature worms, young ones and

8 M. D. Paul

larvae were procured throughout. Breeding, therefore, is throughout the
year. No special seasonal intensity in breeding was observed.

Polyzoa.

Crisia sp. Among the several species of Polyzoa in the Madras Harbour,
a species of Crisia was found most frequently attached to the experimental
objects. This is reddish-brown in colour and grows erect giving rise to
many branches. The colony starts life as a single disc-like body from the
sides of which radical branches are given off in all directions, attaching the
selves to the substratum. Growth is vigorous and in a few days it attains
dimensions in which the zooids are difficult to be counted. The maximum
vertical and horizontal growth of the colony has been measured and taken
to represent the size of the colony.

When the form attains sexual maturity ovicells arise in or in close con-
nection with the fertile zooecia which give rise to the ovary from which later
the embryo is formed. " The ovicell arises in, or is in close connection with,
a fertile zooecium which gives rise to the ovum from which the embryos are

developed The number of ovicells of a given colony correspond to

the number of fertile zooecia" (Alice Robertson, 1911, pp. 226-27). It is
the presence of these ovicells that has been taken as the criterion of sexual
maturity for this species, since, observations have shown that the zooids are
sexually ripe before the ovicells are formed. It is an interesting fact that on
September 4, 1936, a colony of Crisia sp. measuring 21 by 14 mm. in size,
contained more than half a dozen ovicells. This colony was found firmly
attached to a slide which was immersed in water in the Boat Basin on August
25, 1936. It will thus be seen, that this species could attain maturity in 10
days after attachment though several others have been met with, some of
which are mentioned in Table III, where sexual maturity is attained in 16, 19,
20, 24, 25 or more days.

Breeding. Young ones and ripe individuals were met with in the
blocks as well as in different parts of the Harbour all the year round. During
the period of investigation the majority of them settled down and exhibited
very good growth during July, August and September. This form has, thus,
a continuous breeding period lasting throughout the year, with an intensity
in July, August and September.

Membranipora sp. This encrusting Polyzoan which occurs spreading
on shells and other substrata in the Harbour is, met with at times in large
numbers both on the slides and on blocks. After settling down they grow
vigorously and spread on the glass slides into circular or sub-circular colonies,

Growth and Breeding of Certain Sedentary Organisms 9

It has been noticed that on slides of one or two days' immersion one or two
initial zooecia of this colony could be found, which rapidly grow all round
resulting in about a thousand zooecia in a fortnight.

In a colony taken out and examined after fourteen days of immersion,
on February 26, 1937, there were some zooecia which had attained sexual
maturity.

Two measurements across the colony at right angles to each other have
been taken to represent the size of this form. Table IV gives an idea of the
rate of growth of this species.

The breeding of this form was not observed.
Mollusca.

Ostrea madrasensis Preston. Though this is primarily a backwater
form occurring in almost all the backwaters of South India, it is also found
in fairly representative numbers in the Madras Harbour occurring attached
to various objects. The sexes in Ostrea madrasensis are separate and
fertilization takes place in the water. After a free swimming existence of
about a week (Hornell, 191 5 and Moses, 1928) the larra settle down on
any available substrata fixing themselves by their left valve and grow
vigorously. After some days' growth they outgrow the size of the slides
and the bigger forms were usually obtained from wooden racks and buoys.

Sexual maturity is reached in as short a period as 21 days after attach-
ment (Paul, 1937). Slides that were in water from August 25 to September
15, 1936, contained a large number of Ostrea madrasensis attached to them.
The biggest among them (Pl.-Fig. 5) measured 12-5 by 12- Omm. .and its
gonad was found to contain ripe eggs (Text-Fig. 4) and others carried ripe
motile sperms.

Measurements have been taken in two directions, one from the hinge
across the shell to the opposite end and called here as length, and the other,
the breadth, the maximum dimension at right angles to this. PL-Figs. 4 to 9
and Table V give an idea of the growth of this form.

Breeding. The periodicity in breeding exhibited by this species is very
interesting. During the months of April and May were found a large number
of young oysters usually known as spat, attached to the submerged blocks.
This condition continued and plenty of spat settled down during the follow-
ing months till the end of October when it ceased. This clearly shows that
this form breeds during April to October. However, a study of the animals
during the non-reproductive period (November to March) showed that

M. D. Paul

TEXT-FIG. 4. Section of the gonad of Ostrea madrasensis of 21 days' growth (August 25 to
September 15, 1936; size 12-5x12-0 mm.) showing ripe eggs along with others in different
stages of growth

though breeding as such seems to have stopped, reproduction on a minor
scale was taking place, for it was noticed that even during this period they
carried well-developed gonads with ripe eggs (with yolk granules distributed
throughout the egg) and ripe motile sperms. To test the physiological condi-
tion of such sexual products the eggs were artificially fertilized in the Labo-
ratory and were found to give rise to early developmental stages. Unfortu-
nately further stages in the development were not followed. It has to be
mentioned that occasionally even during the non-reproductory period there
were met with here and there on the slides a few young ones.

It is of interest to compare the results obtained here with those of the
same species in the Pulicat back-waters near Madras (Hornell, 1910)
with Ostrea cucullata along the coast of Bombay (Awati and Rai, 1931) and
with Margaritifera vulgaris Schum. on the Ceylon coast (Malpas, 1933). In
the Pulicat lake it has been found by Hornell that there is a maximum
spawning of the oyster in the months of August and September. A second
maximal spawning takes place in March and April and " between this time

ant! August, spawning individuals can always be found**. In the Madras
Harbour the spawning starts about April or May and continues till it reaches
a maximum in September and October, after which it practically stops except
for occasional and irregular spawning, According to Awati and Rai
()\trca cunMaia starts spawning along ihe coast of Bombay in October and
continues upto the end of June, They could distinguish a regular breeding
,vtuv<j/j lasting from March to mid-June with intense breeding and an irregular
.vcuvw from October to i ebruary with a sparse breeding. The Ceylon pearl
oyster ( \fttr gitrititcrit u//:vm Schum.), on tin* other hand, has two spawn-
ing maxima f Malpax l'*,V*K one in July it* August coincident with the height
of the South- West monsoon and the other in December lo January coincident
with the North-l'.ast monsoon. However, 1 understand from Mr Malpas
that during the intervals between the two maxima, irregular spawning takes
place,

,1/viili'v viViJiv I,, Th*nit f ti tins green mussel forms enormously thick
growths on the neighbouring piles it is rarely that the larva* get attached to
flic experimental materials. Slides arc not favourite objects for their attach-
ment. Occasionally they arc met with on the wooden racks, the inner sides
of which aflVifvl f*oatl places for their attachment. The sides of freshly
scraped buoys offer excellent places of attachment for Bahmus and other
organisms and they in turn oiler a foot-holt! for the settlement of Mytilus
larva,'. Here, owing to the large amount of available space and food, they
?*ro\v at a rapid r/.te awl attain large si/c within a -short period,

After attachment the mussel grows rapidly and 4H days later the
fnnii c;ii?u'* n|v i" tl*l -I'ly. 16) or sperms. A mussel found attached
to a ttooilrn i ill ttnJ was immersed on September 6, and removed on
(K'tolvj ,\\ l uir . had in its mantle a large number of ripe eggs. At this
av a h.iil i-%'v':'/il a M/e of 15 .s mm. in length and f )-4mm. in breadth,
\\ 4 Vn 4 ' * d.> . old <!*!,' I i*!. 11.1 a female mussel was observed spawning,
I able \l tMis-. tli,* rale i*!" jM'ovvth of this species. I*l."l*igs. 10 to 15 also
indicate it' rro\ih I lie tnusseK have been measured alom their maximum
Jen th -tiiil breadth cou'ci to lialfa mm. by means of vernier calipers. The
details of the breeding of this farm mill form part of the subject-matter of
a separate paper,*

r^ii'llij t'CVj/finiii A-7'iVii |H, Adams). -The gonad of Patella iCcllana}
wrnit'tt ill Ailamsi, ;i form occurring among the breakwaters in the Harbour
was sluihcvl 0)r*'Uy'luiut the year 1^36 ami i! \vas found that at anytime of the

n on flic gro^ih i.f llvfiliii wtJn I... in the Mailr*w Harbour" (tsnput>!t*hed).

12 M. D. Paul

year fully adult females and males with ripe eggs and sperms were available.
Further, monthly samples of very small individuals (less than 5 mm. in
length) of this species were collected all the year round, which showed that
they were breeding throughout the year. Whether they had any intensity
in breeding during a part of the year or not was not observed.

Crustacea.

Balanus amphitrite Darwin. Of all the forms studied Balanus amphitrite
Darwin, was the one which got attached in very large numbers to the experi-
mental materials covering their surfaces to a great extent. Small cement
blocks immersed in water for 10 to 12 days at any time of the year developed
a dense encrustation of Balanus amphitrite with practically no interspace
between the individuals, A square cm. of the colonised area was noticed
with as many as 14 young barnacles.

The nauplius larvae liberated from mature Balanus lead a free-swim-
ming existence passing through the many stages of development and finally
settle down at the cypris stage. After this they grow vigorously and attain
a size of 10 by 9 ram. in 14 days. PL-Figs. 17 to 27 show the growth of this
form.

Glass slides immersed on February 19 and taken out on March 6, 1936,
contained a number of Balanus amphitrite, the biggest of which measured
8-8 by 7 -3 mm. (PL-Fig. 20). When this was carefully removed from the
slide and examined it was found that within the brood cavity there
were a number of developing nauplii with their appendages not yet fully
formed. This definitely shows that sexual maturity is attained in 16 days
after attachment. Other cases have also been met with, in which the animal >
at an age of 16 days after attachment, carries either ripe eggs or developing
larvas. Table VII and Text-Fig. 5 show the rate at which this barnacle grows.
Text-Fig. 6 and Table XII indicate the growth of this species during the
various months of 1936. The length of the base through the rostrum and
carina is given as the length of the animal and the dimension of the base
at right angles to this as the breadth.

Breeding. The sexual periodicity of this barnacle resembles that of the
tubiculons polychsete, Hydroides norvegica already described. A bi-weekly
examination of the nature of the gonad of individuals attached to
experimental objects and those collected from the Harbour revealed that
ripe eggs and developing nauplii were met with in the mantle cavity through-
out the year. Though their breeding was followed for more than a year,
there does not appear to be any special intensity in the spawning of this form.

nj; ,-

.s /

,,, ,

,/ ..W t v../^ r

I I f /

i !

!i ! !

/. /

1 .< in

\j M M Hi
^ .-, ,! Cm,,

C '" h>1C M

ras plankton
vc any

14 M. D. Paul

Ascidiacea.

Polycarpa sp. The simple ascidian was selected for study. This is
usually found in groups containing individuals of different sizes closely
attached with one another though with no organic connection between
them. Attached on. these are also a large number of Polych&tes and
Polyzoans. The tadpole larve after a free swimming existence attaches
itself to the substratum and after metamorphosis grows rapidly.

This is a hermaphrite form and the gonad is well developed in 17 days
after attachment (PL-Fig. 28) and ripe eggs are met with in 26 days. Thus it
was found that on a slide immersed on October 2, and taken out on October
28, 1936, the ascidian had reached a size of 22-0 mm. in length and 14-0 mm.
in breadth and contained a well-developed gonad with ripe eggs. However,
when the gonads of specimens measuring 10 by 6 mm. were "examined they
were found to contain ripe eggs and these forms would have, in all proba-
bility, reached this size in less than 26 days. It has to be mentioned, that this
species get attached only in certain months of the year and even then not
in anything like large numbers. PL-Figs. 28 to 32 give an idea of the
growth of this form. The distance from the basal portion to the very tip
of the branchial siphon and the maximum side to side dimension have been
taken to represent the length and breadth respectively, of the form. Table
VIII gives the rate of growth of this monascidian.

Breeding. Sufficient material was not available for a complete study
of the breeding of this species but what has been observed is interesting and
is set below. From the beginning of the year till May none of these indi-
viduals were found on the experimental objects but towards the end of May
or the beginning of June there was a rich settlement of tiny specimens. This
continued till the end of October after which practically no individuals
were met with. Examination of the gonad of this species during the non-
breeding period would have been of interest but in spite of repeated efforts
the specimens could not be procured from the Harbour. But from a study
of the organisms settling down on the blocks, it can be said that this species
breeds only during the months of May to October, after which period, if at
all any young ones are produced, it could only be due to irregular spawning.
This form then, seems to have a definite breeding period not lasting the
whole year round.

Diandrocarpa brackenhielmi Michaelsen. The growth of the compound
ascidian, Diandrocarpa brackenhielmi Michaelsen, is interesting when com-
pared with that of the simple ascidian. This encrusting colony contains
ascidiozooids not grouped in systems and is often met with in extensive

'ti\!t!, ". Ihifi.tnj lislu.ifnir, the licc tuttf JH'U*K|% if' %prwic^ Mtulicd in ftm I,abor.t

I mc% *! uiiitnjn* tiiivKiK -. *.h*w *a jfHUioir* liH'cditm without iiiv mtcnsiiv. Where thickcn-
tnf*\ ,H- .biv\n flu."'* i <i?l ntui.M%al liccdm^ .uuv*t\. An intcijijptcvihnc uidkalcs IKXM-
M*n,il wc.il %p..!v,n,iMt: I he lj,Mn*nt|-'.h.ipCiJ Uiu i ;icv, *,hw . inlcuuMcvl pen. t lie sp,iwnui|*,

oilimtcs, The rale t*i" pr*nvi!i iif this k extremely variable and only the
maximum actual measurements of the largest individuals of the specified
age were taken ami are riven in Table IX, and fierce they represent the
m^st rapid growth obtained during the yearn of study. After the settling
itmvti of the tadpole a single individual, the gono/ooid (clearly seen on
the glass slides), develops from if and later on others arise by repeated
budding.

Sexual maturity is attained in 2H days after attachment. My ciforts to
find the nature of the gonad in the living condition and try artificial fertili-
/ation were a failure and hence forms representing different ages were fixed
us Houm's thud, sectioned and. studied after staining in iron-h;cmatoxyJin.
I'orms which were found attached to slides removed on March 2, 1937, after

16 M. D. Paul

an immersion for 28 days contained ripe eggs (PL-Fig. 33) along with others
in different stages of growth.

In 15 days this form reaches a size of 34 by 25 mm. and contains about
110 zooids. It was noticed that this ascidian grows to very large dimen-
sions on the wooden racks but such forms have not been taken into consider-
ation for they generally represent the accumulated growth of a number of
colonies settled down side by side. The maximum size of an individual
colony has been measured and the number of zooids counted and are shown
in Table IX.

Breeding. The sexual periodicity of Diandrocarpa brackenhielmi differs
from that of the simple ascidian mentioned already. All experimental
materials set down at various parts of the year when taken out after a few
days invariably carried a few individuals of this form and sexually ripe
forms were met with throughout the year. Yet, it has been found that
during the summer months they occur on the objects in much larger
numbers than in the other months of the year. For instance, on objects
set and taken in the months of November, December and January these
colonies were not very numerous. Here is again, an instance of a form
which though breeding throughout the year has its intensity in breeding
during certain months only.

Discussion
Rate of Growth.

A study of the tables shows clearly the exceedingly rapid rate of growth
of these organisms. A comparison of the rates of growth of these forms
with those of allied ones in other parts of the world gives confirmatory
evidence. Hydroides hexagonis grows to a length of 7-0 mm. in 16 days and
takes 49 days to reach a length of 45-0 mm. at Woods Hole (Grave, 1933.),
where, according to Fish,* the temperature of the sea- water ranges from
1 to 21-6 C. whereas in the Madras Harbour (temperature ranges from
about 22 to 33 C.) Hydroides norvegica grows to a length of 39 -0 mm. in 16
days and attains a length of 43 -0 mm. in 23 days. Balanus eberneus grows at
Wood Hole to 2-0 by 1-7, 10-0 by 9*0 and 17-0 by 13-0 mm. in 9, 32 and 67
days respectively but its allied species B. amphitrite grows here to a size of
5-2 by 4-6, 14-0 by 13-0 and 20-0 by 20 -Omm. respectively in the three
periods mentioned. Annandale (1906) has observed in the latter species a
growth of 8-0 mm. in 22 days (April 17 to May 9) in the Gulf of Mannar.
A comparison of the growth of B. amphitrite with that of B. balanoides in

* Bull U.S. Bur. Fish., 1925, 41.

Growth and Breeding of Certain Sedentary Organisms 1

different parts of the coast of Europe (according to Orton, 1920, the sea
temperature off Plymouth ranges approximately from 7 to 16 C.) Herdla,
Port Erin and St. Malo reaching an average length of 6-5, 5-3 and 2-5 to
3 mm. * respectively, at the end of one year, also shows the very rapid growth
in the Madras Harbour. Elmhirst (1923) observed Balanus balanoides (L.)
reaching a diameter of 13- Omm. in three months in Clyde Sea-area. Again,
according^ Coe (1932), in La Jolla, California (temperature ranging from
14 to 21 C.) Balanus tintinnahulum. californicus grows to a maximum length
of 20 to 25 mm. within a minimum period of 22 weeks. The same species
has been found to grow to a diameter of 16-0 mm. at the end of 8 weeks
(Coe and Allen, 1937). In the case of B. amphitrite, approximately the
maximum size (20-0 by 20-0 mm., as recorded in Table VII) is reached in
about 68 days.

With regard to Ostrea, however, as mentioned by Hornell (1910),
though the growth is more rapid here than on the European and the North
American Coasts, in such places as in the warmer waters of Nortti Carolina
the rapidity of growth is almost similar. A comparison of the growth of
Mytilus, in the different parts of the world, will be given in a subsequent
paper by the author.

Coe (1932, p. 41) finds that in La Jolla, California, immersion of
experimental materials for at least two we?ks is desirable in order to allow
the young organisms to grow large enough to be scraped off for later study.
Erlanson (1936) working in Cochin Harbour says, " Growth was found to
be so slow that it was unprofitable to examine the blocks more than once in
two months ". It is noticed that the Cochin backwaters 'are very poor in
animl life in species very probably due to the alternation of almost com-
plete marine and fresh-water conditions. It is very natural, therefore, that
only very hardy forms (euryhaline) could survive. Other animals which get
settled down during the salt-water or fresh-water season will perish when the
next season supervenes.

Reference to Text-Figs. 3 and 6 (Tables XI and XII), giving in a graphical
way the rate of growth during 1936 of Hydroides norvegica and Balanus
amphitrite, respectively, two forms found to breed continuously throughout
the year, shows clearly that regular growth takes place during all the months
of the year and there is no appreciable retardation of growth during any
particular period. Here is no adverse season like a very cold winter as in
temperate climates to retard growth-rate. H. S. Rao (1937) working in the

* These figures represent only the average growth and certainly some individuals grow to
bigger sizes at the end of one year.

B2 F

18 M. D. Paul

Andaman Islands records that in Trochus niloticus Linn., there is no slacken-
ing of growth during any season of the year, and no definite period of aesti-
vation; though Moorhouse (1932) studying the same species on the Queens,
land coast, speaks of a definite slackening of shell-growth during the colder
months of the year. Continuous growth is also indicated by Delsman (1929)
as a result of his study on the fishes of the Java Sea. From a study of the
plankton in the Great Barrier Reef lagoon, it has been found that there is
sufficient planktonic food available throughout all the seasons of the year.
Marshall (1933) concludes that " the number of diatoms remains comparatively
constant throughout the year ; there are no great increases such as occurring
in temperate waters. There is no seasonal cycle of diatom growth ". Russell
and Colman (1934) come to almost similar conclusions regarding the zoo-
plankton. It seems, therefore, that there is no difficulty for food for tropical
marine animals during the various months of the year.

It is clear from this study that a very large number of animals in the
tropics attain sexual maturity at a surprisingly early age. Table X gives
a comparison of the age at which related species attain sexual maturity in
different parts of the world. It has to be remembered, however, that it is
impossible from the nature of things, to get data regarding the growth of
identical species in different parts of the world. So we have to content
ourselves with making a comparison of the growth of related forms only.
Reference to the table also shows that the size at which the local
forms attain sexual maturity is very small when compared with that of
others. In a few forms- like Ostrea madrasensis and Mytilus viridis, the
smallest size at which sexual maturity is attained (12-5 by 12 -Omm. and
15*5 by 9 -4 mm. respectively) when .compared with fully grown ones, is
striking. K. V. Rao (1937) working in this Laboratory on the structure
and development of Stiliger gopalai, found that this ascoglossan attains
sexual maturity and giyes off spawn within a fortnight. The rate of growth
of this form has also been found to be extremely rapid for under normal
conditions when food. material is available in abundance, they reach within
15 days their maximum size of 9 to 12 mm.

The constant settling down of generations of organisms even of a single
species, production of several offsprings in a year and their rapid growth result
in an intense competition for food and space and thousands perish in the
struggle. It is interesting to see a wooden rack (PL-Fig. 2) which had been
in water for about a month overgrown with such thick growth of organisms
that there is practically no space in between the two wooden pieces. The
barnacles have settled down in three or four rows one over the other, with

Growth ana Breeding of Certain Sedentary Organisms 19

the result that most of the organisms at the bottom die off for want of
oxygen and food. Compound ascidians also grow to enormous sizes
spreading like a mat over the Balani just leaving sufficient space for the
barnacles to protrude their food-procuring appendages.

This rapid rate of growth and the attainment of sexual maturity at a
very early age are doubtless governed by a number of physical, chemical and
biological conditions such as temperature, salinity, food, sunlight, hydrogen-
ion concentration, oxygen content of the water.

It has long been known that temperature exerts a profound influence
on animals. It is believed that on account of the high temperature the rate
of metabolism in tropical marine animals is extremely rapid. A number of
experimental evidences, such as those on Amphioxus lanceolatus and Beroe
ovata, quoted by Harvey (1928) deafly show that this increased rate of mata-
bolism is due to increase in temperature. Russell (1932) as a result of his
investigations on the breeding and growth of Sagitta elegans in Plymouth
has come to the conclusion that the time taken to reach maturity in colder
months was longer than in the warmer ones. Thus individuals born in
February took 94 days, those born in July 43 days, whereas those born in
September took nearly 165 days to attain maturity. Hjort (1912, p. 762)
has brought forth clear evidence to show " that the growth of fishes has proved
to be largely dependent on temperature, and that generally the growth of cod-
species may be said to decrease, and the age at first maturity to increase the
farther north we go." When the rates of growth of different species of the
oyster in different parts of the world are compared (Hornell, 1910) those
growing under high temperature conditions are found to grow faster. Com-
paring the conditions obtained in the Great Barrier Reef lagoon with those
of the English Channel, Orr (1933, p. 65) says, " the difference in average
temperature between the two areas is large (14 C.) and must result in a very
much higher rate of metabolism in the Barrier Reef lagoon ".

It is generally believed (Aiyar, 1933, p. 242) that as a result of the high
metabolic activities in the tropics there is a consequent rapid rate of growth
and earlier attainment of maturity. It has been found to be N so with regard
to most of the sedentary animals of the Madras Harbour. Delsman, work-
ing on the fish eggs and larvae of the Java Sea, has shown that development
takes place at a very rapid rate. He says (1929, p. 2), " With many kinds
of pelagic eggs development proves to take no more than one day ". " The
high temperature of the water (28-29 C. on an average) accelerates the hatch-
ing of the eggs and the development of the larvae." He cites the example
of Caranx macrosoma, in which the eggs are produced at about 10 or 11 p.m.,

20 M. D. Paul

and the young larvae hatch at 9 o'clock the next morning. Working on the
same problem on the Madras Coast, John* has obtained very similar results,
This rapid development has also been shown by Aiyar as a result of his studies
on the development of Acentrogobius neilli (Gobius neilli Day) (1935 d) and
Salmads bicolor Agassiz (1935 b) in Madras. As a result of this rapid meta-
bolic activity the duration of life of a large number of the organisms is com-
paratively shorter than that of similar forms in other places. There is no
doubt that most of the forms studied pass through several generations in
an year.

Salinity has also got an important part to play on the growth of these
animals. Brandt (1897) as quoted by Field (1922), noted that in Kielwight
the mussel Mytilus edulis grows to a length of 4^ inches, while in the Gulf of
Othnia, where the salinity of the water i| less, the mussel attains only about
half the size of those growing in the saltier regions. In Cochin Harbour,
where the salinity of the water is reduced by flood waters, it has been noticed
(Erlanson, 1936) that growth is very slow. Subramaniam and Aiyar (1936)
have observed that variation in salinity has got a possible effect on the varia-
tion in the size of the eggs of Dasychone cingulata, Salmads bicolor and
Clibanarlus olivaceus occurring in Madras. Hopkins (1931) has noticed
that the increase in salinity helps the development of oyster (Ostrea virginicd)
larvae. It has also been, shown by the same author (1936) that changes in
salinity influence the feeding mechanism of the oyster Ostrea gigas, and con-
sequently the amount of food taken in and the growth of the species.

That abundance of food plays a prominent part on the growth of
marine organisms has been accepted by a number of workers (Moore, 1905 ;
Kellogg, 1910; Field, 1922; Orton, 1928 a). Field discussing the different
factors on which the growth of the mussel, Mytilus edulis L., depends, says
that the chief one is abundance of food. " If .food is scarce, growth is
retarded regardless of all other conditions/' Kellogg has mentioned that
especially in plankton feeding organisms the rapidity of currents with the
consequent abundance of food results in accelerated growth and perhaps
earlier attainment of sexual maturity. It has been observed that in those
places in the Harbour, where wave action is negligible, and consequently

planktonic food limited for instance, the Timber pond (Text-Fig. 1),

the rate of growth is comparatively less.

That sunlight has a marked effect on marine animals is well known.
It has been found that in the temperate waters the great burst of diatom life

* M, A. John, unpublished records.

Growth and Breeding of Certain Sedentary Organisms

in the early winter is almost entirely due to the availability of solar energy.
Allen (1907) has shown that there is a direct correlation between the amount
of sunlight available in February and March in the British Seas and the
mackerel catches in May. From a study of the plankton of the Great Barrier
Reef lagoon, Russell and Colman (1934) show that the ratio between the
, minimum and the maximum of zooplankton available throughout the year
is not so great there as in the Northern waters or in the Massachusetts Bay
and that this is in no little measure due to the fact, that the duration of
daylight at different times of the year is approximately constant there when
compared with other latitudes,

Breeding.

The question of periodicity in breeding of tropical marine animals is
receiving considerable attention at the present day. It has been mentioned
by Semper (1881) that in the tropics, where the temperature variation is
comparatively negligible, there is apparently no periodicity in the breeding
of marine animals, chiefly Invertebrates. He says (1881, p. 135), " During
my stay in the Philippines, nothing struck me more peculiar than the evident
lack of periodicity in the life of animals, peculiar even to the insects, land
molluscs and terrestrial animals. I could always find eggs, larvae and adult
individuals of a species at the same time during winter as well as in summer".
Orton (1920) who showed that breeding in marine animals is correlated with
temperature says that in those parts of the sea, where temperature conditions
are constant, or nearly constant and where biological conditions do not vary
much marine animals breed continuously. However, he suggests that a
thorough investigation of this problem is desirable. Mortensen (1921)
studying the development of tropical Echinoderms, concludes that in the
tropics, though the temperature has an important bearing on the rapid dev-
elopment of the larvae, it need not necessarily result in the continuous breed-
ing of marine animals. He says (1921, p. 246), " I never found the opportunity
for studying the development of Diadema until I came to Tobago, B.W. I.
and there found D. antillarum to have ripe sexual products in the end of
March ; and when, a week later on, I wanted to start a new larval culture
it was impossible to find one specimen containing ripe sexual products, all
were empty ". He mentions a similar experience with Echinometra van Brunti,
Brissus obesus and Stichopus KefersteiniL In some of the Echinoderms he
could detect more than one breeding season in a year. He, however, agrees
with Orton " that where biological conditions do not vary much marine
animals will breed continuously," thereby suggesting that biological condi-
tions may vary in the tropics,

22 M. D. Paul

Fox (1924) working in the Red Sea, on the breeding of some Echino-
derms, Molluscs and crabs and its correlation with the phases of the moon,
infers that breeding is not dependent on temperature. Referring to Orton's
(1920) statement that the European oyster, wherever it is found, begins to
spawn at 15 to 16 C. and continues to produce sexual products as long as the
tempeature remains above this figure, Fox says, " this is not the case with
'Centrechinus, for its breeding season begins at the Suez some months previous
to July at a temperature well below that of July and September, yet from July
onwards, with the temperature still rising, the numbers of individuals reach-
ing maturity decline and in September all the breeding ceases although the
temperature is still above that at which the breeding season was initiated ".
Again the same conclusion is drawn by him for the Alexandria urchins
(Strongylocentrotus lividus).

From the table giving the occurrence and breeding seasons of ascidians
investigated by Berrill (1935), it is seen: (1) that in the Bermudas, where the
average temperature is nearly 25 C. all the forms studied had regular sea-
sonal breeding, (2) in Plymouth (temperature ranges from 7 to 16 C.) ?
though many of the forms observed had regular breeding seasons, others
were not seasonal.

On the Indian coast, Hornell (1910) working on the backwater oyster,
Ostrea madrasensis Preston, in the Pulicat Lake (near Madras), comes to the
conclusion that breeding is not continuous but takes place at a definite period
and is dependent on some factor or factors other than temperature. Dis-
cussing the probable cause of breeding he says, " A heightened temperature
certainly was not the case, as the temperature of the water before the onset
of floods was higher than during their continuance ". Malpas (1933) records
that the breeding of the pearl oyster (Margaritifera vulgaris Schum.), of the
Geylon coast, during two seasons of the year, does not depend on tempera-
ture, As a result of a preliminary survey of the growth and breeding of
marine animals, especially Teredinidse, in Cochin Harbour (Erlanson, 1936)
it is seen that the forms studied have definite periods of breeding and they
do not breed continuously throughout the year.

From what has been recorded by Sewell (1925) with regard to Littorina
scabra (Linn.), Littorina obesa Say and Pyrazus palustris (Linn.) in Nankauri
Harbour, Nicobar Islands, and Mytilus varibilis Krss. on the coast at Tor
in the Gulf of Suez, it is evident that these forms have a particular period
of breeding not lasting the whole year round. However, much emphasis
cannot be laid on these since the breeding period of the forms were not
specially investigated. In the case of Littorina scabra (Linn.) alone he suggests

Growth and Breeding of Certain Sedentary Organisms

that it has two breeding seasons each year, one just before, and another just
after the South-West monsoon.

Working in this Laboratory on the development of Salmads bicolor
Agassiz, a tropical Echinoid, Aiyar (1935 b) could obtain from the Harbour
ripe specimens and successfully fertilize them artificially in all the months of
the year and has thus shown that the form breeds continuously throughout
the year. The same author (1933 a p. 288), found with reference to the
breeding of the Polych&te worm, Marphysa gravelyi Southern, that " though
mature worms are not rare all the year round, there is a distinct reproductive
period immediately after the onset of the rainy weather (October) when the
egg masses are produced in very great abundance " and that " November to
March are crowded months " with regard to the occurrence of Polychaete
larvae in the plankton of the Madras coast (1933 b, p. 2). He also observed
(1935 a) Acentrogobius neilli (Gobius neilli Day) breeding throughout the year
in the Adyar backwaters, Madras, but with an intense breeding period
during the monsoon in October and November. An examination of the
gonad of the brackish-water hermit-crab, Clibanarius olivaceus (Henderson)
showed (Subramaniam, 1935, p. 14) "that they were breeding throughout
the year, though the breeding was observed to be particularly well marked
during the months of September and March ". In his study on the fish eggs
and larvae of the Madras coast, John* found that though the eggs and larvae
of Clupeid fishes are met with throughout the year, there is a definite intensity
in breeding from October to January. Studying the growth of Therapon
jarbua, Ranga Rao J found that this fish spawns with the onset of the mon-
soon in September or beginning of October and continues spawning all
through the rainy season, viz., October, November and December, and stops
breeding in January. He could notice an intensity in breeding in October
when about half of the fish caught contained fully ripe gonads and the other
half spent gonads. K. V. Rao (1937) observed that though specimens of
Stiliger gopalai could be collected from the Adyar and Cooum rivers
throughout the year, a large number of individuals and plenty of spawn were
available only when the salinity of the water was comparatively low due to
the closure of the bar at the river mouth. Menon (1931, p. 491) as a result
of his study of the plankton of this coast comes to the conclusion that "most
of the organisms exhibit a regular seasonal abundance, and corresponding
periods of maxima and minima. This variation, however, is not so clear as
in more northern latitudes, as the record of Herdman and other European

* M. A. John, unpublished records.

| S. Ranga Rao, " A statistical study of growth in Therapon jarbua ". (Unpublished.)

24 M. D. Paul

workers show ". An almost similar conclusion is arrived at by Hornell and
Naidu(1924) as a result of their studies on the plankton of the Malabar
coast. They say (1924, p. 151), " Analysis of the foregoing shows that a
definite seasonal cycle characterises the maximal abundance of the main
classes of organisms of importance in our plankton". It has been recently
recorded (Aiyar and Panikkar, 1937) that the Polychsete worm Platynereis
sp., occurring in the Madras Harbour, exhibits lunar periodicity and has
been found swarming on the New-moon day and the day preceding it or
(and) following it in the months of March, June and September 1935.

In studying the life-history of the Indian Sardine on the Malabar coast,
Hornell and Naidu (1924) found that this form spawns from about the
end of May to the end of August, with a maximal spawning extending
throughout June and July.

Anne Stephenson (1934) studying the breeding of marine Invertebrates
(Coelenterates, Echinoderms, Molluscs and Crustaceans) in the Low Isles
of the Great Barrier Reef observes that breeding takes place in every month
of the year, in summer as well as in winter. She divides the breeding of
animals into four main types: (1) a single breeding period not lasting the
whole year round, (2) continuous breeding throughout the year but more
active in one part of the year than during the remainder, (3) discontinuous
breeding occurring in relation to lunar phases during a longer or shorter
part of the year and (4) two spawning periods in the year with a quiescent
phase between them.

Galtsoff (1934), as a result of his investigations on the oyster, is of
opinion that temperature alone is not the paramount cause in the
breeding of animals but it depends on many factors which may act directly
or indirectly. He says, " Statements often found in text-books that under
the stenothermic conditions of the tropics breeding appears to be
continuous are incorrect". " Even in warmer seas reproduction takes place
as regularly as in cold seas."

Whedon (1936) found Mytilus californianus Conrad in the region of San
Francisco spawning at all times of the year, but with a maximum period of
spawning beginning early in October, followed by two other periods of lesser
degree in January and February and in May and June. He says (1936, p. 39),
" Temperatures, both of water and of air, doubtless play a part in the rate of
development of this spawn, but whether the temperature changes of the region
near San Francisco are severe enough to cause spawning, is questionable."
Water temperature in the region of San Francisco has varied from approxi-
mately 10 C. to as high as 17 C- over a period of more than a year.

Growth and Breeding of Certain Sedentary Organisms 25

H. S. Rao (1937) discussing this problem of breeding in tropical marine
animals puts forth the following questions: (1) Does the small range of
variation in the temperature of tropical seas admit of considering
temperature as the main stimulus for breeding ? (2) If the answer to
this question is in the affirmative, do the maxima and minima of
temperatures attained in various localities act as physiological
constants in the breeding of marine animals ? and (3) Do factors other than
temperature have any correlation with breeding and if so, to what extent ? He,
however, comes to the conclusion that " breeding in tropical marine animals
in relation to temperature changes seems to indicate that the evidence in sup-
port of the view that the tropical marine animals breed continuously tends to
gather weight ". Unfortunately he does not refer to the results of the studies
on breeding by other workers in India (Hornell, Malpas, Erlanson, he. cit.).
But from his study of the habits and growth of Pyrazus palustris (Linne)
(Rao, 1938) he thinks that this form breeds in March or April.

A review of the literature dealing with the breeding of marine animals
clearly shows that though temperature has an important bearing on the
breeding of marine animals in the temperate seas, it is not so important a
factor bearing on the breeding of tropical marine animals. The work of
Mortensen, Fox, Hornell, Malpas, Anne Stephenson, Nicholls, Moorehouse,
Galtsoff, Berrill and others clearly shows that temperature is not the sole
factor influencing the breeding of marine animals in the tropics.

A number of workers in the tropics have correlated breeding in marine
animals with salinity. From a study of the period of breeding of the Ceylon
pearl oyster, Margaritifera vulgaris, Schum., for many years Malpas (1933,
p. 21) concludes, " It has been shown in an earlier paper that the Ceylon
pearl oyster, Margaritifera vulgaris, has two spawning periods each year
which reach their maxima in July-August and in December- January, respec-
tively, the former period being coincident with the height of the South-
west monsoon when a maximum northerly flow of oceanic water of high sali-
nity enters the Gulf of Mannar and reaches the Pearl Banks at the head of the
Gulf, and the latter period being coincident with the height of the North-
east monsoon when a maximum southerly flow of water of low salinity,
produced by the torrential rains of this monsoon, enters the head of the Gulf
from the Palk Strait. This coincidence of the spawning maxima with maxi-
mum and minimum conditions of salinity suggests that the oysters are sti-
mulated to maximum spawning by the changes in salinity."

Hornell (1910) made an experimental study to determine the principal
factors effecting spawning in the oyster, Ostrea madrasensis Preston, in the

26 M. D. Paul

Pulicat Lake and summarising the results obtained, says (1910, p. 30), " the
maximum sexual activity of the edible oyster of the East coast rivers and
backwaters, synchronized with the heavy rains of October and November,
October being apparently the maximum. It also furnishes very strong evi-
dence in favour of the view that a rapid reduction in the density of the
surrounding water and not an increase of temperature as in European and
American waters, is the factor which determines the season at which the
majority of oysters shall spawn ".

K. V. Rao (1937) observed that low salinity condition was favourable
to the breeding of Stiliger gopalal He found that soon after the opening of
the bar at the mouth of the Adyar or Cooum, there was practically no spawn
at all and very few individuals could be found. But when the bar was closed
and the salinity of the brackish-water was lowered there were plenty of spawn
and a large number of young ones, showing thereby, that a sudden fall in the
salinity helps breeding and growth of this species. With regard to
Marphysa gravelyi Southern, Clibanarius olivaceus (Henderson), Acentro-
gobius neilli (Gobius neilli Day), Therapon jarbua and the Clupeid fishes, it
is seen, as already indicated, that breeding or an intensity in breeding takes
place soon after the onset of the rainy season, most probably due to the con-
sequent change in salinity.

Discussing the occurrence of Siphonophora in the plankton of the Great
Barrier Reef lagoon and correlating its comparative absence in it from
February to May, with hydrographical conditions existing there, Russell and
Colman (1935, p. 270) say, " It can at once be seen that the period of absence
of most of the species of Siphonophora from the Barrier Reef lagoon coincided
with that for low salinity, which was a result of heavy rains prevalent at that
time of the year." In this connection the fact that in the Great Barrier Reef
region, the summer months in which " a vast majority of the species investi-
gated was found spawning" (Anne Stephenson) should be the months
of maximum rainfall and the consequent lowering of salinity, seems rather
interesting.

It has been mentioned by Anne Stephenson that the actual breed-
ing season of a particular animal is likely to fluctuate from one district to
another. Thus we find that in Trochus niloticus L., the spawning period
was of at least five months' duration commencing in March in the Low Isles
of the Great Barrier Reef (Moorehouse, 1932), whereas the same species
was found to breed continuously in the Andaman Islands (Rao, 1937).
Centrechinus (Diadema) setosus was found by Fox (1924) to show breeding
correlated with the phases of the moon in the Red Sea, whereas the same-

Growth and Breeding of Certain Sedentary Organisms 27

species does not show in its breeding any correlation to the lunar phases in
the Great Barrier Reef lagoon. It was observed by Moore (1934) that
considerable variation occurs in the period of breeding of Echinus esculentus
at different depths even in localities separated only by a few miles.

It is evident, then, that breeding of animals on the Madras Coast is not
confined to any particular season of the year. While forms like Laomedea
(Obelia) spinulosa were most active during the cooler months of the year, the
majority of the forms were found spawning throughout the year with an
intensity in their breeding during a part of the year. This is shown graphi-
cally in Text-Fig. 7. The breeding of these can be grouped together under
the following types:

(1) Single breeding period not lasting the whole year round, e.g., Poly-
carpa sp. and Therapon jarbua.

(2) Continuous breeding all the year round but more active during a
certain part of it, e.g., Laomedea (Obelia) spinulosa, Crisia sp., Marphysa
gravely I, Ostrea madrasensis, Mytilus viridis, Stiliger gopalai, Clibanarius
olivaceus, Salmacis bicolor, Diandrocarpa brackenhielmi, Acentrogobius neilli
and the Clupeid fishes, probably also Patella (Cellana) cernica.

(3) Continuous breeding- throughout the year without any special
intensity in breeding during any part of it, e.g., Balanus amphltrite and
Hydroides norvegica. And (4) Discontinuous brezding related to phases
of the moon, e.g., Plat y nereis sp.

Comparing the results obtained here with those of Anne Stephenson
(1934) in the Low Isles of the Great Barrier Reef, the breeding seems to be
almost similar. Only, forms which breed throughout the year without any
intensity during any particular period have not been recorded by her. Species
which have two spawning periods in an year have not been so far met with
here, but it is not improbable that such forms do occur on the Madras Coast.

From the result of the studies on the breeding of tropical marine animals
it is clear, that the balance of evidence favours the idea that temperature is not
the all-important factor causing the breeding of tropical marine animals.
After discussing the importance of temperature in affecting the breeding
of animals in the temperate waters and the occurrence of regular breeding in
the animals of a tropical coast like the Great Barrier Reef region (as shown
by the results of the Scientific Expedition to the Great Barrier Reef (1928-29),
Galtsoff (1934) says, " This fact indicates that the temperature is not always
a decisive agent and that there must exist other factors controlling the
rhythm of the life processes in the sea",

28 M. D - Paul

Summary

1 The rate of growth and period of breeding of the following seden-
tary organisms in the^ Madras Harbour have been worked ouiiLaomedea
(Obelia) spinulosa, Hvdroides norvegica, Crisia sp., Membranipora sp.
(growth alone), Ostrea madrasensis, Mytilus viridis, Patella (Cellana) cernica
(breeding alone), Balmus amphitrite,Polycarpa$$., and Diandrocarpa bracken-
hlelmi.

2. The environment and the methods employed in this study are
described.

3. The rates of growth of the different forms studied, have been com-
pared with those of corresponding or related forms in different parts of the
world.

4. The species investigated attain their sexual maturity very early in
their life.

5. There seems to be continuous growth throughout the year.

6. Reference is made to the breeding of other tropical marine animals
and it is found that breeding takes place: (a) once a year, not lasting the
whole year round, (b) twice a year, with a quiescent phase between the two
periods, (c) continuously but with an intensity during a certain portion of
the year, (d) continuously without any marked intensity and (e) dis-
continuously depending on the phases of the moon.

7. The influence of temperature on breeding in marine animals in the
tropics is discussed and it is inferred that temperature is not the only factor
influencing breeding.

Acknowledgement

It is with pleasure that I express my warmest gratitude to Prof. R. Gopala
Aiyar, Director of the University Zoological Research Laboratory, Madras,
not only for suggesting this topic for investigation but also for accompany-
ing me during many of my visits to the harbour and for constant help and
encouragement throughout this work. My thanks are also due to Mr. R.
Velappan Nair for helping me with the sketches and many of the photo-
graphs, and Messrs. S. Ranga Rao and M. A. John for permitting me to
use data from their yet unpublished records. I take this opportunity of
thanking the authorities of the Madras Port Trust for giving me permission
to collect specimens from the harbour and also for giving much useful
information,

Growth and Breeding of Certain Sedentary Organisms
TABLE I. Rate of growth o/Laomedea (Obelia) spinulosa

Period of growth

Age

Height

No. of
polyps

Period of growth

Age

Height

No. of
polyps

days

mm.

davs

mm.

Feb. 19 to Feb. 24, 1936

3-8

Dec. 26 to Jan. 6. 1936

9-0

23 1

Dec. 26 to Jan. 2, 1936

7-5

10*

Jan. 22 to Feb. 3, 1937

10-5

28 {

Jan. 22 to Feb. 1, 1937

5-5

Oct. 12 to Oct. 26, 1936

16-0

351

* No Gonosomes present.
J Gonosome present.

The left-hand column gives the period of growth beginning with the attachment and metamorphosis
of the planula. All measurements are of actual colonies and not averages.

TABLE II. Rate of growth o/Hydroides norvegica

Period of growth

Age

Length
of tube

No.
of seg-
ments

Period of growth

Age

Length
of tube

No.
of seg-
ments

days

mm.

days

mm.

March 15 to March 16, 1936

0-9

. .

Jan. 18 to Feb. 3, 1936

39-0

49*

28 to 30, 1936

1-9

July 13 to Aug. 3, 1936

40-0

44*

Dec. 26 to Dec. 30, 1935

5-1

March 15 to April 7, 1936

43-0

42*

March 15 to March 21, 1936

5-3

July 13 to Aus. 12, 1936

53-0

43*

Dec. 26 to Jan. 2, 1936

5-8

13 to ,r 17, 1936

49*

Feb. 8 to Feb. 17, 1936

14-0

30*

Sept. 6 to Oct. 16, 1936,

43 :

48*

2 to 11,1937

16-0

May 6 to June 20, 1936

54*

Sept. 16 to Sept. 25, 1936

19-0

31*

Sept. 6 to Oct. 23, 1936

42-'o

50*

Jan. 18 to Jan. 29, 1936

20-0

43*

Oct. 16 to Dec. 12, 1936

61*

Oct. 12 to Oct. 26, 1936

23-0

36*

Sept. 16 to Nov. 18, 1936

66*

Sept. 16 to Sept. 30, 1936

26-5

34*

June 22 to 18, 1936

149

90 :

51J

Feb. 10 to Feb. 24, 1937

28-0

22 to March 29, 1937

280

105-0

58J

Sept. 16 to Oct. 2, 1936

33-0

4i*

* This form attains sexual maturity in 9 days after attachment.

if Represent the growth of forms in the Laboratory aquarium tanks.

TABLE III. Rate of growth of Crisia sp.

Period of growth

Age

Size

Period of growth

Age

Size

days

mm.

days

mm.

Aug. 25 to Sept. 4, 1936

21-0x14-0*

July 13 to Aug. 7, 1936

46-Ox 30-0*

July 13 to July 24, 1936

21-0x14-5

Jan. 22 to Feb. 17, 1937

50-Ox 31-0*

13 to 27, 1936

34-0x19-5

July 13 to Aug. 12, 1936

83-Ox 45-0*

13 to 31, 1936

50-0x32-0

13 to 17, 1936

100-Ox 55-0*

June 19 to 8, 1936

24-0x16-5*

13 to 28, 1936

138-Ox 86-0*

July 31 to Aug. 21, 1936

47-0x44-0

Oct. 12 to March 30, 1937

169

180-0x100-0*

31 to Aug. 24, 1936

63-0x35-0

Size measurements have been taken showing the maximum vertical and horizontal growth of the Colony.
* Denotes ripeness of the colony as judged by the presence of ovicells.

M. D. Paul
TABLE IV. Rate of growth of Membranipora sp.

Period of growth

Age

Size of
colony

No. of
zooids

Period of growth

Age

Size of
colony

No. of
zooids

/4o\*e

days

mm.

Julv 13 to July 17, 1936
Jan. 8 to Jan. 15, 1937
July 13 to July 22, 1936

4
7
9

3-Ox 2-5
4-Ox 4-0
15*0x12-5

25
30
over
400

Aug. 25 to Sept. 7, 1936
July 13 to July 27, 1936
13 to 31, 1936

13
14
18

16-0x14-0*
19-0x19-0*
31-0x26-0

over
1000

Aug/25 to Sept. 4, 1936

14-5x14-0

over
460

13 to Aug. 12, 1936

41-0x28-2

over
1000

Jan. 22 to Feb. 3, 1937

16-0x12-0

13 to 17,1936

41-0x40-0

over
1000

Ripe zooecia denoting sexual maturity, were obtained.

TABLE V. Rate of growth o/Ostrea madrasensis

Period of growth j Age

Length

Breadth

Period of growth

Age

Length

Breadth

davs

mm.

days

mm.

Ausj. 25 to Aug. 28, 1936

0-8

0-75

Aug. 25 to Sept. 15, 1936

12-5

12-0*

Oct. 2 to Oct. 12, 1936

4-4

3-0

25 to 25, 1936

15-0

13-5*

Aug. 25 to Sept. 7, 1936

6-3

5-6

May 6 to June 19, 1936

21-5

14-0*

., 25 to 10, 1936

6-5

6-1

July 13 to Oct. 5, 1936

37-0

34-0*

May 6 to Mav 25, 1936

12-0

Feb. 6 to Oct. 7, 1936

243

66-0

71-0*

; Sexuallv mature.

TABLE VI. Rate of growth of Mytilus viridis L.

Period of growth

Age

Length

Breadth

Period of growth

Age

Length

Breadth,

July 13 to Aue;. 10, 1936
Sept. 6 to Ocf. 23, 1936
July 13 to 5, 1936
March 28 to Sept. 8, 1936
Feb. 24 to Aug. 10, 1936

davs
30
48
84
164
167

mm.
14-5
15-5
34-5
52-0
55-5

mm.
9-0
9-4*
19-0
27-0
28-5

Feb. 8 to Aug. 10, 1936
April 1 to Dec. 7, 1936
Feb. 5 to Oct. 7, 19^6
April 15 to March 2, 19^7
May 1 to July 26, 1937

days
184
241
243
321
445

mm.
56-5
77-5
113-0
93-0
103-0

mm.
30-0
36-0
52-O.t
39-5
44-0

* Sexually mature.

{ Represents exceptionally good growth.

Growth and Breeding of Certain Sedentary Organisms
TABLE VII. Rate of growth of Balanus amphitrite

Period of growth

Age

Length

Breadth

Period of growth

Age

Length

Breadth

days

mm.

days

mm.

Jan. 18 to Jan. 20, 1936

0-9

0-7

Feb. 19 to March 6, 1936

8-8

7.3*

June 19 to June 23, 1936

1-1

0-8 -

June 19 to July 6, 1936

10-0

May 6 to May 11, 1936

1-6

1-4

19 to 8, 1936

11-0

Jan. 18 to Jan. 24, 1936

2-9

2-8

Jan. 31 to Feb. 24, 1936

13-5

12-0

May 6 to May 14, 1936

3-0

2-3

Aug. 25 to Sept. 26, 1936

14-0

13-0

Jan. 18 to Jan. 27, 1936

4-8

4-0

Sept. 16 to Oct. 28, 1936

14-5

14-0

Feb. 15 to Feb. 24, 1937

5-2

4-6

July 13 to Aug. 28, 1936

16-5

13-5

Jan. 18 to Jan. 29, 1936

5-4

4-7

Sept. 16 to Nov. 23, 1936

20-0

June 19 to July 1, 1936

5-8

5-0

Feb. 6 to Oct. 7, 1936

243

19-0

19-OJ

24 to 8, 1936

10-0

9-0

Nov. 27 to Aug. 10, 1936

257

21-5

21 -0

Jan. 18 to Feb. 3, 1936

6-2

5-4*

* Sexually ripe with developing Nauplii larvae inside.
J Found attached on a buoy let down into water on February 6, 1936.

Found attached on an oyster shell which was attached to an iron piece tied down on November
27, 1935.

TABLE VIII. Rate of growth 0/Polycarpa sp.

Period of growth

Age

Length

Breadth

Period of growth

Age

Length

Breadth

days

mm.

days

mm.

Oct. 2 to Oct. 19, 1936

12-0

6-0*

Sept. 6 to Oct. 23, 1936

34-0

15-5$

2 to 28, 1936

22-0

14-OJ

April 1 to Aug. 15, 1936

136

54-0

24-Oi

2 to Nov. 2, 1936

29-0

12-OJ

1 to Sept. 8, 1936

160

63-0

33-Oi

July 13 to Aug. 28, 1936

30-0

I6-5J

* Gonad of this form well developed,
i With ripe eggs, sexually mature.

Found attached to the North Buoy, Boat Basin, which was scraped down of all attached organisms,
painted and put in the water on April 1, 1936.

TABLE IX. Rate of growth of Diandrocarpa brackenhielmi

Period of growth

Age

Size of
colony

No.
of indi-
viduals

Period of growth

Age

Size of
colony

No.
of indi-
viduals

days

mm.

days

mm.

Feb. 19 to Feb. 26, 1936

7-Ox 4-1

May 6 to May 28, 1936

62-0x38-0

over

July 13 to July 22, 1936
Feb. 19 to Feb. 29, 1936

9
10

8-4x 6-0
8-lx 7*4

May 6 to May 29, 1936

65-0x26-0

300
over

19 to March 2, 1936
19 to 4, 1936

12
14

16-5x16-5
27-0x23-0

34
60

July 13 to Aug. 7, 1936

65-0x33-0

300
over

2 to Feb. 17, 1937

34-0x25-0

over
100

Feb. 2 to March 2, 1937

60-0x40-0

300
over

Oct. 2 to Oct. 19, 1936

36-0x23-5

over
100

Sept. 6 to Oct. 16, 1936

48-0x42-0

500
over

Aug. 26 to Sept. 15, 1936

46-0x28-0

over
100

6 to 23, 1936

60-0x50-0

500
over

500

Sexual maturity is attained in this species in 28 days.

M. D. Paul

TABLE X. A comparison of the age at sexual maturity of certain sedentary
forms in different parts of the world

Species

Locality

Age at
maturity

Size at
maturity

COELENTERATA

mm.

Clytia Johns to in

Plymouth,

1 month

England

Tubularia

Cawsand Bay,

1 1 days

Plymouth

Companularia flexuosa and C, cal-

Woods Hole,

4 weeks

ceollfera

Mass.

Obelia commisswalis

, ,

Laomedea (Obelia} spinulosa

Madras

8 days

5-6

ANNELIDA

Pomatoceros irigueter and Hydro id-

Plymouth

4 months

es norveglca

Filograna sp. . .
Hydroides hexagon is
Hydro ides norvegica

Woods Hole
Madras

10 V weeks
59 " days

54x2-7
14-19

POLYZOA

Biiguhi flabellata

Plymouth
Woods Hole

8 weeks
30 days

Biigula nerltana

La Jolla,

6 weeks

California

Crisia sp.

Madras

10 days

21 x 14

Membranipora sp.

19x19

MOLLUSCA*

Gahina e.xigua Tergipes

Cawsand Bay,

22 days

Plymouth

Amphorina ?

Madras

Stiliger gopalai

Ostrea edulis

English Coast

1 year

Ostrea lurida

La Jolla '

23 weeks

25-35

Ostrea madrasensis

Madras

21 days

12-5x12

CRUSTALEA

Balanus eberneus

Woods Hole

60 clays

14-5x12-

Balanus tintinnabulum calif or nlcus

La Jolla

12-13

Balanus balanoides

Plymouth

1 year

Herdla,

about

6-5

Bergen

1 year

6-5

Port Erin

5-3

Balanus amphitrite

Madras

16 days

8-8

ASCIDIACEA

Botryllus violaceits

Plymouth

3 months

Leptodinum (Diplosoma) gelati-

3& weeks

nosum

Botryllus gouldii

Woods Hole

30 days

Diandrocarpa brackenhielmi

Madras

60x40

Mo Ig ula manhatensis

Woods Hole

3-4 weeks

70x8

Ascidia conchilega

Essex Coast

Poly car pa sp.

Madras

16-26 days

22x14

Author
and year

Orton, 1914

1929

Grave, 1933

Paul, 1937
Orton, 1914

Grave, 1933
Paul

Orton, 1914
Grave
Coc, 1932

Paul

Orton, 1929

Paul

Rao, K. V.,

1937

Dodd, 1937
Orton, 1921
Coc
Paul

Grave, 1933
Coe, 1932
Orton, 1914
Runnstrom,
1925

Paul

Orton, 1914

Grave, 1930
Paul, 1937
Grave, 1933
Orton, 1914
Paul, 1937

a'/.^ and JiwJiug of G'r

U' XI, Growth mw*/v ^/'Hydroides norvegiea

Pcruui of grew i h

f tube

Fciitnl of grtwih

Length
o!" tube

l:kv,

, days

nun

i!,*vs

mm.

26 to Dec. tu, 1'Hs 4

^ i

May 2

June

10. 1936 W

2(J ^

Vi to JLtlt. 2, 1'J *6 .-'

s-s

2*^, 1*'.<6 I*C)

1 ? 41

6 to .. \ l^ s ' 10

n-s

June

2* tM 16 4

2 4

Vi In ,, f*. 1'Hh 1!

I ' ? "U

l l

2S, l*M6 . V

12 -S

** to , X, |J3<, ! \

24 '0

July

'i, 1^36 1 1

! #* i)

Vi | M ti |(i t j^lft ,, |s

2' ' *

6* i*M6 .. 17

2 ^ o

*e in " i *' 1** U j 'Hi

K "X *^

July

j7 j^t6 ' 4

2 I

^ to " r! I'M** : 22

11 -0

22.*

w If. *i

x- s

V* lo Vf.iUh 14, 1*M6 - ?J

4*MI

24 ,

V36 H

IMJ

IK to Jan. 2''. !*><fi i

12- *

27,

^36 ; 14

1 "." s

is to J4 2'', l**u ' H

JU'U

-U.

9 <6 : I H

2 2 *i

itf t* l-elv t. t*M6 16

I'l

Aug.

t> i|j. 2 1

4!) O

H to , 1 4 I^U ; ^

4- 5

12,

*! <6 : $0

s ru

X Jo ,, 1?, 1*^6 : M

14-0

AUK, 2s

Sept,

M/ JO

1 1 ti

x to !**, 1 4 H6 -. U

i <!

>,
* *

111!

*J'!l! ' \l

j*i-

M to ,, 24, t*M<i ! s

* in .. 26, I 1 '*!. ! 7

4-?

? Sept. 6

Oct.

ill

V 46 , 411

4 1 (1

> to Maivh :\ !*Mf : 1}

IM'O

: < IO

2^,

**3(> ' 4K

^(J- Cl

j f l3 ja <, JW ; 16

J'J

i 1 16 li-

Kept,

2*>

*/ 46 ', V

1 9 C 1

'Mo ,. **, J*M6 ' 1'i

2. 1

1 , 16 to

,Ul 1*M6 * 14

26- <>

- to ,! ji. pMd : i

U 9

i !! t6 u

Oct.

2, !'M6 f 16

U II

"" tO ,, !**, JH6 : 4

.V 1 ^

i C.K;f. 12 U>

26, 1*H6 14

2^-0

*" to ,, ?i, r^?- : 6

5- .1

! ., i? to

\ov.

IS, |*M6 : 36

K-(J

^ !.t ,, / \ !"'*ft S

** I

Nov. 20 to

Ikv,

J2. l*>36 22

1 Vll

' to ,, /4, J'Hri *|

1 2 **

Dec. H to

IK. I.!f* j JO

JO'O

to ,, ? s , l l - 4 ft HI

1 ^ I

X to

2$. 1^36 ; 1 \

1^ i!

1 * ? t* ,. 2 ', J'^ *' 12

JS. J

H X fo

23, I*^'U ! 5

I *

I 1 * to Apiil ", J*M6 ^ $

4^- 1)

H to

2s! 1*^36 ; 20

211 it

;>< in M.u*n Hi, J*iii* _ ,!

I -V

8 to

31, JV36 ' 2^

2S u !\pn! *. r^f* : IU

iM)

M. D. Paul

TABLE XII. Showing the data of growth in Balanus amphitrite

Period of growth

Age

Length

Period of growth

Age

Length

days

mm.

clays

mm.

Dec. 26 to Jan. 2, 1936
26 to 6, 1936

7
11

0-7

2-2

June 19 to June 28, 1936
19 to July 1, 1936

9
12

3-9

5-8

26 to 8, 1936

2-4

19 to 6, 1936

10-0

26 to 10, 1936

4-3

19 to 8, 1936

11-0

26 to 13, 1936

6-7

July 13 to 22, 1936

3-0

26 to 15, 1936

7.4

13 to 24, 1936

3-4

26 to 17, 1936

9-5

13 to 27, 1926

5-3

Jan. 18 to 20, 1936

0-9

13 to 31, 1936

6-8

. 18 to 24, 1936

2-9

13 to Aug. 3, 1936

10*8

18 to 27, 1936

4-8

13 to 7, 1936

1 1-0

18 to 29, 1936

5-4

13 to 12, 1936

13-0

18 to Feb. 3, 1936

6-2

Sept. 6 to Oct. 16, 1936

10 -0

18 to 10, 1936

9-0

6 to 23, 1936

12-0

Feb. 19 to 24, 1936

1-3

16 to Sept. 25, 1936

3-8

19 to 26, 1936

2-9

16 to 30, 1936

9*5

19 to 29, 1936

4-7

16 to Oct. 28, 1936

14-5

19 to March 6, 1936

8-8

Oct. 2 to 16, 1936

7-0

19 to 9, 1936

9-0

2 to 19, 1936

9-5

March 15 to 19, 1936

1-0

2 to Nov. 2, 1936

12-0

;, 15 to 23, 1936

2-2

11 to Oct. 26, 1936

8-0

15 to 27, 1936

2-7

11 to 28, 1936

8-5

15 to 28, 1936

3-7

11 to Nov. 9, 1936

10-0

15 to April 7, 1936

7-2

11 to 16, 1936

11*0

April 1 to 28, 1936

10-5

27 to 12, 1936

7-5

May 6 to May 8, 1936

0-8

27 to 16, 1936

8-0

6 to , 11,1936

1-6

Nov. 16 to Dec. 2, 1936

9-5

6 to , 14, 1936

3-0

16 to 10, 1936

11-0

6 to , 18, 1936

4-2

Dec. 8 to 18, 1936

4-5

6 to , 21, 1936

6-0

8 to 21, 1936

6-5

6 to , 25, 1936

9-0

8 to 23, 1936

8*5

6 to June 19, 1936

11-5

8 to 28, 1936

11-0

June 19 to 23, 1936

1-1

Growth and Breeding of Certain Sedentary Organisms 35

TABLE XIII

Mean temperature of air (maximum and minimum) and amount
of rainfall at Madras during the period of study

(Taken from the Monthly Weather Report of the India Weather Review, published by
authority of the Government of India)

Months

Maximum
F.

Minimum

Rainfall
inches

Surface-water
temp.* F.

1935

November
December .1 \\

85-5
83-4

71-4
70-0

6-40
2-35

1936

January

85-0

68-9

0-10

February .
March
April
May
June

85-4
87-5
94-4
95-5
96-6

73-2
72-9
78-2
81-5
80-2

2-53
0-90
0-46
0-02
2-82

81-73
83-25
85-39
84-25
81-28

July
August
September
October . .
November
December \

94-8
93-9
92-9
89-7
84-8
84-0

79-2
77-2
77-8
76-2
73-4
71-9

4-87
5-99
1-93
8-16
14-53
1-94

79-21
82-01
84-98
84-87
81-04
78-11

1937

January
February

83-9
86-0

68-6
71-9

0-07

79-25
79-33

March

88-9

73-8

Surface-water temperature of the shore near the Laboratory, an average of readings taken
at 9 a.m., and 5 p.m.

Abe, N.

Annandale, N.

Ainsworth, J. R., and Fleure,

H.J.
Aiyar, R. G.

Idem

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WO, !6.

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///.'/, .-f\s//. t r.A*. ..v tl fHM5, .X

<ira\c, B. 1!.* , . " R.itc u{' irmvih ami at.* of s;j\uaf maturity of certain sessile

ir*Mius!ns/' -ln.it. A'<v. t 1924, 29.

. " Mu- natuial hisiorv of t'ttnwwj't tcWtMiih*," Ilfal. /?////., 1<>27,

> ^.

. . " Naiiiial hisiorv t>f Shipwonns. '/*7\W> nuvatis, at Woods Hole,
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A /,*///!,, j*M{) t 4'),

. . " Imhryolotfy ami life-history of r'Vw /*/>/<'/,/ apiadtttt:* Ihhl
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. , " Hate of growth, ae at sexual maturity and duration of life of
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* Urochord.i/ " Bifll, A/W. (ttn-f. A/wv, % w.\. % M<?/. ///\/ IV 1 ?
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. , "On the Growth of the shell of A/rrr/n'v nh'wtn\\ especially with
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/r H\ ,-l, ffenbmiu, /?*>>*;/ ,Vi^\ Ai</. fc 1906, Sup. Rep, 39.
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J, Muiray and .1. Hjort London. 1912.
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logic von lfahttw\" XietMtimfaheex Arc, /Tcw/^. 1876, 3

Hopkins, A. E,

Hornell, J.

Hornell, J., and Naidu, M. R.
John, C C.

Kellogg, J. L,

Lele, S. H., and Gae, P. B.

Leloup, E.
Lo Bianco, S.

Lynge, H.
Malpas, A. H.

Marine Biological Association

of United Kingdom
Marshall, S. M.

- and Stephenson, T. A.

Mayer, A. G.
Menon, K. S.
Moore, H. B.

M. D, Paul

" Factors influencing the spawning and settling of oysters in

Galveston Bay, Tex." Bull. U.S. Bur. Fish., 1931, 42.
" Adaptation of the feeding mechanism of the oyster (Ostrea

gigas), to changes in salinity," Ibid., 1936 a, 48.
" Ecological observations on spawning and early larval develop-
ment in the Olympia oyster (Ostrea lurida)" Ecology, 1936 b,

17.
"The biological results of the Ceylon pearl fishery of 1904,"

Rep. Cey. Biol Lab., 1905, 1.
" Note on an attempt to ascertain the principal determining factor

in oyster-spawning in Madras backwaters," Mad. Fish. Invest.,

1908, 1910, 4.

" A note on the edible oyster," Mad. Fish. -Bull., 1915, 8.
" A contribution to the life-history of the Indian Sardine, with

notes on the plankton of the Malabar Coast," Ibid., 1924, 17.
"Seasonal variations in the distribution of Sagitta of the

Madras Coast," Rec. Ind. Mus., 1937, 39.
" Shell-fish industries," Henry Holt and Co., New York, 1910.
" Common Sagitta of the Bombay Harbour," Rep. Jour. Univ.,

Bombay, 1937, 4.
" Une collection d'Hydropolypes arrartenant ITndian museum

de Calcutta," Rec. Ind. Mus., 1932, 34.
"Notizie Biologishe regulardanti specialmente il periode di

mature sessuale degli animali del Golfe di Napoli," Mith.

aus. der ZooL Station zu Neapel, 1909, Bd. 19.
"IV Marine Lamellibranchiata," Danish Expd. to Siam,

1899-1900, Copenhagen, 1909.
" Further observations on the age and growth-rate of the

Ceylon pearl oyster, Margaritifera vulgar is, with special

reference to oysters of Donnan's Muthuvarathu Paar,"

Cey. Jour. ScL (C), 1933, 5.

" Plymouth Marine Fauna " (Second Edition), 1931 .

" The production of microplankton in the Great Barrier Reef
Region," Set. Rep. Great Bar. Reef Expd., 1928-29, 1933, 2.

" The breeding of Reef animals, Part I Tire Corals," Ibid.,
1933, 3.

" Effect of temperature on tropical marine animals," Carnegie
Inst. Wash., 1914, 6.

"A preliminary account of the Madras plankton," Rec. Ind.
Mus., 1931,33.

" A comparison of the biology of Echinus esculentus in different
habits, I," Jo urn. Mar. Eiol. Assn., U.K., n.s., 1934 a, 19.

"The biology of Balanus balanoides; I Growth rate and its
relation to size, season and tidal level," Ibid., 1934 , 19.

" The biology of Echinocardium cor datum," Ibid., 1935 a, 20.

" The growth-rate of Balanus hameri (Ascanius)," Ibid., 1935 b
20.

Moore, H. B.

Moore, H. F.
Moorhousc, F. W.
Mortensen. Th.

Moses, S. T.

Nelson, T, (",*

Nichulls, A.<.

1 and Breeding of Certain Sedentary Organisms 39

* 4 A comparison of the biology of Echinus esculent us in different

habits, 11," Ibid., 1935 c, 20.
** A comparison of the biology of Echinus esculentus in different

habitats, III," //>/</., 1937 , 21.

** The biology of Llttorinalttlorca: Part I Growth of the shell

and tissues, spawning, length of life and mortality," Ibid.,

1937 /. 21.
" Anatomy, embryology and growth of the oyster,** U.S. Comrn.

i'hli ami Hv/imV.v, Cotnm. Rep. for 1903, 1905.
*" Notes on Twchus niloticus," Set. Rep. Great Bar. ReefExpd.,

192S -29, 1932, 3.
** Studies of the development and larval forms of Echinoderms/'

(/./A f. 1 . Ctihl-Ct>pcri/Mxen, 1921.
*' A preliminary report on the anatomy and life-history of the

common edible back-water oyster, Ostrea madrasensis,"

Jour. Htm, Nu!. Hist. .Vw., 1928, 32.
11 Aids ti> successful oyster culture/' New Jersey Agr. Exp. $tat. t

1921. Nn, 3^1.
** Relation i>f spawning of the oyster to temperature," Ecology,

*' On the distribution of critical temperature for spawning and
for ciliary activity in bivalve Molluscs/* Science, 1928 />, 67.
14 On the breeding and growth-rate of the black-lip pearl oyster,
iJa tnunwittft'rti)" Rt'p. Great. Bar, Reef Comm.,

ighue, C, II.
Orr, A. R.

*' Observations on the early development of Membranipora
vlllastt Hincks," Cont. Canad. Biol. and Fish., 1926, 3.

** Physical and chemical conditions in the sea in the neighbour-
hood of the C i real Barrier Reef," Set. Rep. Great Bar. Reef
/;\/w/., 192K.29, 1933,2.

A preliminary account of a contribution to an evaluation of
the sea," ,/<w. Mar. Biol. /!//., U.K., /.*., 1914, 10.

** Sea-temperature, breeding and distribution in marine animals,"
MM.. 1920, 12.

"An oyster spat (1921) with mature male sex products,"
Mtfii/r, 1921, 108.

** On the rate of growth of Cardium etlulc, Pt. I, Experiment-
al observations," Jour. Mar. BioL Assn., /.&"., n.s., 1927, 14.

" On the rhythmic period in shell-growth in Ostrea edulis with
note on fattening/ 1 Ibid.* 1928 a, 15.

" Observations on Patella vulgata, II Rate of growth of shell,"
Ibid., 19281?, 15.

14 Experiments in the sea on the growth-inhibitive and preser-
vative value of poisonous paints and other substances ,'*
Ibid., 1929, 16,

" Studies on the relation between organism and environment/*
Tram. Liverpool Biol. Assn., 1933, 46.

** Bionomical studies on Cardium edule" James Johnstone Memo-
rial Fa/,, Liverpool, 1934.

Paul, M. D.
Pelseneer, P.
Preston, H. B.
Prytherch, H. F.

Rai, H. S.

Rao, H. Srinivasa

Rao, K. V.
Robertson. A.
Runnstrom, S.

Russell, F, S.

Russell, F. S., and Colman,
J. S.

Semper, K.
Sewell, R. B. S.

hearer, C.

M. D. Paul

" Sexual maturity of some sedentary organisms in the Madras

Harbour," Curr. Sci. 9 1937, 5.
" La duree de la vie et 1'age de la maturite sexuale chez

certain Mollusques," Ann. Soc. Roy., Bslgique, 1933, 44.
" Report on a collection of Mollusca from the Cochin and Ennur

backwaters," Rec. Ind. Mas., 1916, 12.
" Investigations of the physical conditions controlling spawning

of oysters and the occurrence, distribution and setting of

oyster larvae in Milford Harbour, Conn.," Bull U.S. Bur.

Fish., 1929,44.
"The shell-fisheries of the Bombay Presidency," Jour. Bom. Nat.

Hist. Soc., 1932, 35.
" Observations on the rate of growth and longevity of Trochus

niloticm Linn., in the Andaman Is.," Rec. Ind. Mus., 1936, 38.
" On the habitat and habits of Trochus niloticus Linn., in the

Andaman Seas," Ibid., 1937, 39.
"Observations on the growth and habits of the Gastropod

Mollusc, Pyrazus palustris (Linne), in the Andamans,",

Ibid., 1938, 40.
" Habits, structure and early development of a new species of

Stiliger Ehrenberg," Ibid., 1937, 39.
"The cyclostomatous Bryozoa of the West Coast of North

America," Univ. Calif. Publ. in Zool, 1911, 6.
" Zur biologie und entwicklung von Balanus balanoides (LinnS)",

Bergens Mus. Aarbok for 1924-25; 1925, No. 5.
" Uber die thermopathid der forplanzung und entwicklung

mariner Tiere in Bexiehung, zu ihrer Geographischen ver-

breitlung," Ibid., 1927, No. 2.
" On the biology of Sagitta. The breeding and growth of

Sagitta elegans Verrill in the Plymouth area, 1930-31," Jour.
Mar. Biol Assn. U.K., n.s., 1932, 18.
" A comparison of the abundance of Zooplankton in the Barrier

Reef lagoon with that of some regions in Northern Euro-
pean waters," Sci. Rep. Great Bar. Reef Expd., 1928-29,

1934, 2.

" The composition of the Zooplankton of the Barrier Reef
Lagoon," Ibid., 1934, 2.

"The Zooplankton IV, The occurrence, and seasonal distri-
bution of the Tunicata, Mollusca and Coelenterata (Sipho-
nophora)," Ibid., 1935, 2.

"Animal Life," Inter. Sci. Ser., London, 1881.

" Observations on growth in certain Molluscs and on changes
correlated with growth in the radula of Pyrazus palustris " Rec.
Ind. Mus., 1925, 26.

" Geographic and Oceanographic research in Indian waters,"
Mem. Asi. Soc. Beng., 1929, 9.

"On the development and structure of the trochophore of
Hydroides uncinatus (Eupomatus),," Quar. Jour. Micr. $oc.,
1911,56,

Growth and Breeding of Certain Sedentary Organisms 41

Sparck, R.
Stephen, A. C.

Stephcnson Anne
Stott, F. C.
Subramaniam, M. K.

.... linc j Aiyar, R. G.

Visscher,J. P.
Whcdon, W. F.

White, K. M.
Winckworth, R.

*' Studies on the biology of the oyster in the Limford with special

reference to the influence of temperature on sex-change," Rep.

Danish Biol. Stat., 1925, 30.
" Notes on the biology of Tellina tennis da Costa," Jour. Mar.

Biol. Assn., U.K., n.s., 1928, 15.
" Notes on the rate of growth of Tellina tennis da Costa, in the

Firth of Clyde," Ibid., 1929, 16.
*" Notes on the biology of some Lainellibranchs in the Clyde

area," Ibid., 1933, 18.
44 The breeding of Reef animals, Part II-Invertebrates other than

corals," Sci. Rep. Great Bar. Reef. Expd., 1 928-29, 1934, 3.
" The Spawning of Echinus esculentus and some changes in

gonad composition," Jour. Ekp. Biol, 1931, 8,
" Oogenesis of Clihanarius olivaceus (Henderson), with special

reference to a seasonal variation in the cytoplasmic inclusion, "

Jour. Royal Mic. Soc., 1935, 55.
" On the possible effect of the environment on the cytoplasmic

inclusions in'the oocytes and oogonia of Dasychone cingulata,

Salmacis bicolor and Clibanarius olivaceus" Proc. Ind. Acad.

Sci., Ser. B, 1936, 3.
" Nature and extent] of fouling of ships' bottoms," Bull. U.S.

Bur. Fish., 1928, 43.
" Spawning habits of the mussel, Mytilus californianus Conrad

with notes on the possible relation to mussel poison, Mussel

poison I," Univ. Calif. Publ in Zool, 1936, 41.
" Mytilus," L.M.B.C. Memoirs, 1937, 31.
" On the growth of Paphia undulata (Veneridas)," Proc. Malac.

Soc., 1931, 19.

* References marked with an asterisk have not been directly consulted by the author.

EXPLANATION OF FIGURES

FIG. I. Photograph of a wooden rack used in this study. The zinc strip and the glass slides

inside are seen clearly.

FK.I. 2. -The same rack, photographed after an immersion for a fortnight in December 1936.

FIG. 3. Photograph of 3 glass slides with the attached organisms; immersed in water for
(a) 13 days, (/?) 18 days and (r) 22 days (4/5 natural size).

FIGS. 4- -9. Growth of Ostrea madrasensis. (Figs. 4 to 7 J natural size, and Figs. 8 and 9
natural size). FIG. 4. 19 days, 12-0 x 12-0 mm. FIG. 5. 21 days, 12-5 x 12 mm.
(sexually mature). FIG. 6. 31 days, 15-0 x 13-5 mm. FIG. 7. 44 days, 21-5x 14-Omm.
FK;, 8. 84 days, 37-0 X 34*0 mm. FIG. 9. 243 days, 66-0 x 71 -Omm.

FIGS. 10-15. Growth of Mytilus virldis (i natural size). FIG. 10. 84 days, 34-5 x
19-0 mm. FIG. 11. 93 days, 29-0x16-4 mm. (observed spawning). FIG. 12. 135 days,
35-U x 20 mm. FIG. 13. 164 days, 52-0 x 27-0 mm. FIG. 14. 184 days,
56-5 X 30-0 mm. FIG. 15. 243 days, 1 13-0 x 52-0 mm. (exceptionally good growth).

42 M. D. Paul

FIG. 16. Photomicrograph of a section of the mantle of Mytilus vindis 48 days old (15-5 X
9 -4 mm.) showing ripe eggs along with different stages in their growth (10 x 40).

FIGS. 17-27, Growth of Balanus amphitrite. (Slightly bigger than natural size). FIG. 17
9days, 4-8 x 4-Omm, FIG. 18. 12 days, 5-8 X 5-Omm, FIG. 19. 14 days, 10-0 X 9-Omrru
FIG. 20. 16 days, 8-8 x 7-3 mm, (sexually mature), FIG. 21. 19 days, 11-0 x 11-Omm.
FIG. 22. 24 days, 13-5x 12-0 mm. FIG. 23. 32 days, 14-0 x 13-Omm. FIG. 24. 42 days.
14-5 x 14-0 mm. FIG. 25. 68 days, 20-0 X 20-0 mm. FIG. 26. 243 days, 19-0 x 19-0 mm.
FIG. 27. 257 days, 21-5 x 21 -Omm.

FIGS. 28-32.--GrowthofP0/)>c3rpasp. ($ natural size). FIG. 28. -17 days, 12-0 x 6-0 mm,
FIG. 29. 30 days, 29-0 x 12-Omm, (mature). FIG. 30. 45 days, 30-0 x 16-5 mm. FIG. 31.
47 days, 34-0 x 15-5 mm. FIG. 32. 136 days, 54-0 x 24-0 mm. (N. B. Figures reduced
to t of magnification given.)

FIG. 33. Photomicrograph of a section of the gonad of Diandrocarpa brackenhielmi
of 28 days' growth showing ripe eggs. (10 X 40).

Proc. hid, Atari. .SVv M B, v

p 1

THE EXTERNAL MORPHOLOGY OF THE BRAIN
OF SEMNOPITHECUS ENTELLUS

(A Comparative Study)

BY A. ANANTHANARAYANA AVER, B.A., M.B.B.S.

(AtUitwmil /Yfi/J'A.vor of Anatomy, Medical College, Madras)

Received October 29, 1941
f Communicated by Prof. A. Subba Ran)

Introduction

CONSIOI ixr on a study of the brains of some of the old world monkeys
including <Vf/,w//,s\ Cofahus and Cynopiihccus with reference to the exhaus-
tive work of Kufccnthal and Ziehen on the Primate brain, Beddard (1903)
makes the following statement: 4fc I cannot distinguish by any tangible differ-
ences the arrangement of the furrows in the genera Macacus, Cercopithecus,
Cercocclnw and perhaps Papio. It appears to me that among the Cercopi-
thccidie there arc only two plans of cerebral conformation, one confined to
Cercopithecime and the other to Semnopithccinse." It is proposed in this
paper to develop this idea further, by a more detailed study of the external
morphology of typical representatives of these two subfamilies. The
author's own observations on two brains of Scmnopithecus entellus form
the basis for the comparison. Both these brains were practically alike in their
topographical details. T'lv: description of the brains of Macacus and other
monkeys have been taken from studies by Elliot Smith (1902), Beddard
(1903), Duckworth (1915), Tilney (1928) and Mines (1933); and in addi-
tion a personal examination has been made of the brain of Macacus sinicus.

General Features of the Brain of Semnopithecus entellus

Viewed from above the entellus brain has a broad oval outline with the

narrow end forwards. The cerebellum is completely hidden by the cere-
brum. The frontal pole is narrow; the occipital poles are full and rounded.
The orbital surface shows the characteristic keel, medially, and hollow,
laterally. The inferior surface of the cerebrum is not as deeply moulded by
the cerebellum as in Macacus. The broadest part of the brain corresponds
to the region behind the upper part of the parallel sulcus. On the medial
surface of the sagittal section the relatively large corpus callosum is well
made out. The continuity between the septum pcllueidum and the gyrus

44 A. Ananthanarayana Ayer

subcallosus (parts of the original paraterminal body) is seen (Text-Fig. 2 B)
and a flattened band of nervous tissue passes downwards from the lower part
of the septum pellucidum in front of the anterior commissure to become
the diagonal band of Broca.

Measurements of the Brain of a Young Adult Female Entellus Monkey
Maximum length of brain . . . . - 6 85 cm.

Maximum width of brain . . .. .. 5-95,,

Encephalic index . . . . - &7

Length of corpus callosum . . . . . 3-05 cm.

Thickness of corpus callosum at splenium . . 0-4

Precallosal length, i.e., the distance between the genu and

frontal pole . . . . . . . 1-6

Postcallosal length, i.e. 9 the distance between the splenium

and occipital pole .. .. .. 2-35

Vertical measurement of anterior commissure . . . . 0-3

Antero-posterior measurement of anterior commissure . . 0-2 ,,
Weight of brain, with meninges immediately after removal 114 gm.
Weight of brain, without meninges and vessels after pro-
longed hardening in 5 per cent, formalin . . . . 86-5

Weight of forebrain . . . . . . . . . . 73 5

Weight of midbrain .. .. .. .. .. 0-75

Weight of hindbrain .. .. .. .. 12-25

Proportions of forebrain : midbrain : hindbrain . . . . 85:1:14

Fissuration of the Cerebrum in Entellus
The superolateral surface (Text-Figs. 1 A, 2 A)

The central sulcus extends downwards and slightly forwards from a
point near the middle of the medial margin and finally makes a terminal
bend backwards. The length of the central sulcus is 3 -0 cm. On the surface
of the cerebrum in front of the central sulcus three sulci are seen, vtz. 9
s. rectus, s. arcuatus and s. precentralis superior. The sulcus rectus is nearly
parallel to the inferior border near the frontal pole and is 1-9 cm. long.
The sulcus arcuatus is 2-5 cm. long and has a course directed from below
upwards and forwards. The arcuate nature is such that in the lower part
it runs in a nearly coronal direction and then terminates anteriorly in a nearly
sagittal direction. The superior precentral sulcus is a small one in front of
the upper part of the central sulcus.

/ t ;-ii-.t,- Morfiluhgy ,>/ ///, Rrain of Semnnpltheeus cntcllus

1 vr-fi., !. Superior (M and Inferior (B) views of (he cerebral he.imphcrc*

I he .VV/I-W/,./ K .V, W has a genera! oblique direction from below upwards
and backwards. Posteriorly its terminal part does not unite with anv other
sulcus. o,, p crJ ing the lips of the anterior part <>f the Syivian fissure the
insula ts seen, I he submerged part of the insuia is hounded above by the
, whose anterior end extends forwards on the deep aspect

46 A. Ananthanarayana Ayer

of. the operculum, but is not visible on the superficial surface. A pseudo-
sylvian fissure limits the insula below. The anterior limiting sulcus of the
insula (s. front o-orbitalis) is absent and the insula becomes continuous with
the posterior part of the orbital surface without any line of demarcation.
Parallel to Sylvian fissure but more extensive than it inferiorly as well as
superiorly is the superior temporal sulcus (parallel fissure). It does not join
any other sulcus. The inferior temporal sulcus is represented by three small
detached sulci near the lower part of the temporal lobe. The middle one
is the smallest, being just a dimple.

The intraparietal sulcus starts midway between the central sulcus and
Sylvian fissure and passes at first obliquely upwards and backwards arching
over the upper ends of the Sylvian fissure and superior temporal sulcus ;
the intraparietal then gives off a short medially directed transverse branch
in front of the arcus parieto-occipitalis ; then turning downwards and back-
wards the intraparietal joins the lunate ^sulcus. The sulcus postcentralis
superior is seen as a coronal sulcus parallel to and behind the upper part of
the central sulcus. In the inferior parietal lobule in the region between the
intraparietal sulcus above, the posterior part of the Sylvian fissure below
and the superior temporal sulcus behind is a small distinct sulcus of doubtful
identity whose significance will be discussed subsequently.

The lunate sulcus does not touch the superomedial or inferolateral
borders. It comes close to the superomedial border but is separated by a
considerable interval from the inferolateral border. The sulcus is concave
forwards in its upper part and convex forwards in its lower part. The
superomedial portion of the lunate sulcus forms the posterior boundary of
the arcus parieto-occipitalis. The intraparietal sulcus joins the lunate nearly
a third of the way down from its upper end. The lower portion of the sulcus
lunatus and the superior temporal sulcus come close to each other but are
distinctly separated by a gyrus on whose surface a short unidentified antero-
posterior sulcus is seen. The inferior occipital sulcus forms a curve concave
upwards situated about midway between the lower end of the lunate sulcus
and the inferolateral border. The posterior end of the inferior occipital
sulcus has the appearance of being bifurcated. The upper limb of the bifur-
cation, which is the real continuation of the sulcus, passes on to the lateral
surface of the occipital lobe. The lower limb of the bifurcation is, however,
deeply separated from the rest of the sulcus by a submerged gyrus indicated
by two dots in the course of the sulcus in the figures. This deeply detached
sulcus passes medially towards the inferior surface. Its significance will be
discussed along with the collateral sulcus. In the region situated between
the superior temporal .sulcus in front, the lunate sulcus behind and the

External Morphology of the Brain of Semnopithecus entellus 47

5WP.PRECENTRAUS,

^UP.POSTCENTRAL.S,

TEMPORAL.S.

PARACALCARlNE.s.
\ PARltTO-OCCIPlTAt. FISSURE,
PRECUNEAL.S,

PINEAL BOD V'
MAMILLARY BODY-''

ACCESSORY

A NT. C O MMJ 5 SAJ RE .
CHIASMA

,SUPiPOSTCENTRAL.S,

'ARCUS

PARVETO-
OCCIPJTAL1S
PARIETO-

OCCIPJTAL

FISSURE

TEXT-FIG. 2. Lateral (A), Medial (B) and Posterior (C) views of the left cerebral hemisphere.
The posterior view is drawn on a higher magnification and the lips of the lunate sulcus and
intraparietal sulcus are represented slightly, opened out, The arcus parieto-occipitalis is
seen fully exposed,

48 A, Ananthanarayana Ayer

inferior occipital sulcus below, is an unidentified sulcus having a coronal
direction. The significance of this sulcus will also be discussed subsequently.
The lateral surface near the occipital pole shows the superior occipital sulcus
and another small sulcus probably accessory to it.

The medial surface (Text-Fig. 2 B)

In the circumcallosal zone on the medial surface the following sulci are
seen: (1) a rostral sulcus situated midway between the rostrum of the
corpus callosum and the medial orbital border, (2) a genual sulcus in front
of the genu of the corpus callosum, and (3) an intercalary sulcus starting
above the genual sulcus and passing at first backwards parallel to the upper
surface of the corpus callosum and finally taking an obliquely upward course
towards the superomedial border. The termination of the upturned part
of the intercalary is situated along the superomedial border about midway
between the superior postcentral sulcus and parieto-occipital fissure.

In the region corresponding to the precuneus, there is a triradiate pre-
cuneal sulcus (compensatory sulcus) placed above the splenium of the corpus
callosum. It consists of a horizontal element and a vertical element.

The parieto-occipital fissure is a deep cleft beginning on the medial
surface a little behind the splenium and directed upwards and backwards.
It 'does not meet the calcarine fissure inferiorly, being separated from it by
a gyrus. The upper end of the parieto-occipital fissure cuts the superomedial
border and extends on to the lateral surface where it is bounded by the well-
marked arcus parieto-occipitalis (Text-Fig. 2C). The arcus is limited in
front by a transverse limb of the intraparietal, laterally by the posterior part
of the intraparietal and posteriorly by the medial part of the lunate sulcus.
On opening the lips of the parieto-occipital fissure the fossa shows hidden
in its depth a sulcus placed in relation to its floor along the anterior wall
and another very small sulcus on its posterior wall. Posterior to the parieto-
occipital fissure is a small sulcus on the cuneus, probably a paracalcarine
sulcus.

The calcarine fissure begins on the medial surface near the occipital
pole above the superior occipital sulcus and then runs forwards and down-
wards on to the inferior surface. It is a deep single sulcus and shows no bifur-
cation posteriorly and no hidden gyrus in its depth.

The inferior surface (Text-Fig. 1 B)

The rhihal fissure extends backwards from the incisura temporalis and
forms a lateral boundary to the pyriform area. The Collateral fissure begins
posteriorly near the occipital pole ami runs forwards for nearly two-thirds

External Morphology of the Brain of Semnopithecus entellus 49

of the length of the occipitotemporal part of the inferior surface. There is
a submerged gyrus in its depth behind its middle showing that it is consti-
tuted of two united sulci. It also shows two laterally directed rami from the
hinder part of the anterior segment. Mention has already been made in the
description of the inferior occipital sulcus of a small sulcus apparently
united to its posterior part but deeply separated from it by a submerged
gyrus. Between this small sulcus and the collateral fissure is another small
detached sulcus. These two small sulci together probably represent the
transverse collateral sulcus. Between the collateral sulcus and the anterior
end of the calcarine fissure a small sulcus is present. This is probably the
beginning of a paracollateral sulcus. The orbital part of the inferior surface
shows a gently curved S-shaped sulcus, the orbital sulcus, along with small
accessory dimples laterally and medially. The sulcus olfactorius is absent.

Comment on Cerebral Fissuration

Inferior parietal lobule. Even without going into a detailed consider-
ation of the areas of cortical localization and cytoarchitectonics, it will be
generally admitted that the inferior parietal lobule, i.e., the region roughly
between the intraparietal sulcus in front and above, and the sulcus lunatus
behind, is an association area where sensory impressions of touch, hearing
and vision are pooled together. The separation of the terminal part of the
Sylvian fissure and superior temporal sulcus that occurs in entellus indicates
an expansion of this cortical area. In addition certain new sulci have
appeared in this region. (1) A small sulcus has appeared between the intra-
parietal sulcus and posterior part of the Sylvian fissure. It is the superior
parallel sulcus (ascending I of Kappers). This identification is in conso-
nance with the interpretation by Beddard (1903) and Shellshear (1927).
Beddard also considers that the existence of this sulcus differentiates the
brain of Semnopithecus from that of Nasalis. (2) The gyrus that separates
the lunate sulcus from the superior temporal sulcus has a small sagittal
sulcus on it. This is the sulcus prelunatus. (3) In the lower part of the
triangular area between the superior temporal, lunate and inferior occipital
sulci, there is a sulcus in the coronal plane (see Text-Fig. 2 A). This is
probably the beginning of an anterior occipital sulcus (ascending III of
Kappers). The separation of the Sylvian fissure from the superior temporal
sulcus and the appearance of these three sulci in the inferior parietal lobule
of the Semnopithecus for the first time in the evolution of the Primates are
evidences of the expanding growth tendencies of this region.

The intraparietal sulcus. The transverse branching of the intraparietal
in front of the arcus parieto-occipitalis indicates a tendency for the intra-

50 A. Ananthanarayana Ayer

parietal to become more complex. The terminal outward bend of the intra-
parietal sulcus prior to its joining the lunate sulcus is also another feature
in which Semnopithecus differs from Macacus.

The occipital lobe. The definite decrease in the operculation of the
sulcus lunatus and inferior occipital sulcus in Semnopithecus as compared
with Macacus, associated with the constant appearance of a superior occi-
pital sulcus and accessory superior occipital sulcus, shows a backward shift-
ing of the striate area by the expanding peristriate cortex. The formation
of the superior occipital sulcus appears to be a means of accommodating
" pushed back " striate cortex in the limited space available. Transverse
and sagittal sections through the occipital cortex when examined with a hand
lens show the stria Gennari extending up to the posterior lip of the lunate
sulcus but not extending into the cortex in the depth of the sulcus. The
stria Gennari is also made out on the sides of and in the depths of the
superior occipital sulcus and the posterior part of calcarine fissure.

The sulcus cinguli. The sulci that make up the sulcus cinguli of man,
e.g., the rostral sulcus, genual sulcus and intercalary sulcus are all present
in Semnopithecus entellus. But the terminal upturn of the intercalary is
situated midway between the superior postcentral sulcus and parieto-occi-
pital fissure and appears to be far too back from the probable posterior
limit of the paracentral lobule. It is therefore likely that the terminal part
of the intercalary does not correspond to the terminal upturn of the sulcus
cinguli of man.

The precuneus. The precuneus is definitely larger in Semnopithecus
than in Macacus. It is this preponderance of the precuneus over the cuneus
that gives a posterior inclination to the parieto- occipital fissure in entellus
as contrasted with the upward and forward course of the parieto-occipital
fissure in Macacus. In Elliot Smith's diagram of the medial surface of the
brain of Macacus sinicus (fig. 241, R.C.S. Cat., 1902) the precuneal sulcus
is absent; and in her diagram of the rhesus brain, Hines (1933) shows a
small precuneal sulcus, with a query. On the contrary Duckworth (1915)
clearly indicates a compensatory sulcus (sulcus precunei) in Nasalis and it
it equally well marked in Semnopithecus entellus.

The parieto-occipital fossa. The parieto-occipital fissure is really a
complex of submerged sulci, and so it is appropriately called a fossa. The
identification of its constituent elements is based on Elliot Smith's interpret-
ation of the parieto-occipital fossa (1902 and 1904). The sulci a, ,8 and y
described and illustrated in fig. 241 of R.C.S. Cat. (1902) are named incisura
parieto-occipitalis, s. limitans precunei, and s. paracalcarinus, respectively.

External Morphology of the Brain of Semnopithecus entellus 5 1

In the entellus the incisura parieto-occipitalis occurs separated from the
intraparietal by a well-formed arcus parieto-occipitalis and the fossa proper
is formed by the submergence within it of the incisura parieto-occipitalis
and sulcus limitans precunei, due to an overgrowth of the two lips. The
sulcus paracalcarinus, however, is not yet submerged and occurs in the cuneus
behind the parieto-occipital fissure. Of the two opercula of the fossa, the
anterior operculum seems to show greater growth tendencies than the
posterior.

The calcarine fissure. The posterior T-shaped bifurcation of the calca-
rine fissure usually seen in Macacus is absent in Semnopithecus entellus.
But it has been occasionally noted in Semnopithecus (Beddard, 1903; Elliot
Smith, 1902) though not so well visible on the dorsal view of the undivided
brain as in Macacus.

The collateral fissure. This fissure is longer and better constituted in
Semnopithecus than in Macacus. It gives evidence of being a union of two
sulci, by the presence of a submerged gyms in its depth. It also shows a
tendency to develop two laterally directed rami about its middle. The
occurrence of transverse collateral and paracollateral sulci, has already been
noted. These are pointers to an expansion of the area around the collateral
fissure.

Discussion on Cerebral Fissuration of Semnopithecus entellus

From the foregoing comments on the fissuration of the cerebrum, it
becomes clear that the fissural pattern of Semnopithecus entellus in addition
to being different from what occurs in the Cercopithecinae is also definitely
of a more advanced type. Beddard (1903) suggests from the view-point of
cerebral morphology that Colobus "is to be placed with the Cercopithecinas".
The comparison of the brain of Nasalis and Semnopithecus entellus has
already shown that in the formation of the sulcul pattern of the inferior
parietal lobule there is a marked advance shown by the entellus monkey.
Thus the observations herein noted and the relevant comments made on them
lead to the inference that among all the Cercopithecidze the entellus monkey
probably shows the highest cerebral development. In describing the parieto-
occipital fissure of Hylobates hoolock, Elliot Smith (1902) says: " the series
of modifications necessary to convert the brain of a Macacus into that of
a Semnopithecus are carried a stage further in the case of Hylobates ". This
intermediate position of the Semnopithecus between Macacus and Gibbons
is shown by the entellus not only in the region of the parieto-occipital fossa

also in the whole gamut of its cerebral fissuration. More confirmatory

52 A, Anarithanarayana Ayer

evidence will be adduced from the cerebellum and brain stem described
below.

The Cerebellum in Entellus

Nomenclature. The old nomenclature for the parts of the cerebellum
has been purposely ignored. Nor is it considered necessary here to go into
a discussion of the nomenclatures according to various authors (Bolk,
Ingvar, Elliot Smith, Bradley, Abbie, and Larsell) who have worked on the
cerebellum. A summary of the cerebellar subdivisions with the notations
of the various nomenclatures used is found in pp. 788-89 of The Compara-
tive Anatomy of the Nervous System of Vertebrates (Kappers, Huber and
Crosby, 1936). It indicates the complications present. The writer has con-
veniently adopted the method of subdivision and naming followed by
Ranson (1937) in describing the material on hand.

General features (Text-Figs. 3, 4). The cerebellum of the entellus is
4 3 cm. in transverse diameter and presents the characteristic foliated appear-
ance. The dumb-bell shaped form of the human cerebellum is, however,
not present. On viewing it from above, the median part shows a smooth
elevation with gentle depressions on either side and the lateral parts bulging
out beyond. The three pairs of brachia were as usual.

The anterior lobe (Text-Figs. 3, 4). The parts in front of the fissure
prima, the deepest fissure in the cerebellum, constitute the anterior lobe.
On the cut face of the median sagittal section, the anterior lobe appears to
be the largest lobe ; and its sectional area calculated from a tracing on graph
paper is about 48 per cent, of the total sagittal sectional area of the cere-
bellum. The anterior lobe consists of three subdivisions. (1) The lingula
comprises five small folia situated on the anterior medullary velum and
demarcated from the central lobule by, the sulcus postlingualis. (2) The
central lobule consists of six small folia of which the more caudal ones have
expanded laterally. (3) The culmen is situated between sulcus postcentralis
in front and fissura prima behind. Its median part shows five folia and its
expansion on each side forming the anterior quadrangular lobule shows
seven folia on the surface. The whole of the anterior lobe is to be considered
as a median unpaired structure.

The middle lobe (Text-Figs. 3, 4) This is bounded in front by the fissura
prima and caudally by the sulcus prepyramidalis. It consists of four sub-
divisions. (1) The simple lobule is situated immediately behind the fissura
prima. It is also a median unpaired structure though expanding laterally.
It shows three folia on its exposed surface. It is limited caudally by the

External Morphology of the Brain of Semnopithecus entellus 53

postlunate fissure (post clival sulcus). (2) The median part of the rest of
the middle lobe includes the folium and tuber, each showing four folia on
the exposed surface. (3) The ansiform lobules are paired portions of the

CENTRAL.!..

N X
CULM EH X

PETROSAL
LOBULE

^ SIMPLE .L.

POSTLUNATE .

'i F.PRIMA

POSTCgNTRAL.P.

".HORIZONTAL.F.

''INTER CRURAV..F.
V PRETONSILLAR.F.

PYRAMID'/
ANSIFORM.L. /

PARA-
HEOIAN.L

PARAME01AN.L.

ANSiPORM.L.

FLOCCULUS

TEXT-FIG, 3. Superior (A), Posterior (B) and Inferior (C) views of the cerebellum

A. Ananthanarayana Ayer

hemispheres seen partly on superior view (Text-Fig. 3 A) as the expansions
external to the postlunate suclus and better seen on posterior and inferior
views (Text-Figs. 3 B, 3 C). The ansiform lobules include the superior and
inferior semilunar lobules and the biventral lobules. The superior and
inferior semilunar lobules are separated from each other by the horizontal
fissure. The superior semilunar lobule is larger and shows about nine folia
exposed on the surface. The inferior semilunar lobule shows three folia
exposed. Caudal to the semilunar lobule is the biventral lobule with the
fissura intercruralis as the line of demarcation. The biventral lobules show
three exposed folia. (4) The paramedian lobules are also paired parts of the
middle lobe and are placed caudal to the ansiform lobules, the fissure pre-
tonsillaris (retrotonsillaris of some authors) occuning between them. The
paramedian lobules correspond to the tonsils.

UNGULA

^ .POST LING UAL. !

CENTRAL LOBULE

S.POSTCEMTRAMS

CU.LHEM
-V

X SIMPLE

"LOBULE
IMPOST CUVAL.S.

FOLIUM

NODULE'

S.PREPYJfAMiDALlS

UVUL*

TEXT-FIG. 4. Median sagittal section of the cerebellum
Drawing was made with camera lucida

The posterior lobe. This shows two subdivisions : (1) The median part
of the posterior lobe includes the pyramid and uvula. The pyramid shows
three folia and uvula, four surface folia. The pyramid is separated from the
uvula by the fissura secunda and uvula from the nodule by the sulcus uvulo-
nodularis. The uvula has no lateral extension or connection. But the sides
of the cephalic folia of the pyramid apparently show 3, narrow connecting

/V.t A;7*W Morphology of the Brain of Scmnopithecus entellus 55

hand between them and the tonsils. (2) The paraflocculus forms on each
side the lateral parts of the posterior lobe. The paraflocculus is separated
from the ansifoim and paramedian lobules by the sulcus parafloccularis and
from the flocculus by the suleus floeeuloparafloccularis. The paraflocculus
slums seven folia on its surface and has a small foliated petrosal lobule
prolonged front it.

The Hoeeulonodnhir lobe. This consists of the median part, nodule,
shoxsim*. three surface folia and the paired flocculi showing seven folia on
the surface,

Comment on the ivrehellunh The cerebellum of entellus shows a better
development nf the eerebcllar hemispheres and a relative smallness of size

of the flocculus and parulloeeulus compared with Macacus sinicus and

;7?t'\;/A,

Remarks on I he Brain Stem

The anterior commissure of the entellus is relatively smaller than that
iif* Mtiencux. The optic chisma, as in the case of other monkeys, lacks
the clear-cut appearance seen in man and appears relatively more bulky.
The mass;t intermedia is a very broad union between the medial surfaces
of the two thalami. The pineal body is conical in shape. The corpora
mamillaria are distinctly seen as two rounded bodies, whereas in the brain
of .U</<*</w this division into two is not seen. In the midbrain, the inferioi
eollieuli arc smaller, more prominent and slightly more laterally placed.
A section through the midhnun shows a large red nucleus. In the floor
of the fiutrth ventricle the stria accoustici are only faintly made out. The
coiliculuH tacialis is vsell marked in the cephalic part of the paramedian
eminence. In A/<a*</n/v rhesus the colliculus tacialis is not prominent
(Hmes, !^35l. In the lower part of the fourth ventricle, caudal to the trigo-
num hypoplossi and trigomtm vagi, is a posterior funiculus.

The pons is relatively very much larger than that of Macacus; and so
ire the olives and pyramids. Thus there is a greater covering of the corpus
iranc/oiUcum by the overlapping posterior border of the pons than occurs
in 1/ficwin (Te\l-tt?. 5). The maximum width of the corpus trapczoideum
is onlv 2 mm. in Us lateral part. A greater development of the retro-
irii'em'inal part of the pons is indicated by the emergence of the fifth nerve
approximately midway between the two borders of the pons This is a sign
of ' ooluiionarv advance tAbbic, 1934). The facial and abducent nerve
emerge immediately below the pons. The acoustic nerve is an apparent
continuation of ihc corpus trapezoidcum.

A, Ananthanarayana Aycf

VLN

V.M

VII.N

rVHUN

PETROSAL
LOBULE

FLOCCULUS
'PARAFLOCCULUS

PVRAWIO

TEXT-FIG. 5. Ventral view of ports, medulla and upper part of spinal cord
Summary and Conclusions

The brain of the Indian langur, Semnopithecus entellus is described.
The following are some of the significant observations resulting from a
comparative study.

(1) The cerebrum of Semnopithecus entellus compared with that of
Macacus shows a less moulding of its inferior surface by the cerebellum.

(2) The proportions of the weights of forebrain to midbrain to hind-
brain are 85 : 1 : 14 in Semnopithecus entellus and according to Tilney
(1928), 84 : 2 : 14 in Macacus rhesus.

(3) In its cerebral fissuration, especially in the regions of the inferior
parietal lobule, occipital lobe, precuneus, parieto-occipital fossa, and colla-
teral fissure, Semnopithecus entellus shows an intermediate stage between
the other Cerecopithecidae and Hylobates.

(4) In the ceiebellum of the langur there is a relatively greater develop-
ment of the hemispheres and a lesser size of the flocculus and paraflocculus
when compared with Macacus.

(5) The entellus has larger olives, pyramids and pons and the corpus
trapezoideum is covered over by the retro-trigeminal part of the pons to a
greater extent than in Macacus.

(6) This study of external morphology indicates that Semnopithecus
entellus has probably the most advanced brain among the Cercopithecidae.
A study of its internal structure is likely to be an extremely valuable addi-
tion to our knowledge of the Primate brain.

/ Mi>rp/it*hgy of the It rain of Semnopithecus entellus 57
Acknowledgement

It is a pleasure to express my deep indebtedness to Dr. U. V. Nayak,
lor his iinaiiiahlc help and criticism and my thankfulness to Miss Chacko
for facilities to examine the specimen of brain of Macacus sinicits on which

she is \\orktng.

Rf H-RI-'NChS CITI-D

AHne, A. A, ,, "The Projection of the Forcbruin on the Pons and Cere-

bellum," /Vor. Roy. Soc., B, 1934, 115, 504.

IfeiW.ifii, I , f, . , *' On the Brain at* Mastitis Ittrvatux and some other old-world

Primates/* f'roe. Zoot. Soc.< 1903, I, 12, 1903.

I)vk\\iilh, NV . I -II. . . .\ftn-phnltiqy ttml Anthn>iwhx\\ 2nd ed., Vol. I, London, 1915.

I !!ii*i Smith, t. . . Citftikwuf Roytil ('olkw of Surgeons Museum, Physiol. Series,

I oiulon, i l H)2.

"Hie 1-ossii Piiricto-oceipitalis/' J. Anat. and Physiol., 1904

38, 164.

Misir-., M. - "The i:\tennil Morphology of the Brain and Spinal Cord,"

/'// Atuttwiy of the Rhesus Monkey, London, 1933.
K.ippcrs, C , t , A. Hiihcr, Ci, C \, /?/* f^w/;/m//w Anatomy of the Nervous System of Verte-

,5ia! (Vovln, f .(.'., hnttt*\\ New Yt>rk 1936.

Kan Mn, f . \V. , . 'I Iw Anuhwiy of the Nervous System, 5th ed., Philadelphia,

1W.

r, J, I , . . " I tic I .volution of the Parallel Sulcus,"J. Anat. 9 1927,61,267.

v, I*. 't'hi' &Mtu from Ape to Man, New York, 1928.

THE EGG-CAPSULE OF THE MILLIPEDE,

THYROGLUTUS MALAYUS ATTEMS*

(SYN. THYROPYGUS MALAYUS CARL.)

BY M. B. LAL, D.Sc.

(Department of Zoology, Lucknow University)

Received November 11, 1941
(Communicated by Dr. G. S. Thapar)

WHILE studying the ecology of the local millipedes, the author noticed that
the eggs in case of Thyroglutus malayus are not laid in groups or masses but
singly, one in each small capsule. As many as 31 egg-capsules have been
obtained from a single female. These capsules are made of earth and are
so well designed and symmetrical in shape as to attract special attention
from the surrounding soil.

The general external outline of these capsules bears a superficial resem-
blance to a miniature fresh-water mussel, the two valves of which represent
the two convex walls of the capsule. These capsules have practically a uni-
form size, showing very slight variations in their dimensions. The average
measurements of the capsule are : length 9 mm. ; breadth in the middle 6 mm. ;
thickness, i.e., distance between the two convex walls externally 5mm.; the
. curvature of the rim 10 mm.

FIG. 1

Photograph of five egg-capsules of Thyroglutus malayus
The cut capsules show the cavity and the egg in situ

* According to Attems the genus Thyropygus has been restricted and some of the species
originally put under it have been transferred to his new genus, Thyroglutus. Thyropygus malayus
Carl, is now synonymous with Thyroglutus ma fay us Attems.

T!v Kg^Ca^nk ,////,' .I//////,*,/*-, Thyroglutus malayus A items 5

I he capsule has a \cry smooth surface on the inside (in contrast to the
ruiidi external suri-uv), exhibiting the \vonderf, I workmanship of the animal,
that provides a u-heiy sol\ surface for the developing embryo. If an egg-
capsule is cut irunsuTsely in the middle, the egg is seen lying loosely in the
cauty \Utieh is stime\vhut oval ; ihe cut surface measures 5 mm. x 6mm.
The \\ali of the capsule in this region is 1-5 mm. thick, so that the cavity is
realh 2 mm. .* nmi. in cross axes. This cavity does not extend right up
to the uui ends 4*1' the capsule bin stops short by 2 mm. at each tip. These
tip% are merely solid portions of earth as can be clearly seen in a median
loiu'ituiiinul MYtum of the capsule. The entire space inside the capsule at
Ihe disposal of the. developing embryo is more or less ellipsoidal with its
axes weas-uriw 1 $ nun,, 3 nun. and 2 mm.

It has been observed in this case that during the process of egg-laying
the tail and head ends, of the animal are brought nearer to each other by the
bendiftf of the body. As soon as the egg emerges out of the vaginal opening
il is reecived by the open valves of the anal ring where part of the inner
rectal membrane alum-: with a drop of gelatinous fluid protrudes out through
the anus. In some eases cpp-eapsule like pellet of earth is seen adhering' to
the itapiiig anal valves of the female millipede. The egg capsule is shaped

nayranim;tic sketch of the c^-capsulc : () entire capsule injatero-ventral view ; (b) a capsule
mi ti,m%vcrsc!> HI the middle to show the cavity and the egg. .<., convex outer surface; ca.,
vauiv f the tapsule; r,, eg?; r,, rim of Ihe capsule; /., tip of the capsule.

60 M. B. Lai

according to the hollow of the anal valves where it is formed by the accumu-
lation of more and more earth. The rim of the capsule is the portion which
projects out when the anal valves containing the capsule are pressed together.
It has also been noticed that the female millipede spews a large quantity of
mucus during the breeding period and this is spread over the capsule while
it is being formed and is held in position between the anal valves. This
mucus helps in sticking soil on to the external surface of the capsule.

It is difficult to state the exact reasons for the formation of these egg
capsules but it is definite that their formation is a physiological necessity
as otherwise the eggs shrink and do not develop if even slightly exposed to
the air. The capsule further protects the developing embryo from injury due
to exposure or attacks of the enemies.

The embryo with its developing appendages is set free by the breaking
open of the capsule at one of its tips.

It has also been possible to study the optimum egg-laying period in this
millipede. The average temperature and humidity variations during this
period are: maximum temperature 85-95 F.; minimum temperature
62~73F.; humidity 65-95%. Observations extending over a period of
three years show that the most suitable time of the year for egg-laying in this
millipede is middle of September to end of October; but this period may also
vary if the monsoon is late or feeble as in India the heat of the summer simply
bakes the soil and makes it too dry for these delicate processes to take place.

BIBLIOGRAPHY

Attems, C. . . " Diplopoda" in Kukenthal's Handbuch der Zoologie, 1926-30,

Band 4.
Evans, T. J. . . Bionomical observations on some British millipedes," Ann.

Mag. and Nat. Hist., 1910, 6, Series 8.

. . " The egg-capsule of Glomeris" Zool. Anzie., 191 1, 37.

Verhoeff, K. W. .. 43. Aufsatz. Mitteilung, betreffend Okologie, einrollung-

sarten und Metamorphosecharakter bei Glomeris " ibid

1910, 36.

CYTOGENETICAL STUDIES IN DATURA

L Cytology of the Parents and of the F l Hybrid between

Datura fastiiofia and Datura sp.

li^ I'KMI. T. s. RAC;HAVAN, M.A., PH.D. (LOND.), F.L.S.

AND
A. R. SKIMVASAN, M.Sc.

Received October 29, 1941

CONTENTS PAGE

1, f \ 1 |M >ni i 1!0\ . . . . . , _ > ^ gj

II. Dlsi fUJ'flU\ (I! 1Hf. f'AKfc'NrS AND Till: HYBRID .. ..62

HI. {*\minr,irAJ, iHMf\ic)M{ .. .. _ ..63

IV. < ii \i lit AI, . . , . . . , . _ 63

V. (^ intK,y ui iHi; PAKI \js:

(ul <*ytolosy of ttitniru j\wtnosu (black) .. .. ..64

{/) <\toltu f .y of I hit lira sp. ( white) . . . . 65

VI. CYIULOCA* c.n IHI-. HYimii) .. .. .. ..66

VI i, DIM i SSION . . . . . . . . . . 69

Vtll, Si \iw\u\ . . . . . . . . . . 72

IX. ! in H vn.'Rf: CIII-.D .. .. .. .. .. 73

A Introduction

!*IA,MS bclomuitf ti> ilic Sulanaccic have been, of all the families, the
nust witidy in\c%i!;-alcd by the cylogenetieists. Of the dillerent genera, the
jemis Ikiiiira uoukl appear 10 have attracted their attention only compara-
tively recently. H'um the available literature, the earliest cytogenetical work
on Datura seems to be Blakcsiec^s (1922), where he describes the variations
in Ikiiani as correlated to changes in chromosome numbers. From that year
onuarcls. a nuinhcr nf* papers hy Blakeslee and his co-workers on many
speeies o!" Datum have been published, The field that they have covered
is \\kle, hut mainly it can be said that they have studied mutants of Datura,
cither naturally occurring or artificially induced. Haploids of Datura,
chidly of hybrid origin, have been studied by Satina et aL (1937). Blakeslee
and his co-workers (1936) published an account of investigation on Datura,
wheio they have described the various methods by which the study of chro-
dillcrences between species of Datura can be made. Stuart Gager

62 T. S. Raghavan and A. R. Srinivasan

and Blakeslee (1927) have described the nature of mutations in the chromo-
somes and among the genes, caused through exposure to Radium rays.
Colchicine treatment and subsequent formation of chimeras with induced
polyploid numbers, have been studied in Datura stramonium by Satina et al
(1941). Chromosome deficiencies due to the above cause are dealt with in
a subsequent paper by Bergner et al (1941).

The materials for the present investigation are two of the locally avail-
able species of Datura. The character contrasts in these two plants were so
marked, that these formed good materials for a study of the inheritance of
these characters. A detailed description of the parental species and the
'F t hybrid is given below. The cytology of the parents and of the hybrid
is also described.

//. Description of the parents and the hybrid
(a) Datura fastuosa (the black form), PL II, A and B ; 2

A large shrub with crookedly-branching very dark purple stems, growing
to 5-6 feet in height. Leaves large, ovate, sinuate. Flowers are solitary,
large, purple and nearly 8^ inches long, on short pedicels. Calyx, tubular
with five triangular lobes. The base of the calyx is persistent. Corolla
consists of five petals, gamopetalous, trumpet-shaped, purplish outside and
whitish inside, with plicate aestivation in the bud stage. Stamens are also
five, filaments slightly shorter than the corolla, with long longitudinally
dehiscing anthers. Ovary superior, bicarpellary, syncarpous, with fleshy
branched placenta and numerous ovules. The wall of the ovary is thrown
into numerous closely-set papilte, which become straight, sharp and stout
prickles, when the ovary ripens into a fruit. The fruit is a loculicidally
septifragal capsule, big and ovally globose.

(ft) Datura sp. (the white form), PL II, A and B; 1

This parent is a short shrub growing to a height of 2-2 feet. It is
spreading in habit. The stems are green in colour. The flowers are smaller
than those of the other parent, of length varying from 6-6^ inches. The
colour of the corolla is yellowish- white. In the fruits, the prickles are thinner
and shorter and less closely set than in the case of the other parents. The
fruits are globose.

This shows mostly characters intermediate between those of the parents.
The plant grows erect and at the basal portion, the branches show a spread-
ing nature. The stem is, however, dark-purple in colour and the average

Cytogcnctical Studies in Datura /

height of the plant is about 4 44 feet. The flowers are purple in colour
and in length they are shorter than the black parent but longer than those
of the white parent. The fruits show intermediate characters between those

of the two parents.

There is nothing worth any special mention about the technique of
crossing. The technique previously described for Nicotiana (Raghavan and
Srinivasan, A. R., 1941 />) was followed.

///. Cytological technique

Root-tips of the parents and the hybrid were obtained from plants
grown in pots in the Botanical Gardens, Annamalainagar. These were
fixed in Miint/ing's modification of Navashin's fluid. Stages for meiotic
studies were determined through aeelocarminc examination and anther-
smears were fixed in Boiling's Navashin fluid, or fixed in Miintzing's fluid.
The fixed materials were embedded in paraffin following the usual schedule
and sections were taken at thicknesses varying from 10-18 microns. Both
the smears and the sections were stained in Haidenhein's iron-alum haema-

toxylin.

IV. Genet ical

The F! hybrid is intermediate between the two parents as regards most
of the characters. However, a few features of the black parent are found
to be dominant over those of the white in the Fj. generation. The follow-
intx table shows the important features of contrasts between the parents and

their expression in the F-j generation.

TABLE I

Nature of the characters

Black parent

White parent

F! hybrid

Colour of the stem

Dark purple

Green

Dark purple

Habit .,

Tall, 7' and erect

Short, 3' in height
and spreading

Tall and spreading,
4-5' in height

Length of the flowers

Longer

Short, 6"

Colour of the corolla

Purplish outside and
whitish inside

Yellowish- white

Purplish outside and
whitish inside

Nature of fruits-

Ovally globose, 2s*
long

Round, 1 / long

2" long.

Thus features of colour (of stem and of flowers) appear ,to be an
expression of the dominance of the black parent over the white. Other

features of the hybrid are intermediate.

64 T. S. Raghavan and A. R. Srinivasan

V. Cytology of the parents

(a) Cytology of Datura fastuosa (black)

Somatic chromosomes. Fig. 1 represents the somatic metaphase plate
of this parent. The twenty-four chromosomes can be grouped under four
types, which are as follows:

Type A. Longest chromosomes, 4-5 microns in length and with
median constrictions.

Type B. Slightly shorter chromosomes, 3-7 microns long and with
sub-median constrictions.

Type C. Still shorter ones, 3 microns long and possessing sub-terminal
constrictions.

Type D. Shortest chromosomes, with a length of 2-3 microns having
sub-terminal constrictions.

Out of the twenty-four chromosomes, two are of the A-type, six of
the B-type, six of the G-type and the remaining ten of the D-type. Other
features such as secondary constrictions, satellites, etc., could not be seen.
Fig. 2 shows the idiogram representation of the complement.

Meiosis Diakinesis (Fig. 3). During diakinesis, the twenty-four
chromosomes form twelve bivalents. A thorough examination of more than
fifty diakinesis figures revealed that associations of more than two chromo-
somes were absent. Hence multivalent formation can be said to be totally
non-existent. Of the twelve bivalents, one is large, possessing four chias-
mata, two terminal and two interstitial. This probably arises from the two
longest A-chromosomes which have been found to have median constric-
tions. Two other bivalents have three chiasmata, one interstitial and two
terminal. Four ordinary ring-bivalents were met with, each having two
terminal chiasmata. The remaining five bivalents form a single terminal
or subterminal chiasma each. The total number of chiasmata in the black
parent is twenty-three, of which four are interstitial. Thus conjugation is
complete and the formation of bivalents with more than two chiasmata
presumably shows the close relationship of the parental chromosomes.

First division. The nucleolus and the nuclear membrane disappear and
the nucleus after a short prometaphase, enters upon the first metaphase
stage (Fig. 4). Twelve bivalents are arranged in the equatorial plate. No
associations were found between the bivalents. Disjunction is normal.

Second division. A short interphase intervenes between the two divi-
sions, after which the chromosomes which disjoined in the first division

Cytogenetical Studies in Datura / 55

undergo homotypic division. In the second metaphase stage (Fig. 5) twelve
chromosomes were seen at each of the two poles of the pollen mother cell.
The second division is normal and tetrads of both the iso-bilateral and the
tetrahedral types are produced (Figs. 6 and 7).

FIGS. 1-13

FIGS. 1-7. Datura (black)
Somatic plate. X4400. Fig. 2. Idiogram representation of the chromosome com-

M I plate. X2200. Fig. 5. Second metaphase.

Fig. 1.

plement. Fig. 3. Dialdnesis. X4400. Fig. 4.
X2200. Figs. 6 and 7. Tetrads. X1500.

FIGS. 8-13. Datura (white)

Fig. 8. Diploid complement. X4400. Fig. 9. Idiogram representation of the same. Fig. 10.
First metaphase. X2200. Fig. 11. Anaphase I. X1500. Fig. 12. Second metaphase. X2200.
Fig. 13. Anaphase II. X1500.

(6) Cytology of Datura sp. (white)

Somatic chromosomes. The diploid complement of this parent is also
made up of twenty-four chromosomes (Figs. 8 and 9). There -are two
chromosomes of the A-type, four of the B-type, eight of the C-type and
ten of the D-type. There would appear to be difference only in the number
of the two middle types of chromosomes (i.e., B- and C- types of 3-7 and
3 microns length respectively). Whereas in the black parent there are six
of each of these types of chromosomes, in the white parent, there are four
of the B-type and eight of the C-type,

T. S. Raghavan and A. R. Srinivasan

Meiosis. Twelve bivalents are formed by the twenty-four chromo-
somes as shown in Fig. 10, which represents the polar view of the first
metaphase plate. So conjugation in this species also is complete. Ana-
phasic separation (Fig. 11) is regular in the first division and the disjoined
chromosomes organize two nuclei during interphase. During second meta-
phase (Fig. 12) twelve chromosomes are seen at each of the two poles.
Second anaphase is also regular (Fig. 13), and normal tetrads are formed.

VI. Cytology of the hybrid

Somatic chromosomes. The somatic chromosomes of the hybrid are
represented in Figs. 14 and 15. The four types into which the parental
chromosomal complements were analysed, could be recognized here also.
In the somatic complement of the hybrid, there appear the two longest
chromosomes, i.e., of the A-type. Similarly there were ten short chromo-
somes i.e., of the D-type about 2-3 microns long, so that in these two
types there is complete identity between the parents and the hybrid. But
in the nature of the two intermediate types there is some difference, for in
the hybrid, we usually find only five chromosomes of the B-type (3-7 microns
long) and seven chromosomes of the C-type (3 microns long). The different
types of chromosomes and their number as occurring in the two parents and
the hybrid, are shown in the following tabular statement.

TABLE II

Chromosome length and nature
of constriction

White parent
2/t = 24

Black parent
2 /i = 24

F! hybrid
2 n = 24

A-type. 4-5 microns and median con
strictions

B-type. 3-7 microns and sub-median
constrictions

C-type. 3 microns and sub-terminal
constrictions

D-type. 2-3 microns and sub-terminal
constrictions

The occurrence of five B-type and seven C-type chromosomes in the
hybrid is as it should be, if we made a comparative study of the somatic
complements of the parents that have entered into the formation of the
hybrid complement. The haploid complement of the white parent should
consist of the following chromosomes : one chromosome of the A-type,
two of B-type, four of C-type and five of D-type. Similarly the black

Cytogenetical Studies in Datura / 67

parent in its gametes should contain a genome composed of one chromo-
some of A-type, three of B-type, three of C-type and five of D-type. As
these two gametic chromosome sets together compose the diploid comple-
ment of the hybrid, the hybrid complement comes to contain two chromo-
somes of the A-type, five of B~, seven of C- and ten of D-types.

Since the two gametic complements are identical to one another except
in the number of the B- and C- types, we naturally find that in the hybrid
these two types of chromosomes alone show a slight difference in their
constitution. Generally, the prevalence of these odd numbers of chromo-
somes amongst these types may be expected to cause some degree of asynap-
sis resulting in the formation of univalents. But the difference between the
B- and C-types is so slight that they may be expected to conjugate with one
another without difficulty. That is why, we find meiosis regular in the main.
The stray occurrence of trivalents which brings in its trail naturally the
formation of univalents also, justifies this presumption.

Meiosis. Stages earlier than diakinesis were not studied. During
diakinesis, the phenomenon of cytomixis was found to occur frequently.
It is interesting to note in this connection, that cytomixis was completely
absent in the parents while it is so common in the hybrid. This only goes
to support the view expressed in a previous paper (Raghavan and Srinivasan,
1941 a), that this phenomenon is associated with hybridity. Nevertheless
apparently pure species have been found to exhibit this phenomenon as in
the case of Tridax (Raghavan and Venkatasubban, 1941). Less frequent
occurrence of cytomixis has been recently reported in the species, Guettarda
speciosa (Raghavan and Srinivasan, 1941 Z>) and Portulaca tuberosa (Raghavan
and Srimvasan, 1941 c). Nandi (1937) has recorded cytomixis during dia-
kinesis stages in Oryza and is of opinion that it leads to polyploid gamete
production through the formation of bi-nucleate pollen mother cells.
X-rayed materials of pure species show the phenomenon of cytomixis to an
extreme degree, e.g., Capsicum (Raghavan and Venkatasubban, 1940).

Cytomixis in the present case is very interesting. In Fig. 16, nuclear
extrusion takes place into a single cell from two adjacent cells. In Fig. 17,
nuclear matter from a single cell is extruded at the same time into two cells
on either side of it. This recipient cell is found to transfer its chromatic
material to the next cell and so on. Normal cytomixis between two adjacent
cells has also been met with (Fig. 18). In the prometaphase stages also this
phenomenon was found to occur (Fig. 19). Fig. 20 shows pollen mother
cells at first metaphase stage, one of the metaphase plates showing a tendency
to migrate. The relegation of two of the bivalents to a peripheral position

T. S. Raghavan and A. R. Srinivasan

while the rest occupy the equatorial plate (Fig. 25), may also be regarded
as showing an abortive attempt at cytomixis. In the present case the extent
to which cytomixis occurs decreases as the pollen mother cells advance into
the later stages.

30 3T W 33 34 35

FIGS. 14-35. Cytological stages of the F t hybrid

Fig. 14. Somatic plate. X4400. Fig. 15. I diogram representation of the same. Figs. 16-20.
Stages of cytomixis. X 1500. Figs. 21-24. Diakinesis stages. In Fig. 23 a trivalent and a uni-
valent are shown. AH the figures to the magnification X2200. Figs. 25 and 26. Metaphase I.
X2200. Fig. 27. Anaphase I. X1500. Fig. 28. Interphase. X2200. Fig. 29. Second
metaphase. X2200. Figs. 30 and 31. Second telophase. X1500. Figs. 32-35. Furrowing
and tetrad formation. X1500.

Normal diakinesis was also quite common. Fig. 21 shows a P.M.C.
at early diakinesis in which the synapsing chromosomes are long and
slender. Of the twelve bivalents, three are of the ring-type, each with two
terminal chiasmata. All the other bivalents have one terminal chiasma each.
At a later stage, the chromosomes shorten and thicken (Fig. 22). In this
case one of the bivalents was found to have an interstitial chiasma. Three
ring bivalents were met with here also. In one case, a trivalent and a
univalent were found along with ten bivalents, but such cases were very
rare (Fig. 23). At later diakinesis, the configuration of the bivalents
becomes clear (Fig. 24) ? when three ring bivalents and nine rod bivalents

Cytogenetical Studies in Datura / 69

are observed. Thus during diakinesis stages the nieiosis shows normal
conjugation between the chromosome sets of the parents. The total
number of chiasmata is fifteen, interstitial chiasmata being almost absent.
At first metaphase all the twelve bivalents are arranged in the equatorial
plate (Fig. 26). Very occasionally however, one or two of the bivalents are
found to be located away from the euqatorial plate (Fig. 25). First ana-
phase is regular but for the somewhat late disjunction of one of the bivalents
(Fig. 27).

Interphase follows the first division and the nucleus assumes a globular
appearance (Fig. 28). The constricted appearance of the chromosomes is
characteristic of the interphase nuclei. The chromosomes are peripherally
disposed at equal intervals from each other. The second prometaphase
follows, but is of very short duration. Soon the nuclear membrane dis-
appears and these constricted bodies contract and enter upon the second
metaphase. Twelve chromosomes at each end could be recognized as seen
from the pole (Fig. 29). During second telophase, the chromosome groups
are arranged either in the iso-bilateral or in the tetrahedral pattern (Figs. 30
and 31 respectively).

Quadripartition of the tetrads is through furrowing (Figs. 32 and 33)-
The method of formation of iso-bilateral tetrads conforms to that described
for Nicotiana glutinosa (Raghavan and Srinivasan, 1941 a). Normal tetrads
of both iso-bilateral and tetrahedral types are formed.

VII. Discussion

Interspecific hybrids between parents with the same number of chro-
mosomes are not rare. These are interesting in the variety of genetical and
cytological features that they present to us, such as, fertility or otherwise
of the F! hybrids, chromosome conjugation, abnormalities of meiosis, etc.
There is a gradation from, fully fertile hybrids to completely sterile ones.
This only shows that the mere identity in the number of chromosomes is not
enough by itself for the complete pairing of chromosomes. The degree
of fertility is, in almost all cases, determined by the degree of conjugation
between the parental chromosomes. In completely sterile hybrids, either
conjugation is absolutely lacking, or weak conjugation takes place
to such a low extent, as can be considered to be no better than asynapsis.
Hybrids of this latter-mentioned type are many. Ramanujam (1937) obtained
a hybrid between Oryza sativa (n= 12) x O. officindlis (n= 12), in which
during diakinesis twenty-four unpaired chromosomes were met with. In
some cases loosely formed bivalents were found and that too only to a low
degree. This hybrid was found to be sterile. Crosses in Nicotiana, such

70 T. S. Raghavan and A. R. Srinivasan

as those between N. sylvestris (n= 12) x N. glutinosa (n= 12); N. rustica
(=24) x .. tabacum (n =24) and N. Raimondii (n=- 12) x TV". #toc<2
(= 12) have led to similar results (Goodspeed, 1934).

Partial pairing between chromosomal sets of the parent species has been
observed in other cases. Hybrids between species of Lactuca (Whitaker and
Thompson, 1941), L. tatarica (=9) x L. indica (n=9) showed 7 U + 4 l5
while F t of L. saliva x L. vivosa (both n = 9) showed 8 U +2 X . In spite of such
partial pairing, the hybrids were found to be sterile. In hybrids of Nico-
tiana (Goodspeed, 1934), Crepis (Babcock and Emsweller, 1936), Aegilops
(Percival, 1930) and Gossypium (Webber, 1935), such partial pairing between
parental chromosomes resulting in varying numbers of bivalents and uni-
valents were met with.

In the present case however, the parental chromosome sets seem to be
completely homologous to each other. This is proved by the fact that twelve
bivalents are regularly formed. But the number of chiasmata in the diakine-
sis stages of the hybrid is far less than in the parents. This probably signi-
fies that, after all, two different parental sets of chromosomes however
homologous they may be cannot pair as fully as those of the same parental
species. In the present case, the average total number of chiasmata (during
the mid-diakinesis stages) in the parental species is about twenty-three,
of which four are of the interstitial type, the rest being terminal. In the
hybrid, on the other hand, the number of chiasmata at about the same stage
of meiosis, is about fifteen, all of which are terminal. Only very rarely do
we see an interstitial chiasma. There is thus a marked decrease in the
number of chiasmata in the hybrid, as compared to that of the parents.
Also, interstitial chiasmata are conspicuous by their absence in the hybrid.
Similar observations would appear to have been made by Goodspeed
(1934) in interspecific hybrids of Nicotiana and his interpretation on these
phenomena seem to indicate a correlation between these and the degree
of chromosomal identity in the parental species. He says that " with increase
in the number of bivalents (i.e., in the homology of the parental chromo-
some sets), there is a disproportionately greater increase in the total number
of chiasmata, because in certain of the bivalents two chiasmata are formed,
and there is also an increase in the proportion of interstitial chiasmata. With
almost complete pairing, the total number of chiasmata approaches that
in the parent species. ..." Thus according to Goodspeed, the greater the
number of chiasmata formed, the more homologous are the parental chro-
mosomes. Viewed in this light, the chromosome sets of the two parents in
the present case cannot be regarded as being so completely homologous

Cytogeiictical Studies m Datura / 71

For a casual observer the two parents used in the present cross, may
appear to be only varieties of a distinct, fundamental species. On closer
scrutiny however, we find that the external features, such as the size and
nature of the vegetative and the reproductive parts do not go to support
this assumption. The large differences that exist between the two parents
may, by themselves, be sufficient to classify them as distinct species. In
addition to this morphological evidence, we have got cytological data also
to prove the relative distance between the two parents. For example, a
study of the somatic complements shows some differences in the chromo-
some morphology of the two parents. 'These differences have been pointed
out already. Another, fact to reckon with, is that the number of chiasmata
in the hybrid is much less than in the parents: themselves; also interstitial
chiasmata are characteristically absent in the hybrid. This, as has been
pointed out already, suggests the somewhat distant relationship between
the two parents. It will not therefore be wrong to regard them, as two
distinct species.

In spile of the above-mentioned differences, there is no deficiency in the
homology between the parental e'.romosomes. Most probably the genes of
the chromosomes arc related to each other. Consequently, the meiosis is
almost as normal as in the parent species and the F x hybrid is fertile. Such
fertile interspecific hybrids were met with in the case of Nicotiana, between
N. solan ifblla (n 12) X N. paniculata (n 12) (Goodspeed, 1934). Hybrids
of Lactuca, other than those mentioned above, were found to be fertile,
though to a lesser extent than the parents (Whitakcr and Thompson,
1941).

Taxonomica! relationships have recently been determined through
studies in the chromosome behaviour of interspecific hybrids. Species whose
chromosome sets pair freely with each other, arc believed to be nearly
related, the only difference between their chromosomes being of the nature
of certain genetic factors. Partial or no pairing has been taken to represent
distant relationship of the parents. In Nicotiana this principle has been
found to be true. Tints, Goodspeed (1934) remarks "when two chromo-
some complements arc capable of co-operating so that a normal soma is
built, these chromosome sets have at least residual homology and presumably
contain genes in common", lie also says that "there is no justification
for a disbelief in the significance of pairing as indicative of relationships in
Nicotiana, because in other materials the evidence at present may not warrant
such a conclusion ". We find that this principle is applicable to the present
hybrid in A/mm. No abnormality of any serious nature having been
observed in the hybrid, the two species may be looked upon as being very

72 T. S. Raghavan and A. R. Srinivasan

nearly related to each other and can be traced back to identical origin. So,
the above said principle that a close correlation exists between chromosome
pairing and taxonomic relationship, is found to be true in the case of
Datura also.

However, cases have been found where this principle presents obvious
difficulties. Crosses by Clausen (1931) in Viola are examples of this. Viola
arvensis (n = 17) was crossed with V. tricolor (n = 13) and the F x hybrid
showed 13 U +4 1? according to the Drosera type of chromosome conjuga-
tion. Thus the thirteen chromosomes of V. tricolor are homologous to
thirteen chromosomes in the haploid set of V. arvensis. Another cross
between V. tricolor (= 13) and V. nana (n = 24) also showed perfect pairing
between the tricolor and the nana chromosomes. If the hypothesis that
chromosome pairing is indicative of the relationship between species be true,
thirteen chromosomes of V. arvensis which paired with the chromosomes
of tricolor, should conjugate with the thirteen chromosomes of V. nana,
with which also the tricolor chromosomes exhibited pairing. This however
was not the case and two to six bivalents were only formed in the hybrid
between V. nana and V. arvensis. Even this bivalent formation might have
been due to autosyndesis. In Nicotiana digluta, where the chromosomes
are obviously homologous, non-pairing has been reported (Miintzing, 1935).
Ramanujatn (1937) is of opinion that chromosome conjugation may be
influenced by genetic and environmental factors. According to him " while
conjugation of chromosomes indicates a kind of homology between them,
non-conjugation does not necessarily always mean non-homology ".

The above instances show that much caution should be exercised while
interpreting partial or no pairing with reference to relationship between
parents of interspecific hybrids. But there seems to be no difficulty in inter-
preting complete pairing (as in the present case) as indicative of taxonomic
nearness of the parental species.

VIII. Summary

Two species of a local Datura were crossed and the hybrid compared
with the parents.

The somatic chromosome complements of the parents were analysed
and compared with that of the hybrid.

Meiosis in the parents and the hybrid is also described. Chromosomal
pairing in the hybrid is discussed as throwing light upon the relationship
between the parents.

L \togenctical Studies in Datura /

Babcock, E. B., and Hmsweller,
S. L.

Bergner, A. !>., Avery, A. G.
and Biakcslec, A. F.

Blakeslee, A. I ; .
--.-.- and co-workers

Clausen, J.

Goodspeed, T. H.

Miint/ing, A.
Nandi.H. K.
Navashin, M.
Pcrcival, J.

Raghavan, T. S., and Srinivasan,
A, R.

....,.- and Vcnkatasubban, K, R

Ramanujam, S.

IX. LITERATURE CITED

" Meiosis in certain interspecific hybrids in Crepis and its
hearing on laxonomical relationship," Univ. Calif, publ-
AgrL *SV/., 1936, 6, 325.

" Chromosome deficiencies in Datura stramonium induced by
colchieinc treatment," Attwr. Jour. Bot. t 1940, 27, 676.

'* Variations in Datum due lo changes in chromosome num-
bers;* Attwr. M//., 1922, 56.

'* Datura investigations," Report of the Director of the Depart-
ment of Genet, of Canwgie Institution of Washington,
1^36, 36.

*' ("ytogenctic and taxonomic investigations in Melanium
violets," lleredit. Lund., 1931, 15, 219.

*' Nicotiantt pliylesis in the light of chromosome number,
morphology and behaviour," Univ. Calif. Publ. Bot. t 1934,
17, 369.

'* C'hromosomc bcliaviour in Nlcotiana hybrids," Heredit.
Lund., 1935, 20, 251.

" C'ytological investigations of the rice varieties," Cytologia,
1937, 8 t 277.

" C'hronuvsomc alterations caused by hybridization and their
bearing on certain gcnctical problems," ibid., 1934, 5, 169.

** Cytological studies of some hybrids of Aegilops sp. Y-. wheat
and of some hybrids between different species of Aegilops"
Jtwr. (tenet.* 1930, 22, 201.

49 (ytogenetical studies in Nicotiuna. T. Cytology of TV. taba-
cum var. macrophylla, N. glutinosa and the F! hybrid
between them, 1 ' Jour. Ind. Bot. Soc. 9 I94lc, 20, 307.

" C'ytogenctical studies in Nlcotiana, II. Morphological
Features of N. j?/w//V/.v and the hybrid between N. gluti-
nasa and A^. tuhacum" Proc. Ind. Acad. Set., 1 941^,
14, 33.

" Studies in the Rubiacex. II. Spermacoce hispida, Guet-
tarda xpeciosa and some cyto-morphological considera-
tions," ibid., 1941c, 14, 412.

** Cyto-morphological features of Portulaca tuberosa Roxb.,'*
ibid., 194h/, 14, 472,

"Studios in the South Indian Chillies. L A description of
the varieties, chromosome numbers and the cytology of
some X-rayed derivatives in Capsicum annum" ibid., 1940,
12, 29.

" Contribution to the cytology of Tridax procumbens" ibid.,
1941, 13, 85.

' Cytogonctical studies in Oryzcsc. II. Cytological behaviour
of an autotriploid in rice/* Jour. Gen., 1937, 35, 183.

T. S. Ragliavan and A. R. Srinivasan

Satina, S., Blakeslee, A. F., and
A very, A. G.

Sturt Gager, G. 5 and Blakeslee,
A. F.

Whitaker, W. T., and Thompson,
C. R.

Webber, J. M.

"Balanced and unbalanced haploids in Datura" Jour. Here.,
1937, 27, 193.

* Demonstration of three germ layers in the shoot apex of

Datura by means of induced polyploidy in periclinal chime-
ras;' Amer. Jout., Bot. 1941, 27, 895.

" Chromosome and gene mutations in Datura, following
.exposure to Radium rays," Nat. Acad. Set., 1927, 13, 75.

4 Cytological studies in Lactuca" Bull. Ton: Bot. Club, 1941
68, 388.

* Interspecific hybridization in Gossypium and the meiotic

behaviour of the F x plants," Jour. Agri. Res., 1935, 51,
1047.

7". *Y.

A, A\ AV////W.

A, Photographs of whole plains <f: I. 1 hi* white p,u rut .. .? I lie bJ.uk p,ucni .

and .V liu* I', hvhrnt

I J J

B. Photographs of ikwcri and fruits of- I, The white parent
2, The black parent; and 1. The !', hybrid

NEW CONCEPTS OF THE SOLID STATE*
BY SIR C. V. RAMAN

MY first duty is to thank the authorities of the Nagpur University on
behalf of the Indian Academy of Sciences for the invitation which has
enabled the Annual Meeting of the Academy to be held this year under the
auspices of the University. The Fellows of the Academy deeply appreciate
the labours of the Chairman and members of the local Committee and
of Prof. Moghe in making the arrangements for the meeting and are
grateful for the hospitality which has been generously provided for the
occasion.

# 5fc * *

For good or for evil, we live in an age of science. No one who is
familiar with the history of science would fail to recognize the great influence
which has been exercised on its progress by the work of the various national
academies of science, as for instance the Royal Society of London and the
Academy of Sciences at Paris. The publications of these academies are the
primary records of scientific discovery and invention in their respective
countries. To no small extent, also, the Academies have been responsible
for the promotion and encouragement of research work and for the co-ordi-
nation of the research activities of the Universities. During the seven
years the Indian Academy of Sciences has been in existence, it has striven
to fulfil these functions in our country. The Proceedings of the Academy
which have appeared punctually, month after month, embody the best
part of the research work done in most of the Indian Universities. It is
greatly to be desired that these Universities appreciate what the Academy
is doing for them and help the Academy to carry on under the present
difficult conditions.

$ * * *

I propose to devote my address this year to an exposition of the new
ideas concerning the solid state of matter which have emerged from recent
investigations made at Bangalore. The vast majority of actual* solids are
crystalline in structure and are either single crystals or else consist of
polycrystalline aggregates. The gateway to an understanding of the solid

* Presidential Address to the Indian Academy of Sciences at the Annual Meeting held at the
Nagpur University on the 24th of December 1941.

75
Bl

76 C. V, Raman

state is therefore to be found in the study of crystals. The most effective
starting point for such a study is, again, the ultimate structure or atomic
architecture of the solid. The physics of the solid state of matter indeed
concerns itself largely with the relationship between the atomic grouping
in space which characterizes a crystal and the physical behaviour of the
solid in various circumstances.

As is well known, crystals often possess beautiful external forms with
specific geometric features. The symmetry characters of these geometric
forms stand in the closest relation to the physical properties of the solid,
such relationship being most evident when we consider those properties
which vary with direction. The study of the geometric forms and of the
physical properties of crystals resulted in the classification of crystal forms
into six or seven systems and their further sub-division into thirty-two
classes of crystal symmetry. It is natural that crystallographers were led
by such studies also to speculate on the features in the internal architecture
of crystals to which could be ascribed the external symmetry properties
manifested by them. The theoretical investigations which dealt with this
problem resulted in the recognition that a crystal is essentially a repetitive
pattern in space and that the material particles of the solid are arranged
in regular geometric order in a three-dimensional space-lattice. The dis-
covery of the 14 possible kinds of space-lattice and of the 230 possible
ways of grouping the atoms, each in its own appropriate type of space-lattice
and coming under one or another of the 32 possible symmetry classes, gave
the necessary precision and completeness to such general notions of crystal
architecture.

The ideas of the mathematical crystallographers of the nineteenth
century found a spectacular confirmation in Laue's great discovery made
in 1912 of the diffraction of X-rays by the space-lattice of crystals. During
the thirty years which have elapsed since that discovery, a vast amount
of detailed knowledge regarding the structure of individual crystals has been
built up by the labours of the X-ray crystallographers. Around such know-
ledge, again, there has been a great deal of discussion regarding the nature
of the forces which held together the atoms, ions or molecules in a crystal
in the form of a coherent solid.

It must be recognized that the concept of a regularly ordered assem-
blage of atoms, ions or molecules in a space-lattice is only a static descrip-
tion of crystal structure and does not suffice to give a complete view of
the solid state. That the density and many other physical properties of

New Concepts of the Solid State 77

a solid vary with temperature is clear Indication that the atomic positions
in a crystal are subject to disturbance by thermal agitation. A description
of the possible atomic movements in a crystal is thus as important for
crystal physics as a knowledge of the static structure. In other words,
a dynamic picture of the crystalline state is required as a complement to the
static picture furnished by the space-group theory. The possible modes
of atomic vibration would evidently he determined by the atomic groupings
in the crystal lattice and the forces that come into play when such grouping
is disturbed. It follows that the static and dynamic aspects of crystal
architecture should stand in the closest relationship to each other.

A dynamic concept of the solid state is necessarily the starting point
in any consideration of the thermal properties of a crystal, e.g., its specific
heat, thermal expansion or thermal conductivity. It is equally fundamental
in any attempt to elucidate such physical properties of solids as are notably
influenced by temperature, <'.#., the electrical resistivity of metals. The
subject of eiystal dynamics assumes a special importance in considering
the effects arising from the propagation of electromagnetic waves through
crystals, <>.#., the scattering of light or the diffraction of X-rays. Spectro-
scopic and X-ray studies on crystals indeed afford us a penetrating insight
into the problems of the solid state,

* * * *

The theorists who have handled such problems in the past have pro-
ceeded by carrying over notions derived from the classical theory of vibrat-
ing elastic solids into the domain of atomic dynamics. The history of
physics during the present century suggests that all such extrapolations from
macroscopic to atomic concepts must be regarded with caution. The
extrapolations made in the Debye and Born theories of crystal dynamics
do not, however, appear to he justified even from a purely classical point
of view. It is not surprising, therefore, that the conclusions derived from
these theories fail to survive the test of comparison with the experimental
facts in several different branches of research. Before we proceed to
consider evidence of this kind, it appears desirable to examine the founda-
tions on which these theories rest.

* # * *

We may, in the first instance, comment on the well-known specific heat
theory of Debye which has had the run of the text-books of physics for
many years and even yet seems to be in favour. The theory assumes that
the thermal energy of a solid may be identified with the energy of elastic

waves travelling within it, and gives an expression for the energy in

78 C. V. Raman

of the velocities of these waves. That these assumptions are unjustifiable
is evident from Debye's own formulae. For, the calculation shows that
a very large proportion of the elastic vibrations must be assumed to possess
wave-lengths comparable with the lattice spacings of the crystal. Their
frequencies also become comparable with those of the vibrations of the
individual atoms. Even according to the classical principles, vibrations of
such short wave-lengths and high frequencies could scarcely be expected
to travel through the crystal with the assumed acoustic velocities. Indeed,
the familiar fact that thermal energy does not travel at all but only diffuses
with extreme slowness in solids is a clear disproof of the basic assumptions
of the Debye theory. Far from supporting the postulates of the theory,
the facts point to exactly the opposite conclusion, namely that no sensible
part of the thermal energy of solids consists of the elastic vibrations of
macroscopic physics.

The so-called postulate of the " cyclic lattice " on which the crystal
dynamics of Born is based was introduced by him as a mathematical device
to escape the difficulties which he believed to arise from the unspecified
conditions at the external boundary of the crystal. The postulate in effect
prescribes " wave-lengths " for the atomic vibrations in the crystal which
bear no relation to its internal architecture but are related to its external
dimensions in exactly the same way as the elastic vibrations of macro-
scopic physics. The postulate of the cyclic lattice has no theoretical justi-
fication and its introduction makes Born's approach to the problem of
crystal dynamics wholly unreal and no less open to criticism than the
theory of Debye.

The fallacy of the basic ideas underlying the Debye and Born theories
becomes evident when we consider the nature of the vibrations within
a solid indicated by the classical theory of elasticity. The form and size of
the external boundary of the solid determines the possible modes of elastic
vibration. In each individual vibration, the motion at all points within the
solid has a specifiable frequency and a coherent phase-relationship. But
there would be an immense number of such modes with varying frequencies.
The superposition of all such modes, assumed to be co-existent, would
therefore result in the agitation within the solid being of a completely chaotic
character, varying from point to point and from instant to instant without
any recognizable periodicity in space or recurrence in time. Thus, in effect,
the assumptions made in. the Debye and Born theories are equivalent to the
assertion that while the static arrangement of the atoms in a crystal is
one of perfect order and regularity, the dynamic character of their move-
ments is one of perfect chaos and disorder, indeed exactly of the same

New Concepts of the Solid State 79

kind as the movements or vibrations of the molecules of a gas. This con-
clusion is obviously so improbable that we may well feel justified in reject-
ing without hesitation the premises on which it is based. *
# _ # * #

A crystal, as we have seen, is a periodic array of similaj: particles,
similarly situated and capable of influencing each other's movements. It
follows that the vibrations of such an assemblage should exhibit a high
degree of orderliness, approaching the ideal of a perfectly co-ordinated
vibration in which the frequency, amplitude and phase are identically the
same throughout the crystal. . To picture such a vibration, we may first
consider the group of the atoms present in an individual cell of the space-
lattice. The internal vibrations of such a group would comprise several
distinct modes determined by the number of atoms present. Each such
vibration may then be pictured as occurring in identically the same way in
every cell of the crystal lattice. Geometrically, such an oscillation could be
represented as a periodic movement, relative to each other, of the inter-
penetrating simple lattices of similarly placed atoms of which any crystal
may be regarded as built up. Such a vibration would have a uniquely
definable frequency, and the vibration spectrum of the crystal would therefore
consist of a finite number of discrete monochromatic frequencies.

Thus, instead of an infinite array of choatic movements varying arbi-
trarily in phase from cell to cell of the crystal, and having a continuous
spectrum of frequencies, we obtain a finite group of vibration modes with
space-patterns coinciding with the lattice structure of the crystal and having
a set of discrete monochromatic frequencies. These vibrations are essen-
tially periodic changes in the fine structure of the crystal and do not involve
mass movements of the substance of the solid. Hence, neither the existence
of an external boundary nor the conditions restraining its movements can
have any influence on such vibrations.

The most appropriate choice for the space unit of the three-dimensional
repetition-pattern of the atomic vibrations is evidently that which enables
all the modes possible to be included without redundancy. Hence the
appropriate choice is not the cell having the smallest dimensions or includ-
ing the least number of atoms, but one which is fully representative of the
crystal structure and symmetry. In the majority of crystals, the number of
atoms included in such a space-unit would be fairly large. Hence, the
internal vibrations of the group of atoms contained in it would comprise
the largest proportion of the available degrees of freedom of movement,
indeed all except a small residue representing the translatory movements

80 C. V. Raman

of the chosen cell. To enable these latter to be included in the scheme,
we may consider the internal vibrations of a group of atoms contained in
the cells of a super-lattice having cells of twice the linear dimensions and
therefore of eight-fold volume. Proceeding in this way by successive
steps, the vibration spectrum of the crystal could be developed with all
desirable completeness as a set of monochromatic frequencies.

* * # *

It will be realised that the geometric characters as well as the frequency
distribution of the atomic movements in crystals obtained in this way
would be radically different from those indicated by the Debye and Born
theories. It is evident also that the new concepts involve striking differences
in the spectroscopic., X-ray and thermal behaviour of crystals as compared
with those derived from the older ideas. The issues arising between the
new and the older concepts are thus capable of being brought to an exact

experimental test.

* # * *

The atomic vibrations in crystal lattices are accessible to optical and
spectroscopic investigation in several different ways. A method which
makes the entire frequency range conveniently accessible to observation
is the spectroscopic study of the scattered radiations emerging from a
crystal traversed by monochromatic light. The most striking feature
revealed by such studies with crystals is the extreme sharpness of the
displaced lines appearing in their spectra. Even in those cases where the
lines are somewhat diffuse, they sharpen into the finest lines when the
crystal is cooled down to low temperatures. The monochromatism of the
lattice frequencies thus indicated is especially significant when the vibrations
are observable only in the crystalline state, in other words when the lines
disappear in the molten or dissolved material. These facts are wholly
inconsistent with the Debye and Born theories. Indeed, it may be said that
the character of the spectra observed even with the simplest of crystals
bears no resemblance to the diffuse continua suggested by these theories.
Evidence confirmatory of the new concepts of crystal dynamics is also
furnished by the absorption and luminescence spectra of crystals observed
. at low temperatures, e.g., diamond. Here again, the lattice spectrum is
revealed as a set of discrete monochromatic frequencies stretching down
to low values, in startling contrast with the conclusions of the Debye and
the Born theories.

* * # #

As already explained, the new concepts indicate a close correspondence
between the static structure and the dynamic behaviour of a crystal,

New Concepts of the Solid State 81

in other words that the atomic vibration patterns are either identical with
or closely related to the lattice structure of the crystal. As an immediate
consequence of this relationship, it follows that the lattice planes of a
crystal should give two distinct types of X-ray reflection a dynamic reflec-
tion with altered frequency in addition to the static reflection of unmodified
frequency discovered by Laue. The more perfectly co-ordinated is the
oscillation of the lattice structure, the more perfect would be the geometric
character of the dynamic X-iay reflections. Hence, these reflections should
be shown in the most striking way by diamond-like structures in which the
entire crystal is practically a single molecule and less perfectly by other
crystals in which the lattice structure is of a more open kind.

That the lattice planes in crystals do give the new type of dynamic
X-ray reflection here indicated and that such reflections are incapable of
being explained on the older theories was discovered and announced by
myself and Dr. Nilakantan in March 1940. In a symposium of fifteen
papers published in the Proceedings of the Academy for October
1941, the theory of these new X-ray reflections, their relation to quantum
mechanics and the experimental facts as observed with diamond and
numerous other crystals have been thoroughly explored. It has been proved
that the experimental facts are, on one hand, fatal to the Debye and Born
theories and that on the other hand, they give the strongest support to the
new concepts of the solid state.

He * * *

To the pioneer investigations of Einstein, we owe the basic principles
of the quantum theory of the specific heat of solids. He showed clearly
that the thermal energy of a crystal stands in the closest relation to its
optical properties and could, in fact, be expressed in terms of the charac-
teristic frequencies of atomic vibration appearing in the infra-red region
of frequency. In his earliest paper, Einstein suggested that the atomic
frequencies could be assumed to be monochromatic. Considering one such
characteristic frequency in the case of diamond, he evaluated the same from
the specific heat data. It will be seen from our present discussion that
the basic assumption of monochromatism was justified, and that the only
amendment needed in Einstein's theory was the inclusion of the full number
of discrete monochromatic frequencies demanded by the lattice structure
of the crystal with the appropriate statistical weights. It is also seen that
the application of the macroscopic theory of elastic vibrations due to
Debye, successful though it seemed at the time, was, in reality,
a false step.

82 C. V. Raman

In a symposium of seven papers published in the Proceedings of the
Academy for November 1941, the problem of the thermal energy of
crystalline solids has been discussed fully from the new point of view
and compared with the experimental data for a variety of substances.
In several cases where the necessary spectroscopic data were available, these
have been effectively made use of. In other cases, e.g., metals, the specific
data themselves have been utilised to evaluate the atomic frequencies.
The most significant fact which emerges from the symposium is that the
experimental facts in several cases which refused obstinately to fit into the
Debye and Born theories find a natural explanation in the new concepts
without the aid of any special hypothesis.

Summary

The postulates on which the Debye theory of the specific heat of
solids and the Born crystal dynamics are respectively based have been
critically examined and shown to be theoretically untenable. Since a
crystal is a three-dimensionally periodic grouping of similar oscillators
coupled together, it follows that the modes of vibrations possible would be
also space-periodic, the geometric modes being determined by the
characters of the atomic space-grouping in the crystal. They would further
form a finite and enumerable set of monochromatic frequencies. The
spectroscopic, X-ray and thermal behaviours of a crystal would on these
views be radically different from those consequent on the Debye and Born
theories. The experimental facts are found to contradict the conclusions of
these theories and on the other hand, to be in full accord with the new
concepts.

A CONTRIBUTION TO THE LIFE-HISTORY OF

VAHLIA VISCOSA, ROXB., AND VAUL1A

OLDENLANDIOIDES, ROXB.

BY PROF. T. S. RAGHAVAN, M.A., Pn.IX (Losi>.). KL.S.

AND

V. K. SRINIVASAN, M.Sr,

Received October 2*). W!

CONTi'NTS

I'Mvl

I. INTRODUCTION . . . . . . . . . . ''*

II. MATERIAL AND Mi'.i'Hoi) .. .. . . . . s-l

HI. I'V//;//V/ v/Amw, ROXH.

(V/) Flora! Organogcny

(A) Microsporogcncsis , . , . . , , . s**

(r) Megasporogcncsis

(d) Tapctum . . . . . . . . f| t

ff) Fcrtili/ution . . . . , . . . 4 O

(/) Endosperm . . . . . . . . . . 1 J.'

(^) Embryo , , . . . , . . . . 4 M

IV. Vahlitt ohhmlandioidw* ROXB, . . , . . . **

V. DISCUSSION

(a) The Endosperm .. ., ., . . "'

(/>} 'The Nucellus and the Tapetum ., ,. 1^1

VI. SUMMARY . . . . . . , . . . , . KM

VII. LlTHRATURh CiTIiD . . . . . , . . 104

/, Introduction

THE family Saxifragacccc is a fairly well worked family, 1 1 must he described
as a heterogeneous family for the various genera of this natural order c\htl.>u
a variety of characters, and the systematic position of some of the j.*enera has
been questioned; for example. Pace (1912) having studied the lite-hisiury of
a few species of Saxifruga, ParmiMia jwlnstris and Droscru n*funt{ift*lii*. s
of the opinion that Parnassia has greater afltnities to Drowni than u* Sit\i-
fraga and concludes that it should be included in the Droseraeea:, IXihli?rca
(1930) gives an account of the development of the endosperm in a number of

B2 t

84 T. S. Raghavan and V. K. Srinivasan

genera of the Saxifragaceas and draws a scheme of endosperm development
in five genera, where he distinguishes three important types of endosperm
development, namely, nuclear endosperm, cellular endosperm and an inter-
mediate type in which both kinds of endosperm development are combined.
Chapman (1933) gives a summary of the more important of the work already
done in the family. In 1933, Mauritzon made one of the best contributions
to our knowledge of the development of the embryo-sac, endosperm and
embryo in a number of genera of the Saxifragaceae. In a critical study of the
closely allied families of Crassulaceae and Saxifragaceas, he classifies the
various genera of the Saxifragaceae into two broad groups, the " Krassi-
nucellate " and the " tenuinucellate ", on the basis of the nature of the
nucellus. Among the tenuinucellate, there are two groups, those in which
there is a single layer of nucellar cells and those in which the nucellus is
two cells thick just above the megaspore mother cell. He found that
some genera were characterised by the possession of a single integument,
while the others had two integuments. He also describes the nature of the
ovary and ovule, and gives the developmental stages of the embryo-sac, the
endosperm as also the embryo in a a number of genera. Like Dahlgren
(1930), Mauritzon (1933) also groups the various genera investigated till
then under three heads, mentioned by Dahlgren (1930), the basis of grouping
being the nature of endosperm development.

So far as we are aware, the work on the genus Vahlia is very meagre.
Skovsted (1934) gives the haploid chromosome number of Vahlia olden-
landioides as six, which number is confirmed in the present investigation.
Mauritzon (1933) has given a few stages in the development of the embryo-
sac, the endosperm and the embryo in Vahlia oldenlandioides. No
reference could be found in the available literature to any study of
the species Vahlia viscosa, Roxb. The present paper describes the
entire life-history of Vahlia viscosa, and also that of V. oldenlandioides,
for purposes of comparison.

//. Material and Method

Plants of Vahlia viscosa and F. oldenlandioides were grown in the Uni-
versity Botanical Gardens, Annamalainagar. Different stages of ovary were
fixed in corrosive sublimate formalin- Acetic- Alcohol fixative. For determin-
ing the chromosome number, flower buds after acetocannine examination
(to see whether they showed pollen mother cell division stages) were fixed
in hot corrosive sublimate-formalin-Acetic-Alcohol fixative. Sections were
cut at thicknesses varying between 6 and 14 microns. All the materials
were stained in Haidenhain's Iron-alum Haematoxylin.

Life-History ofV. viscosa, Roxb. & V. oldenlandioides, Roxb. 85

///. Vahlia viscosa Roxb.

(a) Floral Organogeny.Flowexs occur usually in pairs (Fig. 4, A and B)
in the axils of leaves. Each flower is initiated as a small conical protuberance
(Fig. 4, A). The sepals are the first to be initiated. Each sepal arises as
a fold from the sides of the conical body (Fig. 1, se\ The flower primordium,
which to begin with, has a convex free end, at the time of the differentiation
of the sepals becomes flattened and in later stages becomes progressively
depressed, in the centre. The next floral organ to be initiated is the stamen
and not the petal. The stamens arise as protuberances from near the rim
of the depression 6r cavity which is being formed in the centre of the free
end of the thalamus (Fig. 2, st.). The primordia of the petals appear be-
tween the .stamens and the sepals (Fig. 3, pe). By this time, the depression '
in the free end of the thalamus is very deep. This is the ovarian cavity and
from its sides near the top, twoprot uberances arise (Fig. 3, cd). These are
the placenta. They grow downwards into the ovarian cavity into which they
hang. When they are fully formed, the ovules appear as papillate protuber-
ances from all round the two placentai. There can be no doubt that the
gynsecium is made up of the fusion of two adjacent carpellary margins.
This is quite clear when transverse sections are taken of very young gynaecia
(Fig. 7) where the bi-carpellary nature of the ovary is quite evident especially
at the top. The two carpel ends have not fused completely so that their
individuality is unmistakably clear. At a later stage, they fuse together
to form a single-celled ovary with two parietal placentas (Fig. 6).
On account of the peculiar disposition of the placentas, the latter appear
as two oval bodies unconnected with either one another, or with the wall
of the ovary, if a transverse section of the ovary is taken in the middle region.
That the gynaeciuin is bi-carpellary and the placentation parietal, there can
be no doubt about, as sections near the top of the oVary reveal the clear
fusion of the adjacent carpellary margins. It is likely that the two carpellary
margins by whose fusion the parietal placentas are primarily formed, have
failed to form any such normal placentas except at the top of the ovary where
on account of the massiveness of the placentas so formed and the concentra-
tion in one region, they hang as it were into the ovary cavity. In Fig. 5, a
transverse section taken at level A, may reveal a position as depicted in
Fig. 7, but a section taken lower down will present Fig. 6.

(b) Microsporogenesis. Transverse sections of very young anthers re-
veal the primary archesporium. The primary archesporium is hypodermal
and usually consists of a plate of two cells (Fig. 8). The primary wall cell
cuts off towards the periphery a layer of primary parietal cells and a layer
of primary spojogenous cells towards the interior. The primary parietal

86 T. S. Raghavan and V. K. Srinivasan

cell again divides twice periclinally. As a result, the anther wall including
the epidermis becomes four cells thick (Fig. 9). The innermost ^yer of he
parietal cells functions as the tapetum. The tapetal cells are of differing

14 15 ^ 16_ 17

FIGS. 1-26. Vahlia viscosa Roxb.

Fig. 1. Origin of sepal at se. X150. Fig. 2. Origin of stamens at st. X150. Fig. 3.
Origin of petal at pe X 150. Fig. 4. Shows the origin of two flowers A. and B in the axil of
a leaf, and also that of the carpels at ca. x 150. Fig. 5. L. S. of ovary showing the pendulous
parietal placentas. xl50. Fig. 6. T. S. of ovary showing uni-locular and bi-carpellary ovary.
Five vascular strands are seen along the fruit wall. X150. Fig. 7. T. S. of very young gynae-
cium showing the bi-carpellary nature of the ovary. x!50. Fig. 8. Hypodermal archesporium
of two cells in the anther, x 800. Fig. 9. Shows the anther-wall which is four cells thick, the
innermost of which is the tapetum. X1500. Fig. 10. Mitotic division of the tapetal nucleus.
X2200. Fig. 11. Bi-nucleate tapetal cell. X2200. Figs. 12-18. Various stages in the form-
ation of the pluri-nucleate tapetal cells. X2200. Fig. 19. Pollen mother-cell nucleus in diakine-
sis; nine rod bivalents are present. X2200. Fig. 20. Metaphase I, n = 9. X2200. ' Figs. 22
and 23. Polar and side view respectively of Metaphase II. X2200. Figs. 24 and 25. Tetra-
hedral and isobilateral arrangement of the pollen tetrads. X2200. Fig. 26. Two-celled mature
pollen grain. Note the three germ pores, X2200.

Life-History ofV. viscosa, Roxb. & V. oldenlandioides, Roxb. 87

sizes and stain brightly, and to begin with are uni-nucleate (Fig. 9). Often
small vacuoles appear in the tapetal cells. The pollen mother cells which
are closely packed together are surrounded by the tapetal layer. When the
pollen mother cells are in the early propbase, the nuclei of the tapetal cells
divide and become bi-nucleate (Fig. 11). This division is distinctly mitotic
(Fig. 10) ; the mitotic nature of the division of the tapetal nucleus has been
observed in a number of families like Capparidaceae (Raghavan, 1938), Scro-
phulariaceae (Srinivasan, 1940), Acanthaceae (Rangaswamy, 1941), etc. This
is followed by a number of nuclear divisions and their immediate fusion,
as a result of which the tapetal cells become pluri-nucleate (Fig. 15).
Various stages in the formation of the pluri-nucleate condition have been
noticed (Figs. 12-18). The possible significance of this has been dis-
cussed recently by Raghavan and Srinivasan, A. R. (1941). Fig. 19 re-
presents the pollen mother cell nucleus in the diakinesis stage. Nine bi-
valents, all of which are of the rod kind can be clearly seen ; two of them
are attached to the nucleolus (Fig. 19). In Fig. 20 is represented the polar
view of metaphase I, and nine bivalents are seen. Anaphasic separation is
normal and the two groups of chromosomes as soon as they reach the poles,
organise themselves into interkinesis nuclei. The nucleolus appears and the
chromosomes which are more or less uniformly spaced are connected by
thin strands (Fig. 21). In Fig. 22 is shown the second metaphase polar
view. There are two groups of nine univalents each. Fig. 23 shows the
side view of the second metaphase. The tetrads are formed by cell plate
formation. They may be either iso-bilateral (Fig. 25) or tetrahedral (Fig. 24).
At the time of shedding, the pollen grains are two-celled (Fig. 26). There
are three germ pores.

(c) Megasporogenesis. The ovary of Vahlia viscosa is bi-carpellary and
uni-locular and the two parietal placentae are pendulous and hang into the
ovarian cavity (Fig. 5). The numerous anatropous ovules are arranged all
around the placentas. At a very early stage, a hypodermal archesporial
cell is differentiated (Fig. 27). Sometimes a plate of two archesporial cells
is also found (Figs. 28 & 29). They may be either one below the other
(Fig. 29) or side by side (Fig. 28). Multicellular archesporia are not un-
common in the Saxifragaceae. Pace (1912) in Parnassia palustris reports
the presence of archesporial plates composed of two, three or four hypo-
dermal cells. The four cells were arranged in a linear row. Multicellular
hypodermal archesporia have also been reported in Jamesia americana
(Mauritzon, 1933). No wall cell is cut off by the archesporial cell. It
begins to increase in size and functions directly as the megaspore mother
cell (Fig. 30). Thus the genus Vahlia belongs to the " tenuinucellate "

B2a E

T. S. Raghavan and V. K. Srinivasan

FIGS. 27-48. Vahlia viscosa, Roxb.

Fig. 27. Hypoderma! archesporiura. XlSOO. Figs. 28 and 29. Two kinds of two-celled
archesporial plate. xlSOO. Fig. 30. Megaspore mother cell invested by the nucellus xlSOO
Fig. 31 Heterotypic division of the M.M.C, X350. Figs. 32-35. Various stages in the form
ation of the linear tetrad. X350. Figs. 36-39. One, two, four, and eight-celled embryo-sac

Life-History of V. viscosa, R.oxb. & V. oldenlandioides, Roxb. 89

In the 8-nucleate embryo-sac, the tapetal tissue does not surround the embryo-sac completely.
X1500. Fig. 40, The polar nuclei are fusing; the synergids with the " synergidenhaken ".
Note the darkly-staining spherical body between the egg-cell and the polar nuclei. X15QO.
Fig. 41. Fertilization. The spherical body is present here also. X15QO. Fig. 42. Division of
the endosperm nucleus. X1500. Fig. 43. Two-celled endosperm. Note the dark body in the
micropylar endosperm cell and the persisting antipodals. xlSOO. Fig. 44. Longitudinal division
of the micropylar endosperm nucleus. In this case, the dark body is present in the chalazal
chamber. X1200. Fig. 45. Four-celled endosperm. X1200. Fig. 46. Transverse division of
the two micropylar endosperm nuclei and the longitudinal division of one of the two chalazal
endosperm nuclei, x 1200. Fig. 47. Shows four chalazal, two middle and two micropylar endo-
sperm cells, x 1500. Fig. 48. Ovule showing later stage in the development of the endosperm
and degenerating tapetum. X750.

Saxifragaceae. In the " Krassi-nucellate " genera of the Saxifragaceae, the
archesporial cell cuts off a parietal cell which builds up a massive nucellus.
When there is a plate of two archesporial cells, one of them alone functions,
for no case of two megaspore mother cells or two tetrads or two embryo-
sacs was noticed in hundreds of sections examined. Chapman (1933), how-
ever, found that the occurrence of two embryo-sacs in the same ovule was
not rare in Saxifraga virginiensis. In all these cases, the extra embryo-sacs
were developed from a megaspore resulting from the division of a second
megaspore mother cell. In three out of five cases, each of the embryo-
sacs was surrounded by its own micellus, and in these, the embryo-sac was
in the two or the four-nucleate condition. In the other two cases, he found
two megaspore mother cells, one of which was in the prophase of the first
division, while in the other, the nucleus was in the metaphase of the first
division. In one case, the two megaspore mother cells lay side by side,
while in the other, they were separated by two or three layers of nucellus.
This kind of archesporium which functions directly as the megaspore mother
cell has been reported in Jamesia americana, Philadelphus coronarius
(Mauritzon, 1933) and in Parnassia palustris (Pace, 1912). Just at the time,
when the primary archesporium increases in size, to assume the functions of
the megaspore mother cell, the primordia of the integuments arise. The inner
integument is the first to be initiated, while the outer soon follows the inner.
The inner integument is longer than the outer and it alone takes part in the
formation of the micropyle (Fig. 35). As the integuments grow, the ovule
curves to assume its anatropous nature (Fig. 30). At this stage, the mega-
spore mother cell could be seen to be invested almost to its base by a layer
of cells, the nucellus (Fig. 30). This nucellus is derived by repeated anti-
clinal divisions of the epidermal cells just above the primary archesporium.
In the Saxifragaceas both single and two integumented ovules occur. The
genera Ribes, Vahlia, Brexia, Parnassia, etc., have two integuments, while
the genera Saxifraga, Kirengeshoma, Hydrangea, etc., possess a single integu-
ment. With the development of the megaspore mother cell, the cells of the

90 T. S. Ragliavan and V. K. Srinivasan

nucellus get flattened and finally disorganise and disappear completely, in
the early stages of the development of the embryo-sac. When the mega-
spore mother cell has increased in size considerably, the heterotypic divi-
sion sets in, as a result of which a dyad is formed (Fig. 32). Fig. 31
represents the telophase of the heterotypic division. The cells of the dyad
undergo the homotypic division to give rise to a linear tetrad (Fig. 35). The
dyads do not divide simultaneously. Figs. 32 to 34 show the various stages
in the formation of the tetrad. In Figs. 32 and 33, the dyad cells are dividing
simultaneously. In Fig. 32, the two nuclei are at metaphase. In Fig. 33,
while the chalazal dyad is in the metaphase of the division, the micropylar
dyad cell is in the telophase of the same division. In Fig, 34, the chalazal
dyad has divided to form two cells, while the micropylar cell is still in the
anaphase of the first division. Such a non-simultaneous division of the
dyads has been recorded in Parnassia palustris (Pace, 1912). Here, while
the chalazal dyad cell has only formed the chromosomes, the micropylar
cell has already formed the spindle for the second division. Though the
linear arrangement of the tetrads is more common in the Saxifragaceas, T--
shaped tetrads have also been reported in some cases. 'Mauritzon (1933)
reports the occurrence of T-shaped tetrads in Bergenia crassifolia, Ribes
aureum, Tiarella cordifolia, Heuchera sanguined and Chapman (1933) in Saxi-
fraga virginiensis. The chalazal megaspore is the functional one and the
rest degenerate (Fig. 36). The functional megaspore divides to give rise
to the 2-nucleate embryo-sac (Fig. 37). The four and the eight-nucleate
embryo-sacs (Figs. 38 & 39) are formed in the usual manner. Fig. 39
shows the mature embryo-sac, where the egg apparatus has been organised.
There are two prominent synergids, an egg cell, two polar nuclei situated
usually in the middle of the embryo-sac and three antipodals towards the
chalazal end. The filiform apparatus described by Pace (1912) as being
usual in Saxifraga virginiensis was not evident in the present case. The
" Synergidenhaken " however are seen, though not very markedly, as broad-
ened bracket-like bases of the synergids (Fig. 40). In the Saxifragaceae,
these structures have been reported in Hydrangea petiolaris, Francoa appendi-
culata, Ribes stenocarpum, R. nigrum, R. grossularia, Mitella nuda (Maurtizon,
1933), etc. Fig. 40 shows the two polar nuclei fusing in the centre. The
two nucleoli can still be seen. The secondary nucleus is the largest and
most prominent nucleus in the embryo-sac.

The mature embryo-sac is roughly elliptical in shape. The other genera
of the Saxifragaceae have embryo-sacs of differing shapes (Mauritzon, 1933).
The embryo-sac is long, narrow and cylindrical in Francoa appendiculata ;
highly enlarged in the micropylar region, while the chalazal portion is

Life-History ofV. viscosa, JRo.vd. & V. oldenlamlioicles, A* At/. 91

narrow, wherein the untipodals are situated as in Heuchem pubescent and
M itella nuda ; more or less elliptic as in Itca virgitrica* Ewallonht nnnrttnffta,
Parnassia ovatai the cliala/ul portion of the embryo-sac may be bent at an
angle to the micropylar portion as in Hydrangea petiolaris. In Pitlyswna
ilicifoliwn, the embryo-sac is obovato in shape, the chala/al end being
broad and blunt, while in Rihcs missoiirkmM* the sides of the embryo-sac
dilate and grow towards the elialazal end of the ovule like a haustorium.

At about this stage, when the embryo-sue is fully organised and the
polar nuclei have fused, a spherical, darkly-staining nueleus-Iike body makes

its appearanee just above the fusion nucleus. This appears to be a feature
of constant oceuirenee, for in about sixty or seventy embryo-sacs examined,
we found this body always present. It cannot, however, be a nucleus since
there appears to be no definite nuclear membrane and also there is no
nucleolus. This body persists till after fertilization. In Fig. 41, where the
remains of the pollen tube arc seen and the egg cell is undei going fertilisa-
tion, we find this spherical body in a line with the fusion nucleus and the
fertilized egg. In Fig, 43, whoie fortili/ation of the egg cell has been com-
pleted as could be seen from the degenerated synergids and where the endo-
sperm has formed a 2-eellecl structure, we find this body again. In
Fig. 44, which is a later stage showing 3-eelIed endosperm, we find this
body below the chalazal endosperm nucleus* Later than this stage we have
not met with this body. We are not at present able to offer any interpreta-
tion as to the exact nature of this spherical body,

(d) Tapctwn.-M a result of the breakdown of the single layer of
nueellus investing the rnegaspore mother eelL the innermost layer of the
inner integument comes into direct contact with the sides of the embryo-sac.
This layer of cells, the lapetum, becomes conspicuous on account of the
regularly arranged rectangular cells and they soon come to possess rich cell
contents and hence stain rather darker than the other cells of the integu*
mcnt. Often, however, small vacuoles could be noticed in the tapteal cells,
The cross walls are oblique. The tapetum is thus of integumentary origin
and its differentiation from the inner integument commences simultaneously
from either end of the embryo-sac. The tapetal cells are uni-nucleate
often bi-nucleolated (Fig. 39). Bi-nuclcatc tapteal cells hive been occasion-
ally recorded in the Solanacex (Bhaduri, 1932} and in the Orobanchaccae
(Srivastava, 1939), The tapdurn docs not surround the entire embryo-sac,
At the chalazal and micropylar ends, only ordinary cells are found. A
distinct tapetal tissue surrounding the embryo-sac has been reported in
various genera of the Scrophulariaccse (Srinivasan, 1940), Solanaeca* (Bhaduri,

92 T. S. Raghavan and V. K. Srinivasan

1935), Labiatese (Billings, 1909), Lobeliaceae (Kausik, 1938), Orobanchaceae
(Srivastava, 1939), Verbenaceae (Tatachar, 1940), and in many other sym-
petalous families, as also in Crassulaceae (Mauritzon, 1933) of the polypetate
and in Parnassia palustris (Pace, 1912), P. ovata and Brexia madagascariensis
of the Saxifragaceae (Mauritzon, 1933). In Escallonia rubra, and Hydrangea
petiolaris also belonging to the Saxifragaceae, the tapetal tissue is confined
to the chalazal half, which is often bent at an angle to the micropylar half
(Mauritzon, 1933). The function of the tapetum is essentially nutritive.
For, as the endosperm in the embryo-sac increases in size, the tapetal tissue
graudally becomes thinner and thinner (Fig. 48) and finally disappears.

(e) Fertilization. Fig. 41 shows the male nucleus about to fertilize the
egg. The darkly staining pollen tube enters the embryo-sac through the
micropyle. The male cell would appear to be spherical. Though vermi-
form and spiral-shaped male cells are by far the commonest in Angiosperms,
spherical cells have however been occasionally reported, e.g., Weinstein
(1926) in Phaseolus vulgaris, Madge (1929) in Viola odorata, Newman (1934)
in Acacia Bailey ana Raghavan (1937) in Cleome chelidonii, and Raghavan and
Srinivasan (1941) in Ilysanthes parviflora. The male nucleus and the egg
nucleus seem to be in a resting condition at the time of contact. Such a
condition is not only common in Angiosperms but also in the Coniferales
(Guilliermond, 1933) and some Cycadales (Lawson, 1926). No phylogenetic
significance can be attributed to this, as this phenomenon is found in such
widely separated families as Oenotheraceas (Ishikawa, 1918), Hydrocharitaceas
(Wylie, 1923), Orchidaceae (Pace, 1907), Capparidaceas (Raghavan, 1937),
and Scrophulariaceae (Raghavan and Srinivasan, V.K., 1941). The synergids
are ephemeral and degenerate soon after fertilization. The antipodals often
show a tendency to persist, though in a rather degenerated form, and are
to be seen in embryo-sacs, in which the endosperm has become two to four-
celled (Figs. 43 and 46). In the- Saxifragaceae, antipodal haustorium is known
to occur in Kirengeshoma palmata (Mauritzon, 1933) ; though all the three
antipodals which are arranged one above the other in a linear fashion persist,
the one towards the extreme chalazal end is elongated and is haustorial in
its function.

(/) Endosperm. The fusion endosperm nucleus is the largest nucleus in
the post-fertilization embryo-sac. The position of the endosperm nucleus
is always in the middle of the embryo-sac. In the other genera of the Saxi-
fragaceae, the position of the. endosperm nucleus varies considerably. In
Mitella pentandra (Dahlgren, 1930), the endosperm fusion nucleus is more
towards the antipodal end. The fusion nucleus undergoes a period of rest
before it divides. The first division is transverse and is accompanied by

Life-History fV. viscosa, Rox6. & V. oldenlandioides, Rozb. 93

wall formation. Fig.. 42 shows the fusion nucleus in the anaphase of
the transverse division. The transverse wall divides the embryo-sac into
two more or less equal halves (Fig. 43). In the other genera of the Saxi-
fragraceae that exhibit cellular endosperm, the first wall is also transverse,
though the two endosperm chambers that result in the embryo-sac are not
equal as in the present case. Usually, in these, the chalazal endosperm
chamber is considerably smaller than the micropylar one. Such a difference
in size of the two cells of the two-celled endosperm stage has been figured for
Mitella pentandra, Boykinia occidentalis (Dahlgren, 1930), Boykinia Jamesii,
Bergenia ligulata, Saxifraga micranthidlfolia, Tiarella polyphylla and Ribes
bureiense (Mauritzon, 1933). Of the two endosperm cells thus formed, the
micropylar one is the next to divide. This division is longitudinal followed
by wall formation (Fig. 44). Following closely on this, the chalazal cell
also divides longitudinally (Fig. 45). As a result of these two divisions,
four endosperm cells are formed in the embryo-sac. This bears a close
resemblance to the sequence of division of the endosperm nucleus in a few
genera of the Scrophulariaceae (Srinivasan, 1940). In the Other genera of
the Saxifragaceae, however, the second and the third divisions are not longi-
tudinal as in Vahlia viscosa. The second division is frequently transverse
resulting in a row of three cells as in Mitella pentandra (Dahlgren, 1930).
The two micropylar endosperm cells (Fig. 46) then divide transversely fol-
lowed by wall formation. As a result, six endosperm cells are formed in the
embryo-sac. The two cells in the middle of the embryo-sac by repeated
divisions form the cellular endosperm tissue. The two chalazal and micro-
pylar cells also cut off cells towards the centre of the embryo-sac and contri-
bute to the endosperm. The two chalazal cells often undergo a longitudinal
division resulting in four cells arranged in two juxtaposed tiers one tier below
the other (Fig. 47). These cells become very rich in cytoplasm and hence
stain brightly and -the cells abutting on the chalazal end of the embryo-sac
begin to exhibit signs of degeneration. These four chalazal endosperm cells
thus appear to be haustorial in function, though they do not show any well-
marked growth or haustorial protuberance penetrating the tissue around it.
Though the endosperm cells towards the micropylar end also take a deep
stain, they do not seem to be haustorial as the adjoining tissue does not
show any signs of disintegration. As the endosperm tissue increases in
size in the embryo-sac, the tapetal layer surrounding the embryo -sac be-
comes thinner and thinner and in very late stages, it completely disappears.
This is as it should be because the tapetum is essentially nutritive. As the
endosperm develops rapidly, it absorbs nutrition required for its growth
from the tapetum, which consequently becomes shrivelled up gradually.

T. S. Raghavan and V. K. Srinivasan

FIGS. 49-60. Vahlia oldenlandioides, Roxb.

Fig. 49. T. S. of ovary showing three placentas. x75. Fig. 50. Hypodermal archesporium.
X1500. Fig. 51. M.M. cell with nucellus. X350. Fig. 52. Dyad in division showing six
chromosomes, x 350. Fig. 53. Formation of the linear tetrad, x 350. Fig. 54. Two-nucleate
embryo-sac, x 1500. Fig. 55. Eight-nucleate embryo-sac, the polar nuclei fusing with the darkly
staining body situated near it. X1200. Figs. 56 and 57. Two- and four-celled endosperm.
X1200. Figs. 58 and 59. Ovules showing later stages of endosperm development. x750.
Fig. 60. Metaphase II, n = 6. X2200.

(g) Embryo. The oospore (Fig. 61) undergoes a long period of rest
before it begins to develop into the embryo. The oospore divides long
after the endosperm nucleus has divided. A stray case where in one embryo-
sac, the oospore divides before the endosperm is reported by Pace in
Parnassia palustris (1912). Here, he observed a 5-celled embryo with the
endosperm nucleus still undivided and somewhat amoeboid in shape. After
a considerable amount of endosperm tissue has been formed, during which

Life-History of V. viscosa, Roxb. & V. oldenlandioides, AW//. 4 *5

time, the oospore has elongated to some extent; it divides, Consequently
the oospore is surrounded by the endosperm tissue. The first division of
the oospore is transverse and is immediately followed by a cross-wait
(Fig. 62). Of the two resulting cells, the lower one, i.e., the one farther from
the micropyle, gives rise to the embryo proper, and the upper, to the sus-
pensor. The lower or apical cell is the next to divide. This division is
anticlinal (Fig. 63). Following closely upon this division, the upper one
also divides, but transversely, both the divisions being followed by the
formation of cross-walls (Fig. 64). Within the Suxifragace;e, a similar
behaviour of the 2-celIed embryo has been figured in Purnassia palustrix
(Pace, 1912). In this also, while the apical or lower cell divides longitudi-
nally, the basal or upper cell divides transversely. There is however some
difference in the timing of the two divisions between the two-celled embryo
in Parnassia and in the present case. For, while in Vahlia viscosa* the lower
(embryonal) cell divides before the basal cell, in Parnassia ptilustris, from
the figure given by Pace (1912), it would appear that the upper cell begins
to divide a little earlier than the apical cell. In Fig. 64, the lower cell has
divided while the upper is in the late telophase of the transverse division.
As a result of these two divisions, a four-celled pro-embryo is formed, in
which the two lower cells are placed side by side, while the tipper two arc
placed one above the other (Fig. 65) and as such the 4-celled pro-embryo is
only three cells long. This type of arrangement of the four cells of the
pro-embryo is common in families like Cmeifene, Rununculacce (Sougcs,
1913, 1919), Capparidaceae (Raghavan, 1937), etc. In the other type, the four
cells of the pro-embryo are arranged in a linear fashion. Such an arrange-
ment of the four cells of the pro-embryo is characteristic of the Rtihiacc;e
(Raghavan and Rangaswamy, 1941), Solanacea? (Souges, 1922), Leguminoscw
(Cooper, 1933), Orobanchaccas (Srivastava, 1939), Serophulariaca? (Srinivasan,
1940), and in the Acanthaccas (Rangaswamy, 1941). In the Saxifraguecx,
both types of arrangement of the four cells in the pro-embryo arc met with.
For instance, in Ribcs divancatum, in Astilhoidcs tahuhiris and in Hcwhcra
sanguinea (Maurilzon, 1933), the four-celled pro-embryo is linear, while in
Parnassia palustris (Pace, 1912), the arrangement of the four-celled embryo
is similar to that found in the present investigation. Besides these two types
of arrangement, often, a linear pro-embryo 'of five or more cells in length is
also to be found as in Boykinia tcllimoides (Dahlgren, 1930), and Ticirr/JV?
polyphylla (Mauritzon, 1933). The four-celled embryo stage is an important
one, as each one of these four cells gives rise to a definite region in the mature
embryo. For purposes of description, these four cells beginning from the
uppermost or basal cell will be designated A, B, C and D. The cell B is the

96 T. S. Raghavan and V. K. Srinivasan

first to divide among the four cells of the pro-embryo. It divides by a
transverse wall (Fig. 66) into B x and B 2) as a result of which the suspensor
becomes three cells long (Fig. 67). Often this division is followed by an
oblique cross-wall as shown in Fig. 68. The suspensor becomes four cells long

FIGS, 61-71, Vahlia viscosa, Roxb.
Various stages in the development of embryo. Figs. 61-70. X1500. Fig. 71. xllOO.

by another transverse division of the cell A. The cell B 2 divides transversely
into two cells B 2)1 and B 2 , 2 of which the latter is the hypophysis. The cell B 2 , 2
cuts off a cell which abutts into the embryonal sphere and is itself divided
into two cells by a vertical wall (Fig. 70). Figs. 69 and 70 are those of later
stages in the embryo development and show the differentiation of the pri-
mary tissues, dermatogen, periblem and plerome. In the mature embryo,
the suspensor is about 5 to 6 cells long and is uni-seriate (Fig. 71). Multi-
seriate and massive suspensors especially at the base, are a common feature of
most members of the Saxifragacese. Multi-seriate suspensors have been
recorded in Mitella dyphylla, Sullivantia sullivantii, etc., and in Ribes aureum,
the suspensor is very massive and irregular in shape,

IV. Vahlia oldenlandioides, Roxb.

The two species of Vahlia investigated here, are characterised by parietal
placentation. The placentas are pendulous and hang from the roof of the
gynaeceum into the ovarian cavity as has already been described. In
K. oldenlandioides, however, often three parietal placentas and three styles

Life- His to ty of V. viscosa, Roxb. & V. oldenlandioides, Roxb* 97

are to be found, and Fig. 49 shows the transverse section of such an ovary.
The development of the ovule, embryo-sac, the endosperm and of the embryo
bears a striking resemblance to that already described for J 7 . viscosa. The
primary archesporium which is hypodermal may consist of a single cell
(Fig. 50) or a plate of two cells. This species of Vahlia is also characterised
by the possession of two integuments, the inner of which alone takes part
in the organisation of the micropyle. The nucellus is also similar to that
in Vahlia viscosa. Fig. 52 represents the dyad. The nuclei of the dyad
are in the metaphase of the homolypic division and in the miropylar dyad,
the haploid chromosome number of six is clearly seen. A linear tetrad is
formed (Fig. 53), the chalazal one of which develops into the mature embryo-
sac in the usual manner (Fig. 55). Fig. 54 shows the bi-nucleate embryo-sac.
The mature embryo-sac bears a close resemblance to that of V. viscosa and
is surrounded by a tapctum of integumentary origin. The tapetal cells are
uni-nucleate. Even in this species the darkly staining spherical body de-
scribed for the other species was found to be a feature of constant occur-
rence. The endosperm cells towards the chalazal end have a haustorial
function as in the other species already described.

Thus we find that the life-histories of the two species of Vahlia are very
similar and it would appear that the genus Vahlia is characterised by the
possession of two or three parietal and pendulous placentas, ovules with
two integuments and a single layer of nucellus which degenerates very soon
giving place to a tapetal layer of integumentary origin, the tapetal cells being
uni-nucleate. The chalazal megaspore of a linear tetrad always forms the
embryo-sac, which is normal, the degenerated antipodals often persisting
till the two-celled endosperm stage. Endosperm is cellular, and in both
the species, the plan of cellular endosperm development is different from
the other genera of the Saxifragace so far studied, but is similar among
themselves. In the four-celled pro-embryo, the two apical cells are placed
side by side while the two basal ceils are one over the other and the sus-
pensor is uni-seriate and about 5 cells long.

The haploid chromosome number has been determined to be 6 and this
finding corroborates the number already recorded by Skovsted (1934). Fig. 60
is that of M II in polar view and shows two groups of six univalents each.

V. Discussion

(a) The Endosperm. The family Saxifragaceae is interesting especially
from the point of view of endosperm development. Within the family,
three distinct types of endosperm development are found. Some genera
are characterised by the cellular type ; in some others the free

98 T. S. Raghavan and V, K. Srinivasan

nuclear type of endosperm development is the rule. The third or the
" Helobiale type " is a combination of the cellular and the free-nuclear
type of endosperm development. In this last mentioned type the first
one or two divisions are followed by the formation of cross-walls
while successive divisions produce free nuclei, wall formation com-
mencing later. Of the two cells resulting from the first division,
in one the development of the endosperm will be cellular, while in.
the other cell it is exclusively free-nuclear. Mauritzon (1933) has Classified
the plants investigated till his time into three groups on the basis of the
nature of endosperm development. According to him, the genera Brexia,
Parnassia, Tetilla and Francoa have the " nuclear type " of endosperm.
The genera Astilbe, Astilboides, Bergenia, Boykinia, Cardiandra, Decumaria,
Deutzia, Heuchera, Hydrangea, Jamesia, Kirengeshoma, Lithophragma, Mitella,
Peltiphyllum, Philadelphus, Ribes, Rodgersia, Tellima, Tiarella, Tolmiea
and Vdhlia exhibit the " cellular type " of endosperm development, while
the genera Boykinia, Chrysosplenium, Mitella, Ribes, Saxifraga, Sullivantia
and Tiarella possess the " Helobiale type " of endosperm in which endo-
sperm development is cellular to begin with but later becomes free-nuclear.
There are, as will be seen from the examples cited, a number of cases, in
which the two types of endosperm development are found in the same genus.
For example, in the genus Mitella, the species M. diphylla exhibits the
"Helobiale type" of endosperm development, while in M. pentandra its develop-
ment is exclusively " cellular ". Again in Boykinia, B. aconitifolia, B. Jamesii
and B. occidental, show the cellular type of endosperm; B. tellimoides,
however, is characterised by the " Helobiale type " of endosperm. Similarly
in the genus Tiarella while some species show the " Helobiale type " of endo-
sperm, a few other species exhibit " cellular " endosperm. Thus we find
that in the Saxifragaceae endosperm development varies considerably from
genus to genus and even within the same genus, different species exhibit
different type of endosperm development. In the two species of Vahlia
investigated, cellular endosperm is the rule. Taking into consideration the
cellular type of endosperm development in the Saxifragaceae, we find that
all the species showing cellular endosperm do not follow the same plan or
sequence of divisions in the building up of the endosperm. Even here, we
find that different genera show different plan or sequence of divisions in the
formation of the endosperm tissue. In the Saxifragaceae, among those
exhibiting cellular endosperm the more usual method seems to be the forma-
tion of three endosperm cells by two transverse walls laid across the
embryo-sac, and during the further development of the endosperm tissue
a longitudinal wall is usually laid in the chalazal chamber to begin with,

Life-History ofV. viscosa, Roxb. & V. oldenlandioides, Roxb. 99

and similar longitudinal walls are formed in the other two endosperm cells
also. This type of cellular endosperm development has been noted by
Mauritzon (1933) in Astilboides tabularis, Mitella nuda, Bergenia ligulata,
etc., while in others like Boykinia occidentdis (Dahlgren, 1930), the second
division takes place in the chalazal cell and is longitudinal. In the present
investigation, the two species of Vahlia studied present an altogether different
plan of endosperm development from the others. The first division is trans-
verse, while the second division is longitudinal and takes place in the micro-
pylar endosperm cell. The third division is also longitudinal and takes
place in the chalazal cell. The two micropylar cells then divide transversely.
Such a plan of division of the endosperm nucleus has not so far been reported
in any other member of the Saxifragaceae. A similar scheme of endosperm
development has been found in Dopatrium iobelioides, Stemodia viscosa and
Vandellia Crustacea all belonging to the Scrophulariaceas (Srinivasan, 1940).

While in the closely related family of Crassulaceas (Mauritzon, 1933)
haustoria of diverse origin, like megaspore haustorium, suspensor haustorium,
chalazal and micropylar endosperm haustorium are common, in the Saxi-
fragaccae, however haustoria are of rare occurrence. In Kirengeshoma
palmata (Mauritzon, 1933) antipodal haustoria are present. Micropylar
haustorium of endospermal origin has been recorded by Mauritzon (1933)
in Corokia cotoneaster, in which is figured a single uni-nucleate micropylar
endosperm haustorium. Four uni-nucleate vermiform micropylar haustoria
occur in Philadelphia* coronarius (Mauritzon, 1933). The function of these
haustoria is the usual one of supplying nutrition to the developing endo-
sperm and embryo.

An effort was made to find out whether a correlation could be established
between the division of the family on the basis of the type of endosperm
development and the division of the family by taxonoraists on morphological
grounds. We also endeavoured to discover whether chromosome numbers
that are known so far in this family could, in any way be employed for the
elucidation of this. In this connection, we gathered most of the available
data regarding this family, both morphological and cytological. Engler
recognises seven sub-families and in trying to find out if a relationship could
be established between the type of endosperm formation as available from
previous work and this classification, we found that within each sub-
family there occurred almost all the three types of endosperm development.
In some cases, as in the genus Ribes* even within a genus, different types of
endosperm development are reported. The statement given below will
show this point clearly. For instance, in the sub-family Saxifragoidae,
Yalilia shows the "cellular type," so also Astilbe; Parnassia, shows "nuclear ",

10 T. S. Raghavan and V. K. Srinivasan

Chrysosplenium and Saxifraga show the ' helobiale type ** of endosperm
development. The Hydrangeoide* is a bit more uniform where the three
about which information is availabe show the cellular type of
endosperm but no generalisation is possible since we have no itrforniation
about the other sixteen or seventeen genera comprised in this sub-lanuiy.
In the tabular statement presented, only important genera included m the rat-
families are shown; it will be seen that practically no information is availabe
about the method of endosperm formation in the sub-families Petrostemo-
noide*, Escallonoide*, and Baueroide*. But the information available
at hand shows clearly that there could be no question of the division of the
family on the basis of the method of endosperm formation. The chromo-
some numbers of a comparatively large number of genera included in this
family are known. But these are confined principally to the sub-families
Saxifragoides, Hydrangeoideae and Ribesioideae. Some of the numbers
known are noted against the respective genera. It could be seen that aneu-
ploidy has played, presumably, a very important part in the evolution of the
genera and that no support could be had from these numbers for tackling
the problem cyto-taxonomically. We also endeavoured to see if the chromo-
some numbers and the type of endosperm development could in any way be
related. For example, the species showing the " cellular type " of endo-
sperm exhibit numbers: 6, 7, 8, 9, 13, 16, 18, 38, 39, 52, etc. Similarly, the
"nuclear" forms exhibit 9, 10, 20 and those belonging to the "Helobiale type"
show also a similar range of chromosome numbers: 8, 9, 11, 13, 16, 18, 20,
24, etc. It is obvious from these data that have been presented, that it may not
be easy to classify the family easily, from these points of view. It has already
been said that it is a rather heterogeneous family ; the position of some
members is even doubtful, as for example that of Parnassia already mention-
ed. If the problem is. to be tackled cyto-taxonomically, it would be better to
arrange the different genera and species according to the respective chromo-
somal series : those belonging to the 8 series, 9 series and so on. The
genus Deutsia for instance would appear to fall in with the 13 series; so also
Philadelphus. Hydrangea belongs to the 9 series. In Saxifraga we get an
aneuploid series 8, 9 5 11, 13, 18, and so on. Very likely, a more critical
cytological examination may throw some light upon the basic number of the
genus and also that of the sub-family. In this, phenomenon like secondary
association coupled with some genetical data will undoubtedly play a very
large part. It may be that there is a primary basic number from which there
might have arisen other secondary basic numbers, each of which might have
produced their own polyploid series. How these secondary polyploid series
arose can again be inferred only by a cytological and genetical study. In this

Life-History ofV. viscosa, Roxb. & V. oldenlandioides, Roxb. 101

way, if data are gathered, it may be possible to tackle the problem cyto-
taxonomically. Pending this, it can only be said that the family is indeed,
in the words of C.B. Clarke (1879) "very difficult of definition".
I. Saxifragoidece:

H Saxifraga (6, 7, 8, 9, 13, 14, 15, 16, 18, 36, 39, 52, 65).

Z Vahlia(6, 9).

H Chrysosplenmm (12, 24).

Z Heuchera (7, 8).

N Parnassia (9, 10).

Z Astilbe (7).
Z&H Tiarella(9).

II. Francooidece :

N Francoa(20).
N Tetilfc.

III. Hydrangeoidece :

Z Hydrangea (18, 36).

Z Philadelphia (13).

Z Deutzia(13, 39, 52, 65).

IV. Pterostemonoidece :

Pterostemon.

V. Escallonoidece:

Escallonia.
Phylloma.
Polysoma.
Itea.

VI. Ribesiodea :

Z&H Ribes(8, 16).

VII. Baueroidece :

Bauera.

N.B. The numbers given in brackets on the right-hand side of the genera are the chromo-
some numbers prevalent in the respective genera, and the bold letters on the left-hand side denote
the type of endosperm development found in the respective genera :

Z *= Cellular endosperm.

N = Nuclear endosperm.

H = Helobiale type of endosperm.

(b) The Nucellus and the Tapetum. The 'family Saxifragaceae is interesting
not only from the point of view of endosperm formation but also from that of
presence of the tapetum. In this connection, we have tried to gather avail-
able information regarding the tapetum, endosperm formation and the

102 T. S. Raghavan and V. K. Srinivasan

nature of the nucellus and the possible correlation between these. Genera-
ally speaking, nucler type of endosperm formation is associated with a
massive nucellus. This is widely prevalent amongst the apetalae and poly-
petate of Benthem and Hooker. In these, there is no tapetum. formation.
This statement does not mean, however, that this is a rigid rule. There are
some families amongst this group of Angiosperms where we get reduced
nucellus " Tenuinucellate " (single layer of nucellus) as for instance in
Sarraceniaceze, Podostomaceas, Pittosporaceae, etc. There are also
a few cases in this group, where the massive nucellate condition " Krassi-
nucellate " is associated with cellular endosperm. All that the statement
implies is that in the vast majority of the families comprised in this group,
massive nucellus and nuclear endosperm coupled with the absence of any
tapetum seems to be the rule. In the Sympetalae, the reduced nucellus is
the prevailing condition. As in the previous case, there are found a few fami-
lies in which this reduced' nucellate condition (tenutnucellate) is associated
with nuclear endosperm for example, Gentianaceae, Apocyanaceae, Logania-
cese, Asclepiadaceae, Rubiaceae, Goodeniaceas, etc. It is amongst this
group, that we get the tapetum and it is in some <of the families
comprised in this group that the endosperm haustorium is prevalent. The
pertinent question arises, whether there is any factor which governs the
appearance of the tapetum. The tapetum such as occurs in the ovule, is
almost always integumentary in origin, and must be regarded as nutritive in
function even as the microsporangial tapetum. While there can be no doubt
as to the nutritive character of the latter, because of its 'Universal occurrence,
the same cannot be said of the integumentary tapetum, because it is confined
only to some families. In order, therefore, to find out its true role, and also
if possible, the conditons under which it usually occurs, we tried to see if a
correlation could be discovered between this and the other associated tissues
like the nucellus and the endosperm. Such an investigation has revealed as has
been indicated, the existence of some relationship from which could be drawn
a few inferences which for the moment must obviously be regarded as tenta-
tive. The first observation of importance is that the tapetum almost always
is integumentary in origin. That is, it occurs only where there is no parietal
tissue. A possible inference from this is that where there is* no massive
(Krassi-nucellus) parietal tissue, the nutrition of the embryo-sac is presum-
ably defective, however much its place may be taken up by the integument.
Naturally, in order to strengthen the nutritive mechanism, the tapetal layer
is present. Support to this can be gathered from the fact that the occurence
of the tapetum is mostly associated with cellular endosperm, many of which
exhibit some type of haustorium or other. This means that because the

Life-History ofV. viscosa, Roxb. & V. oldenlandioides, Roxb. 103

nutritive mechanism is not perfect, recourse has been taken to these supple-
mentary devices, by which to make up for- the deficiency. The next question
is in what way does the tapetum discharge its nutritive role ? If an analogy
is to be established between this tapetum and the anther tapetum we must
naturally look for the pluri-nucleate condition of the tapetal cells and the
subsequent usage of this nuclear material for the nutrition of the embryo-sac.
Though this multi-nucleate condition is widely prevalent so far as the anther-sac
tapetum is concerned, this appears to be rather the exception than the rule so
far as the integumentary tapetum is concerned. This leads to the question
whether the tapetal cells directly contribute to the nutrition of the embryo
sac or does the tapetal layer merely act as a sort of a liaison tissue, merely
helping in the transference of nutritive material from the surrounding integu-
mentary tissue. To our mind the latter alternative seems to be the more
possible, for as has already been said, this tapetum occilrs only where the
massive (Krassi) nucellate condition does not exist. In this " Krassi-
nucellate " condition, the embryo-sac is closely surrounded by the parietal
tissue and naturally there can be no difficulty whatsoever in the matter of
the supply of food material by this closely enveloping tissue to the embryo-
sac. But in the " tenuinucellate " condition, the embryo-sac is left severely
alone. The single layer of nucellus very soon perishes in the ontogeny of the
ovule. There is a gap between the developing embryo-sac and the developing
integument. There can be no question of a close contact of these two, -such
as exists between the parietal tissue and the embryo-sac in the Krassi-nucellate
condition. Naturally, when contact, ho.wever imperfect, is established,
between the developing embryo-sac and the integument, some sort of an
intermediary tissue is found necessary in order to facilitate the free transference
of -nutritive material from the integument to the embryo-sac. Presumably,
even this device is not sufficient, for in many cases, the endosperm haustorium
makes its appearance as a post-fertilization .structure. The tapetum after
performing its function disintegrates, when the endosperm tissue is begin-
ning to take up the nutritive role.

VI. Summary

The haploid chromosome number of Vdhlia oldenlandioides has been
confirmed to be 6, and that of V. viscosa has been determined to be 9.

The origin and development of the microsporangium, the embryo-sac,
the endosperm and the embryo is described in detail; both Vahlia viscosa
' and V. oldenlandioides are found to be quite similar.

In both the species of Vahlia investigated, the endosperm is cellular and
there are four unirmcleate chalazal endosperm haustorial cells in both.

T. S. Raghavan and V. K. Srinivasan

No relationship could be established between the divison of the family
on the basis of the type of endosperm development with (i) division of the
family by taxonomists on morphological grounds and (ii) the chromosome

numbers known in the family.

For a cytotaxonomical approach, cytological details like secondary
association, etc., of which no information is now available, are suggested to
be a necessary prerequisite.

* The role of the integumentary tapetum is discussed in the light of its
correlation to the nucellus and the endosperm.

VII. LITERATURE CITED

Bhaduri P N . . " The development of ovule and embryo-sac in Solatium wdon-

gena L.," Jour. Ind. Bot. Soc., 1932, 11, 202-24.

_ " Studies on the Female Gametophyte in Solanacese," Ibid.,

1935, 14, 133-50.

Billings, F. H. ' . . " The Nutrition of the Embryo-sac and Embryo in certain Labia-

te*," Kansas Univ. Sci. Bull., 1909, No. 5.

Chapman, M. . . " The Ovule and embryo-sac of Saxifraga virginiensis" Amer.

Jour. Bot., 1933, 20, 151-58.

Clarke, C. B. . . " The Flora of British India," by Hooker, J. D., 1 879, 2, 388.

Cooper, D. C. . . " Macrosporogenesis and embryology of Melihtus" Bot. Caz.*

1933, 95, 143.

DaWgren, K. V. O. . . " Zur Embryologie der Saxifragoideen," Sartryck w Svensk

Botanisk Tidskrift, 1930, Bd. 24, H. 3, 429-48.
Guilliermond, A. G., Mangenot, Traite de Cytologie Vegetale, Paris, 1933, 788.

G., and Plantefol, L.
Ishikawa, M. . . " Studies on the embryo-sac and fertilization in Oenothera"

Ann. Bot., 1918, 32, 279.
Kausik, S. B. . . " Gametogenesis and embryology in Lobelia nicolianae folia

Heyne," Jour. Ind. Bot. Soc., 1938, 17, Nos. 1 and 3,

161-68.

Lawson, A. A. .. "A contribution to the life-history of Bowenia" Trans. Roy.

Soc. Edinb., 1926, 54, 357.
Madge, M. A. P. . . " Spermatogenesis and fertilization in the cleistagamous flowers

of Viola odorata" Ann. Bot., 1929, 43, 545.
Mauritzon, J. ' . . " Studien tJber die embryologie der familien crassulaceae und

Saxifragaciae," Lund. Hakan Ohlssons Buchdrickeri, 1933.
Newman, L V. . . " Studies in the Australian Acacias, IV. The life-history 8 of

Acacia Baileyana, Part IT.," Proc. Linn. Soc. N.S. Wales,

1934, 59, 277.

Pace,L. .. "Fertilization in Cypripedium" Bot. Gaz*, 1907, 44, 353.

v " Pamassta and some allied genera," Bot. Gaz., 1912, 54, 306-29.

Raghavan, T. S. . . " Studies in the Capparidaceae, I. The life-history of Cleome

chelidonii Linn.," Jour. Linn. Soc. Lond., 1937, 51, 43-72.

Life-History ofV. viscosa, Roxb. & V. oldenlandioides, Roxb. 105

Raghavan, T. S.

- and Srinivasan, A. R.

and Srinivasan, V. K.

and Rangaswamy, K.

Rangaswamy, K.

Skovsted, A.
Souges, R.

Srinivasan, V. K.

Srivastava, G. D.
Tatachar, T.

Weinstein, A. I.
Wylie, R. B.

44 Morphological and Cytological Studies in the Capparidaceae,

II. Floral morphology and cytology of Gynandropsis penta-
phylla DC.," Ann. Bot. New Series, 1938, 2, 75-96.

44 Cyto-morphological features of Portulaca tuberosa Roxb.

Proc. Ind. Acad. ScL, 1941, 14, No. 5.
"Morphological and Cytological studies in the Scrophulariaceae,

III. A contribution to ^the life-history of Ilysanthes parvi
flora Benth.," Ibid., 1941, 13, 24-32.

" Studies in Rubiaceae, I. The development of female gameto-
phyte and embryo in Dentella repens Forst and Oldenlandla
alata Koch., and some cytotaxonomical considerations,"
Jour. Ind. Bot. Soc., 1941, 20.

44 Cyto-morphological studies in Asteracantha longifolia Nees.
(Hygrophila spinosa, T. And.)," Proc. Ind. Acad. Sci., 1941,
14, 149-65.

4t Tabulae Biologicae Periodicae," 1934, Band V-VI.

44 Recherches sur 1 ' embryogenie des Renounculacees Renoun-

culees (Genre Rananculus)", Bull. Soc. Bot. France, 1913,

62, 237 and 542.

44 Les premieres divisions deFoeuf et les differentiations du sus-

penseur chez le Capsella Bursapastoris" Ann. Sci. Nat. Bot'.,

1919, 10, Ser. Ipl.
44 Recherches sur Fembryogenie des Solanacees Nicotianees,"

Bull. Soc. Bot. France, 1922, 69, 163.
4 'Morphological and Cytological studies in the Scrophulariaceas ,

II. Floral morphology and embryology of Angelonia grandi-

flora, c. Morr. and related genera," Jour. Ind. Bot. Soc., 1940,

19, No. 4, 197-222.
44 Contribution to the morphology of Orobanche aegyptiaca

Pers.," Proc. Nat. Acad. Sc. India, 1939, 9, Pt. II, 58-68.
44 The development of the embryo-sac and formation of haus-

torium in Lantana indica, Roxb., and Stachytarphaeta indica,

Vahl.," Jour. Ind. Bot. Soc., 1940, 19, Nos. 1-3, 45-52.
44 Cytological studies in Phaseolus vulgar is" Amer. Jour. Bot.,

1926,13,248.
44 Sperms of Vallisneria spiralis" Bot. Gaz., 1923, 65, 191.

TRYPSIN-KINASE IN BLOOD

BY N. K. IYENGAR, M.Sc.

(From the Biochemical Standardization Laboratory, Government of India, Calcutta)

Received January 21, 1942
(Communicated by Prof. G. Sankaran)

ScHMiiz 1 has isolated a trypsin-inhibitor from beef blood and has shown
that it is probably a poiypeptide of low molecular weight. This inhibitor
exists in combination with trypsin in the circulating blood, thus inactivating
most of the enzyme. The author makes the statement that this trypsin-
inhibitor is present in blood in excess of the amount required for the complete
inactivation of trypsin present in blood. If this were the case, the acetone
precipitate of the plasma should not possess any proteolytic activity-
lyengar 2 and Scott have reported a definite, though small, tryptic activity
in the plasma proteins obtained by precipitation with acetone. This
however does not disprove the existence of the inhibitor, the presence of
which has been substantially confirmed by us in our experiments on the
proteolytic activity of various blood fractions. Trypsin circulating in
plasma is only partially neutralised by the inhibitor and a small portion
of the trypsin is left free to exert its activity. The significance of this
free plasma-trypsin in physiological and pathological conditions, is under
investigation.

The proteolytic system in blood has been the subject of extensive investi-
gations by Schmitz, who has experimentally demonstrated a combination of
trypsin with inhibitor to form a tryp sin-inhibitor compound which as such
is inert. The enzyme is liberated in an active form by treatment with
trichlor-acetic acid bringing the pH of the plasma to 3 and breaking the
compound into its constituents. If the plasma-trypsin is to play a role in the
physiological processes, there must be a mechanism in the body to bring
about such a breakdown of the trypsin-inhibitor 'compound. The pH of
the blood in vivo is at no lime as low as 3, and hence the mechanism of acti-
vation of plasma-trypsin in vivo if present must be other than by acidifica-
tion. Schmitz envisages the possibility of the presence in blood of a * kinase '
similar to the trypsin-kinase present in the intestinal secretions 'bringing
about the activation of trypsinogen of the pancreas. No direct evidence
has been adduced to support the above hypothesis. According to this author
106

Trypsin- Kinase in Blood 107

the ' kinase ' also combines with the trypsin-inhibitor present in the blood,
and when present in such a combination, it is not capable of liberating the
trypsin from the trypsin-mhibitor compound. Summing up the above
observations, it is seen that there is present in plasma a trypsin-inhibitor
compound and a kinase-inhibitor compound, both of which are unable to
exert their respective actions. It is presumed that the inhibitor is the same
chemically in both the compounds.

Pope 3 during the course of his work on the purification and
concentration of diptheria anti-toxin, has detected the presence of a
proteolytic enzyme in fibrin clots. Since this enzyme lyses the fibrin, Pope
named it fibrinolysin. The enzyme fibrinolysin is also reported to be pre-
sent in the culture medium in which Streptococcus is grown. Judging from
the properties of this enzyme, I am of opinion that the nomenclature, fibrino-
lysin, can be employed only to the enzyme which digests specifically the
fibrin protein, and does not attack other proteins. The proteolytic
enzyme present in the fibrin clot appears to resemble trypsin in its pH
optimum and in its action on other proteins. Trypsin, it may be stated, can
also digest the fibrin clot. I am of opinion, therefore, that the so-called
fibrinolysin reported by Pope is nothing but plasma-trypsin in its free and
active state.

Schmitz has put forward a very interesting hypothesis to explain the
presence of trypsin in the fibrin clot.

According to Schrnitz, during clotting, the trypsin-inhibitor compound
is adsorbed on the fibrin clot. The kinase-inhibitor compound which is
also present in plasma, is broken up into its constituents with the result that
only kinase which is probably of a protein nature gets adsorbed on the clot
while the inhibitor which is a polypeptide of low molecular weight remains
in the serum. The components of the proteolytic system present in the
fibrin clot are : (i) trypsin-inhibitor compound and (ii) trypsin-kinase.
Under these circumstances, it is reasonable to visualise an interaction of the
two components if the fibrin is suspended in a buffer suitable for kinase
action. This reaction can be represented by the following equation:

Trypsin-inhibitor compound + Kinase z? trypsin + Kinase-inhibitor.

It can be seen from this equation that trypsin is liberated in an active
form during the process of clotting by the mechanism described above. This
fascinating possibility is based on the assumption that the trypsin-kinase is
present in the blood, for which no direct evidence has been presented as yet.
As I was engaged in a fairly detailed study of the proteolytic system present
in the whole blood, it occurred to me that it might be worth while to

B3 ?

103 N. K. lyengar

investigate the various blood fractions for the presence of this kinase.
In pursuance of this idea, the following constituents of blood have been
examined :

(i) Red Blood Corpuscles,
(ii) Platelets.

Experimental

(1) Red blood cells from horse were laked with distilled water and
precipitated with 4 volumes of acetone at a temperature of 10 C. ? centri-
fuged and washed with acetone.

(2) Citrated horse blood was kept in the refrigerator and red blood cells
were allowed to settle. The plasma was then taken off and centrifuged at the
rate of 3,000 revolutions per minute. The sediment was taken in a jar and
allowed to settle in the refrigerator. The supernatant was siphoned off and
the sediment resuspended in citrates; this suspension was precipitated with
4 volumes of acetone in the cold, centrifuged and the precipitate again washed
with acetone. The precipitate was finally dried at room temperature.
This is the platelet preparation,

(3) Trypsin-inhibitor Compound. This is present in plasma as a protein
and hence is precipitated by acetone. 100 c.c. of citrated plasma were
precipitated by the addition of 400 c.c. of acetone. The precipitate was
first washed with aqueous 90% acetone, then with pure acetone and finally
dried at room temperature. This preparation has been shown by lyengar
and Scott, to possess slight proteolytic activity.

(4) Trypsin from Plasma freed from the Inhibitor. 100 c.c. of citrated
plasma were precipitated with 11 volumes of 2-5% trichlor-acetic acid. The
precipitate was centrifuged thoroughly freed from the residual nitrogen by
washing with 2 5% trichlor-acetic acid. The still moist precipitate is dis-
solved in 500 c.c. of water and this solution was mixed with two litres of
acetone. Upon the addition of a small amount of sodium acetate solution,
the plasma was again precipitated and now washed with acetone. This
precipitate was dried at room temperature. This preparation has a greater
proteolytic activity than preparation 3, sine i the trypsin-inhibitor compound
has been broken up by treatment with trichlor-acetic acid.

1 gm. of each of the above preparations were suspended in 20 c.c. of
M/15 phosphate buffer of pH 8-4 and incubated for 18 hours at 37 -5 C.
The increase in non-protein nitrogen in each case was as follows;

Trypsin-Kinase in Blood
TABLE I

109

Red Blood Cells

Platelets

Trypsin-inhibitor
Compound

Plasma-Trypsin
freed from the
Inhibitor

*ng.

2-2

mg.
9-48

mg.
1-57

mg.
3-51

If the kinase is present in red blood cells, it should be able to liberate
the trypsin from the trypsin-inhibitor compound when incubated with the
latter in M/15 phosphate buffer of pH 8-4. The following experiments were
carried out to ascertain the presence of kinase in red blood cells.

1 gm. of the red blood cells was mixed with Igm. of the trypsin-inhibitor
compound preparation, the mixture suspended in 20 c.c. of M/15 phos-
phate buffer of pH 8-4 and incubated for 18 hours at 37-5 C. In another
experiment 1 gm. of red blood cells was mixed with 1 gm. of the plasma-
trypsin preparation freed from the inhibitor, the mixture also suspended in
20 c.c. of M/15 phosphate buffer of pH 8-4 and incubated for 18 hours at
37 -5 C. The increase in non-protein nitrogen in each case was as follows:

TABLE II

Increase in

N.P.N.

1. Red Blood Cells

2. Trypsin-inhibitor Compound

3. Red Blood Cells and Trypsin-inhibitor

Compound preparation

4. Plasma-Trypsin preparation freed from the

inhibitor

5. Red Blood Cells plus Plasma-Trypsin freed

from the inhibitor

mg.
2-2
1-57

3-9
3-51
5-95

If kinase was present in red blood cells, the non-protein nitrogen in
the case of (3) should be approximately the same as in (5), since the latter
represents the combined proteolytic activity of red blood cells and the
plasma-trypsin freed from the inhibitor by treatment with trichlor-acetic
acid. The increase in non-protein nitrogen in (3) is 3-9mg. which is
approximately equal to the increase in N.P.N. of (1) .and (2) added together.
The above results definitely show that trypsin-kinase is not present in red
blood cells.

Attention was then directed to the platelet preparation. Similar experi-
ments as in Table II were conducted with the only difference that in place of

110 N. K. lyengar

red blood cells, the platelet preparation was employed. The results of

these experiments are ;

TABLE III

Increase in

N.P.N

mg.

1. Platelet preparation .. .. .. 9-48

2. Trypsin-inhibitor compound preparation .. 1-57

3. Platelet preparation plus Trypsin-inhibitor

compound preparation .. .. 13-05

4. Plasma-Trypsin preparation freed from the

inhibitor .. .. . . .. 3-51

5. Platelet preparation plus Plasma-Trypsin

preparation freed from the inhibitor .. 13-65

The results reported above lend direct experimental evidence for the
presence of trypsin-kinase in platelets. The proteolytic activity of (3) is the
same as (5) within the limits of experimental error. If kinase was not
present in the platelets, the proteolytic activity of (3) should have been
represented by an N.P.N. increase of (l)+(2) (i.e., 9-48+ 1-57= 11-05
mg.) whereas the actual increase is 13-05mg. The increased tryptic
activity is due to the extra amount of trypsin released from the associate
inhibitor by the kinase that might be present in platelets. The fact that the
proteolytic activity of (3) is approximately the same as in (5), further shows
that, the trypsin-inhibitor compound is practically completely broken up by
the platelets.

It may be argued that the increased tryptic activity of (3) may be due to
the addition of an increased amount of protein, since the trypsin-inhibitor
compound added to the platelet preparation, is merely plasma proteins
directly precipitated by acetone. Such a possibility does not however exist,
because the platelet preparation itself contains a very large proportion of
substrate compared to the quantity of associated trypsin, and the percentage
protein broken up by auto-digestion is only 6%. Besides, the enzyme being
simultaneoulsy precipitated with the platelet proteins by acetone, the
trypsin is already adsorbed on the platelet proteins and will therefore have a
preference to digest the associated protein which is present in plenty beyond
the capacity of the enzyme present to digest. lyengar (I.J.M.R., July 1941)
has shown that, if to an enzyme preparation containing a large quantity of a
susceptible protein substrate another protein which is less susceptible is
added, the latter remains practically unattacked. When insulin was added
to a platelet preparation (obtained by acetone precipitation) and incubated
for a period of 24 hours, the hormone remained practically intact which was
demonstrated by the complete retention of its physiological activity. The

Trypsin-Kinase in Blood 111

plasma proteins obtained by acetone precipitation if incubated in M/15
phosphate buffer undergoes auto-digestion to the extent of only 1%. So, in
this case also, the associated trypsin has a plentiful supply of substrate
already and the addition of the platelet preparation should not make any
difference so far as the tryptic activity of the plasma proteins are concerned.
Therefore if the tryptic activity of the mixture of platelets and plasma
proteins (obtained by acetone precipitation) is significantly more than the
added value, the only possibility is that some intei action between (he two
has taken place giving effect to this increased activity. This interaction is
between, kinase probably present in platelets and the trypsin-inhibitor
compound known to be present in acetone precipitated plasma proteins as
hypothelically visualised by Schmilz. It maybe stated that the above results
may not be direct convincing evidence for the presence of kinase in platelets,
since the kinase has not been isolated from the platelet-proteins and the
associated trypsin, hut the results reported in. this paper lend strong experi-
mental support for the presence of kinase in blood.

Summary

. A clear picture of the proteolytie system existing in blood has been
presented.

The possibility of the presence of a trypsin-kinase in blood has been
discussed.

The red blood cells and the platelets have been examined for the pre-
sence of trypsin-kinase.

Red blood cells do not contain the kinase.

The experiments reported in. this paper strongly suggest the presence
of trypsin-kinase in platelets, which is capable of liberating the trypsin from
the inhibitor compound present in acetone precipitated plasma proteins.

BIBLIOGRAPHY

1. Schmitz .. Zeit.f.PhysioLChem., 1937,250,37.

2. lycngar, N. Kn and Scott, Tram. Roy, Sac. Canada, 1940, Sec, V, 34, 45.

D.A.

3. Pope, B. J. . . Expt. Pathol., 1939, 20, 2, 132.

4. lyengar, N. K. . . Indian Journal of Medical Research, July 1941.

ANTI TRYPTIC COMPONENTS OF BLOOD
BY N. K. IYENGAR, M.Sc.

(From the Biochemical Standardization Laboratory, Government of India, Calcutta)

Received January 13, 1942
(Communicated by Prof. G. Sankaran)

IT is well known that the blood serum has the property of retarding the
digestive activity of trypsin. Landsteiner 1 reported that this effect was
associated with the albumin fraction of serum. Hedin 2 ' 3 found many
similarities between the inhibition of trypsin by adsorption on charcoal and
inhibition of trypsin by serum albumin and therefore came to the conclu-
sion that the equilibrium between trypsin and serum albumin was governed
by adsorption phenomena, i.e., the trypsin is adsorbed by the serum albumin
as it is by the charcoal. The correctness of this interpretation is questioned by
Hussey and Northrop 4 after a detailed study of the mechanism. They have
adduced evidence which suggests that the inhibitive agent in serum combines
with trypsin to form an inactive but dissociable compound. The conditions
of equilibrium are apparently goverend by the law of Mass-Action.

It has been shown by Schmitz 5 that the enzyme trypsin present in plasma
is blocked by an inhibition body, whose action is in no way related to the
anti-tryptic action of serum albumins. This inhibitor could be separated
from the serum albumins by precipitation of proteins with trichlor-acetic
acid whereby the inhibitor remains in solution. This can also be prepared
by ultra-filtration of an acid solution of plasma proteins obtained by acetone
precipitation. By this process, the inhibitor passes into the albumin-free
filtrate. This inhibitor is thus strongly analogous to the trypsin- inhibitor
obtained by Kunitz and Northrop 6 from the pancreas. The reaction between
Schmitz plasma-inhibitor and crystalline trypsin shows that it runs quanti-
tatively as in the case of Northrop-inhibitor from the pancreas. The one
important difference between the two inhibitors is that plasma-inhibitor
does not inactivate chymo-trypsin from the pancreas a opposed to the pan-
creas-inhibitor which reacts with chymo-trypsin bringing about a gradual
fall in activity. The inhibitor associated with the serum albumin also
inactivates chyrno-trypsin and hence is similar only iu this property to the
inhibitor from pancreas. The inhibitor present in native serum is however
differentiated from either the inhibitor from pancreas or the inhibitor
isolated from plasma by Schmitz. According to Hedin 7 the inhibition of
trypsin appears only when ferment and serum are mixed first and after an
interval the substrate is added. On the other hand, the other two inhibitors

112

Anti Tryp.tic Components of Blood 113

act only if they are added to the ferment substrate mixture when the diges-
tion is in progress. The destruction of trypsin by serum (albumin) is there-
fore sharply differentiated from the inhibition by the substance isolated
from, blood plasma. In blood plasma, there are therefore two inhibition
mechanisms side by side, of which one is probably (according to Schmitz) by
unspecific adsorption of the ferment on serum albumin and difficulty of
elution and the other by a quantitative combination with the ferment speci-
fically. In order to detect the presence of the second inhibitor, either free
inhibitor should be present in plasma or it should be separated from the
trypsininhibitor compound.

During the course of a search for a simple substance which might retard
the activity of specific proteases, it was discovered by Horwitt 8 that heparin,
the physiological anticoagulant is a trypsin-inhibitor. The effects of heparin
on the hydrolysis of casein by crystalline trypsin and chyino-trypsin isolated
by beef pancieas, have been studied. No inhibition, of trypsin is obtained
unless the heparin is allowed to remain in contact with the alkaline trypsin
solution for about 30 minutes before .the two are added to casein. This
inhibition of trypsin. by heparin has many similarities with the mechanism
of inhibition of trypsin by the inhibitor isolated from plasma. The heparin-
trypsin addition complex like the trypsin-inhibitor compound may be dis-
sociated by acidifying to pH 3 for about 30 minutes to recover all the
tryptic activity. Like the plasma-inhibitor, heparin does not inhibit chymo-
trypsin. This property of heparin is very interesting since trypsin catalyses
the clotting of recalcified plasma while chymotrypsin does not. It has
been reported by Horwitt that trypsin and heparin are mutually antagonistic
in a sample of plasma and that clotting will not occur unless the amount
of trypsin added is more than enough to neutralise the effect of heparin.
It is evident from the foregoing account that there are present in blood,
three different substances having the property of retarding the activity of
trypsin. The presence of these substances acquires an added significance
in the light of my (lyengar, N. K.) 9 observations on the presence of trypsin in
plasma and its possible role as the physiological thrombo-plastic substance.

The part played by trypsin in blood in the inactivation of insulin has
been discussed by lyengar and Scott. 10 In view of this dual role of trypsin
in blood, the action of the anti-tryptic components of blood in retarding
coagulation or in prolonging the hypoglyceimic effect of insulin is worth
investigating. The olject of this paper is to study first of all the in vitro
effect of these trypsin-inhibitors on the action of trypsin in (i) catalysing
the coagulation of blood and (ii) in the destruction of insulin. The action
of heparin as a general anti-coagulant is well known and therefore need not

N. K. lyengar

be considered. The action of heparin in checking the destruction of insulin
bv irypsin has not been reported, although the possibility is indicated by
Horwitt (Science, 1940). Long before Hewitt's' work was published, while
I was working in the Cohnaught Laboratories on the destruction of insulin
in blood, the way in which heparin would behave in a reaction between
trypsin ind Insulin, attracted my interest, firstly, because the work on
heparin was going on in the laboratory and secondly because Ferguson 11
has shown that different amounts of heparin can inhibit clotting of citrated
dog plasma by crystalline trypsin under varying conditions of calcium
and cephalin mobilization. A study of the action of the Schmitz-inhibitor
on the coagulation of blood plasma by trypsin, would be exceedingly inte-
resting. The effect of adding this inhibitor to a mixture of insulin and trypsin
on the course of the destruction of the hormone has also been studied.

Materials and Methods

Preparation of the trypsin-inhibitor from plasma (according to Schmitz).
5 Litres of oxalated blood after keeping overnight in a refrigerator are mixed
with 15 litres of 0-25NH 2 SO 4 to dissociate ferment inhibitor compound.
The dark coloured solution is kept overnight on ice. 242 gm. of (NH 4 ) 2 SOi
per litre (40% saturation) are then added. The total albumins are
precipitated. The precipitate is filtered and thrown away. To this yellow
coloured filtrate, 205 gm. of (NH 4 ) 2 S0 4 per litre are added. When this
is kept for a few days in an ice-chest a fine flocculent precipitate separates.
It is filtered under suction on a hardened filter-paper. The precipitate which
is very small in quantity is dissolved in a few c.c. water (6 c.c.), the turbid
solution is naked with 4 c.c. saturated (NHJoSO* solution and filtered. The
filtrate is salted out by the addition of 2 gm. of (NH 4 ) 2 SO 4 per 10 c.c.
The white precipitate is filtered by suction on small hardened filer-paper.
The, precipitate is dissolved in 5 c.c. water and the solution is mixed with
equal volume of 5% trichlor-acetic acid. This is kept for 30 minutes and the
precipitate formed during this period is filtered and thrown away. The
clear filtrate is brought to pH 3-0 by the addition of a few drops of 5 N
NaOH. The inhibition body is then salted out by the addition of 5-6 gm.
ammonium sulphate per 10 c.c. The precipitate is filtered through a hard-
ened filter-paper. A fairly good quantity of the inhibitor (about 100 rag.)
was obtained by undertaking the preparation in four different batches.

Method of insulin assay employed. The mouse convulsion method was
employed. Since we were concerned only with comparative effects, a large
number of animals were not considered necessary for the test. On an
average, 50 mice were used, 25 for test solution and 25 for standard.

Ant i Tryptic Components of Blood

115

Method of study of the prolongation of hypoglyceimlc effect. These
tests were made by studying the effect of the test solution on the blood sugar
of rabbits and comparing with the blood sugar reducing effect of a similar
dose of standard insulin at the ead of the same period. The mean blood
sugar figure for each hour is calculated both for rabbits injected with the
standard and for rabbits injected with test solution. Each figure is then
converted to a percentage of the mean initial figure. The point at which
the blood sugar has returned to normal in the case of rabbits injected with
standard insulin is taken. At this point the blood sugar of rabbits receiving
the test solution is noted. The percentage of this value to the initial blood
sugar gives an idea of the prolongation of the hypoglyceimic effect when
compared with standard insulin.

The effect of the addition of the inhibitor isolated from plasma, on the
coagulation of citrated plasma by trypsin, has been studied first. In order
to be able to study this, it is imperative to determine the optimum concen-
tration of trypsin required to bring about coagulation in the shortest time.
The importance of this has been pointed out by Eagle and Harris 12 who
have determined such an optimum concentration using Digestive Ferment
Company's trypsin 1: 110 and in the absence of calcium. Ferguson and
Erickson" have shown that the enzyme trypsin is much more potent in the
presence of ionised calcium. As it was intended to study the effect of the
trypsin-inhibitor on the coagulation of citrated plasma by tryspin in the
presence of calcium, it was considered of interest to find out the optimum
concentration of the trypsin employed, (B.D.H.) that is necessajry to bring
about coagulation of citrated plasma in the presence of calcium. Accordingly
the following experiments were undertaken :

TABLE I

Coagulation time of 1 c.r, plasma -varying quantities of trypsin solution

(B.D.H. trypsin 10% in salt solution)

Quantity
citrated
plasma

Quantity of
CaC! t N/10

Quantity of
trypsin solution

Clotting
time "

C.C.

c.c.
0-25
0-25
0-25
0-25
0-25

c.c.

0-75
0-60
0-50
0-40
0-30

sees.

32
19
35
80
95

The total volume was made up to 2 c.c. in each case.

116 N. K. lyengar

It is evident from the above table that 0-6-c.c. of a 10% solution of
the trypsin used, coagulates 1 c.c. citrated plasma in the shortest interval
of 19 seconds in the presence of CaCl 2 . Having thus determined the opti-
mum amount of trypsin, we proceeded to study the effect of the Schrmtz-
inhibitor, isolated from plasma on the above process.

TABLE II

B.D.H. Trypsin 10% solution in 0-85% Nad. 5 rug. of the
tryps'in-inhibitor dissolved in 10 c.c. of normal saline

Citrate plasma

CaCl 2 N/10

Trypsin

inhibitor solution

Clotting time

C.C.

c.c.

sees.

0-25

0*6

0-25

0-6

0-3

0-25

0-6

0-5

0-25

0-6

0-65

0-25

The volume was made up in each case to 2 5 c.c.

In the above experiments, the solutions were mixed in the following
order : Plasma Trypsifi Inhibitor Calcium.

There was thus no interval allowed for the combination of trypsin and
the inhibitor. The inhibition brought about in the above experiments does
not appear to be complete. The maximum inhibition that has taken place
(column 4) is still far from complete as can be seen from the blank experi-
ment carried out with neither trypsin nor inhibitor. As it is known that the
inhibition of the trypsin brought about by this inhibitor is not instantaneous,
and a certain amount of time is required to effectively block the enzyme,
it was decided to incubate the enzyme solution and the inhibitor solution
for a period of 1 hour at 30 C. This incubated mixture was used in the
following experiments.

TABLE III

Plasma CaCl 2 N/10

Trypsin -f Inhibitor
solution (a)
(5 c.c. Trypsin + 5 c.c. Jnhib

Trypsin
(10% Sol.)
itor sol. containing

Clotting time
2-5 mg. inhibitor)

c.c. ! c.c.
1 | 0-25
I ! 0-25
1 i 0-25

c.c.
i-2

c.c.
6 : 6

sees.
95
22
70

0-25

Trypsin -{-Inhibitor
solution (b)

1-2

(5 c.c. Trypsin-f-5 c.c. Inhibitor sol.
containing 5 mg.)

Trypsin -{-Inhibitor
solution (c)

(5 c.c. Trypsin +5 c.c. Inhibitor sol.
containing 10 mg. )

0-25

1*2

Aiiti Try pile Components of Blood

When the inhibitor is incubated with trypsin before use, the blocking
of the effect of trypsin on coagulation is more effective. The amount of* the
inhibitor required to check the trypsin completely can be calculated from
solution (b). 0*6 mg. will prevent practically completely, 0-6c.c. of I0" t ,
B.D.H. trypsin fiom exerting its catalysing effect on the coagulation of
citrated plasma. It is therefore clear that the plasma-inhibitor lias eot the
property of inhibiting the coagulation action of trypsin. Ferguson 11 has
reported that heparin can inhibit clotting of citrated dog plasma by crys-
talline trypsin. The plasma-inhibitor can therefore be considered analogous
to heparin in this respect.

The close analogy of thrombo-plastin and trypsin has been observed
by Ferguson and fresh evidence has been adduced in support of this t"v
lyengar in his work on plasma-trypsin and prothrombin. In view of this
similarity of the behaviour of thromo-plastin and trypsin in the process
of coagulation, and in the light of the above results, the question whether
the plasma-inhibitor can inhibit the thrombo-plastie action of the Russcl
viper venom, suggested itself to the author,

The following experiments were carried out to test this possibility: -

TABLE IV

Oxaiatcd plasma

c,c.

0-2

0-2
0-2

Russcl Viper Venom

\ CaCIj.N/40 Clotting time

I in 20,000

ujiscl Viper Venom I in 20*000 plus
inhibitor (5c.c. Venom soL p/wv

2mg. inhibitor) kept for one hour
at 25" C.

0*2 c,c.
nil

c.c.

0-2

0-2
0-2

15
80

it is evident from the above table that the plasma-inhibitor cannot
prevent the thrombo-plastic activity of Rtissel viper venom. In this respect,
the similarity between trypsin and thrombo-plastin breaks down.

The destruction of insulin by blood has been investigated by a large
number of workers (Schmidtz, 14 Karelitz, 13 Fraudcnbcrg 16 and Black 17 ).
The destructive principle has been shown to be a proteolytic en/ymc of a
tryptic nature (lyengar and Scott 10 ). The presence of the anti-tryptic
agents in blood assumes an added significance in the light of this properly
of blood. Advantage of the anti-tryptic effect of blood serum has been
taken by Murlin and Hawley 18 to protect the insulin from destruction by
trypsin and they claim that the hormone can be absorbed from alimentary
tract of depancretized dogs, Harncd and Nash 1 * have reported that when

P 4

118

to. 1C. lyengar 1

insulin is mixed with anti-trypsin prepared from the round Worm of swine
(Ascaris lumbricoides\ and given by stomach tube, it causes a marked
decrease in sugar output. These authors have also been able to demon-
strate that the physiological activity of insulin can be protected from
destruction by trypsin, if the enzyme has been previously incubated with this
anti-trypsin.

The possible prolongation of insulin action by inhibitors of proteolytic
activity has not been studied by any of the above workers, but is merely
mentioned by Horwitt in his work on heparin as an anti-tryptic agent.
Since the inhibitor we are working with occurs in blood, it would be exceed-
ngly interesting to study the (1) in vitro effect of a mixture of trypsin and
inhibitor on the course of the destruction of insulin, and (2) the in vivo
effect of injecting into rabbits a mixture of insulin and the inhibitor and
observe the prolongation if any, of the hypoglyc^mic effect as compared
with standard insulin of an identical dosage. The following experiments
have therefore been conducted.

TABLE V

Inhibition of the tryptic digestion of Insulin by plasma-inhibitor
The solutions were incubated for 2 hrs. at 38 C.
The pH of the solutions were maintained at
pH 8 by the addition of Phosphate buffer

Increase in

Destruction

N.P.N

of Insulin

mg.

Protein

Activity

Trypsin -f Insulin (5 mg. or 1 10 units)

0-32

Trypsin + Inhibitor (3 mg.) incubated

for 1 hr. and then Insulin (5 mg.) added . .

0-08

The influence of the inhibitor on the course of the destruction of physio-
logical activity of insulin and also on the digestion of the insulin protein
has been studied in the above table. The digestion of the insulin protein
is only 10% in the presence of the inhibitor whereas 40% digestion takes
place if the trypsin is not previously incubated with the inhibitor. While
there is a definite inhibition of the tryptic destruction of the physiological
activity of insulin, the inhibitor has not been able to prevent it completely.
It is generally recognised that a large part of the activity of insulin is
destroyed during the early stages of diges-ion of insulin protein. The fact
that 40% of the activity is destroyed even in the presence of the inhibitor
shows that the plasma-inhibitor is not capable of blocking the earliest stages
of tryptic action. Nevertheless the results obtained clearly show that the
checks the tryptic digestion of insulin protein.

Anti Tryptic Components of Blood

Having thus established that the plasma-inhibitor can partially block
the tryptic destruction of insulin, the possibility of the practical applications
of this finding was next investigated. If the hypoglyczemic effect of insulin
could be prolonged by the simultaneous injection of the inhibitor, it would
be of great practical utility in the treatment of diabetic subjects. The
amount of the inhibitor circulating in the blood is not enough to block all
the trypsin present in blood. In addition the trypsin present in platelets
(lyengar and Scott) is released into the blood. If therefore an additional
amount of the inhibitor is administered with insulin, the hormone may not
all be destroyed so quickly as it normally happens. The method of study of
the prolongation of the hypoglycsemic effect has already been described.

TABLE VI

Effect of Plasma-inhibitor in the prolongation of the
Hypoglyccemic effect of Insulin

Expt.

No.

Blood sugar 6 hrs.
after injection
expressed as percentage
of the original
blood sugar level

1 1 Unit Insulin injected
21,,
31,,
41,, + 2 mgs. Inhibitor
51,*, + 4
61,, + 5

100
90
94
95
100
90

There is practically no prolongation of the kypolgycawnic effect since
in all cases with varying amounts of the inhibitor, the blood sugar has been
restored to the original level within a period of 6 hours after the injection
of the mixture.

Similar experiments were conducted using heparin in place of the inhi-
bitor, in view of similarity of its behaviour towards trypsin.

TABLE VII
Inhibition of tryptic d'gestion of Insulin by Heparin

Approximate

Percentage

percentage of

Solutions were incubated for

Increase in

of destruction

the destruction of

2 hrs. at 37 C

N.P.N .

of Insulin

physiological

Protein

activity

mg.

Trypsin -f- Insulin (5 mg.)

0-30

Trypsin ~f Heparin (3 mg.) incubated for
1 hr. at 37 C. and then Insulin (5 mg.)

added

0-18

120

N. K. fyengaf

TABLE VIII

Study of the effect of Heparin in the prolongation of the
hypoglyccemic effect of Insulin

Blood sugar 6hrs.

Expt
No.

after injection
expressed as percentage
, of the original

blood sugar level

1 Unit Insulin . . . .

100

1 ,

1 , ,+2 mg. Heparin

1 -f-4

1 > r ^ >

1 , , +6

Heparin is also not able to prolong the hypoglycaemic effect of insulin.
Although the trypsin-inhibitors are able to check the in vitro destruction of
insulin by trypsin, they are practically useless in slowing down the process
of physiological destruction of insulin in the body. This is probably due to
the fact that the trypsin present in blood or generated in the blood comes
into contact with the insulin first and the destructive process starts before
the enzyme can combine with the added inhibitor to form the inactive trypsin-
inhibitor compound. If the substrate and the inhibitor are allowed to come
into contact with the enzyme simultaneously, the competition between the
two for affinity with the enzyme comes into play. Irf this process of compe-
tition, the substrate gets the upper hand. If on the other hand the inhibitor
alone is added to the tryspin and incubated for some time an inactive trypsin-
inhibitor compound is formed.

Crafford and Jorpes 20 observed that a larger doze of heparin is rendered
inactive in the blood shortly after a surgical operation than is the case with
the same patient before the operation. This is regarded by them as a clear
expression of the tendency to the formation of clots which cause thrombo-
embolic complications post-operatively.

The observation that trypsin and heparin are mutually antagonistic
and that heparin inactivates trypsin both in its digesting and coagulating
action, can be linked up with the* finding of Crafford and Jorpes. So far
the only known atiheparin agents present in the blood are prothrombln
and trypsin. During the early stages of the post-operative period prothrom-
bin is known to decrease if at all any change takes place in the prothrombin
content of blood. The changes in the trypsin content of plasma have not
been studied. The following table gives the tryspin content of plasma both
immediately before and after the operation.

An ft Try p tic Components of Blood
TABLE IX

121

Nature of
operation

Plasma-trypsin before
operation expressed as
increase in N.P.N for

After operation

100 c.c. plasma

Obstructive

mg.
22-5

mg
35-8

Jaundice

18-4

31-9

24-8

41-2

There is a significant increase in plasma-trypsin immediately after an opera-
tion. The increased inactivation of heparin under this condition may
reasonably be ascribed to the trypsin content of plasma.

Summary and Conclusions

A review of the anti-tryptic components of blood has been made. There
are present in blood three different substances capable of inactivating
trypsin. There are (1) A factor in serum associated with the albumins.
(2) An inhibitor isolated from plasma which according to Schmitz is a poly-
peptoid of low molecular weight. (3) Heparin, the physiological anti-co-
agulant.

The in vitro effect of the plasma-inhibitor on the action of trypsin in
catalysing the coagulation of blood has been studied in detail. This sub-
stance has been found to inhibit this property of trypsin also.

The course of tryptic digestion of insulin in the presence of this inhi-
bitor and heparin has been studied both by following the increase in N.P.N.
and also the physiological activity of insulin. The digestion of the insu in
protein is not completely checked although a definite inhibition is observed.
Since the digestion of insulin takes place even in presence of the inhibitor,
or heparin, quite a considerable amount of physiological activity of insulin
is destroyed by trypsin even in the presence of the inhibitor, or heparin. This
destruction is however very much less than the inactivation of insulin by
trypsin under identical conditions but without inhibitor or heparin. The
plausible reasons for this observation have been enumerated.

In view of the close analogy between trypsin and thrombo-plastin in
their behaviour towards the process of coagulation, the action of the
plasma-inhibitor on the thrombo-plastic activity of Russel Viper Venom
was studied. The thtombo-plastin of the venom remains quite active even
after incubation with the inhibitor. In this respect therefore the analogy
between trypsin and thrombo-plastin breaks down,

122

N. K. lyengar

Attempts have been made to ascertain whether prolongation of the
hypoglycasmic effect of insulin can be obtained by injecting a mixture of
insuLn with the plasma-inhibitor or heparin. A large number of experi-
ments have been carried out to test this important practical application.
The results obtained are not very encouraging and there is practically no
prolongation of the effect either with the inhibitor or with heparin.

The trypsin content of plasma have been estimated both before and
after operation in a number of cases. A significant increase in the enzyme
content of the plasma has been observed immediately after operation. It
is suggested that this high trypsin content might be responsible for the
increased inactivatioa of administered heparin, observed by Crafford and
Jorpes.

Acknowledgment

The author's thanks are due to Sir R. N. Chopra, C.I.E., I.M.S. (R) and
Dr. B. Mokerji, M.D., D.SC., for their kind interest in this invesdgaiion.

1. Landsteiner

2. Hedin

4. Hussey and Northrop

5. Schmitz s

6. Kunitz and Northrop

7. Hedin

8. Horwitt

9. lyengar, N. K.

- and Scott

10. -

11. Ferguson

12. Eagle and Harris

13. Ferguson and Erickson

14. Schmidtz, et al.

15. Karelitz

16. Fraudenberg

17. Black

18. Murlin and Hawley

19. Harned and Nash

20. Crafford and Jorpes

BIBLIOGRAPHY

. . Centr. Bakt. ite abst., 1900, 27, 357.

. . /. Physiol., 1904-05, 32, 390.

.. Biochem.J., 1906,1,485.

. . /. Gen. Physiol, 1922-23, 5, 335.

.. Zt-schrift Physiol Chemie, 1937, 250, 37;

1938, 255, 234.

. . /. Gen. Physiol, 1936, 19, 991 .
. . " Grundzuge der Physik Chemie " in ihrer

Bezeihung Zur Biologic, Munchen, 1924.
. . Science, 1940, 92, 89.

. . Sent for publication. Proc. Ind. Acad. ScL,
1942.

. . Trans. Roy. Soc. Canada, Sec. V, 1940, 34, 45.

. . Proc. Soc. Exptal Biol. and Med., 1939, 42,

33-37. ,

. . J. Gen. Physiol., 1936-37, 20, 543.

. . Am. J. Physiol, 1939, 126, 661.

. . Ckem. Abst., 1930, 24, 4859.

. . Arch. Int. med., 1930, 45, 546.

. . Zt. f. Physiol Chemie, 1931, 202, 159. '

. . Bt. J. Ext. Pathol, 1933, 14, 318.

. . Am. J. Physiol, 1927-28, 83, 147.

.. J.B.C., 1932,97,443.

,. J.A.M.A., 1941, 116,2831,

PROTHROMBIN AND PLASMA TRYPSIN

BY N. K. IYENGAR, M.Sc.

(From the Biochemical Standardization Laboratory, Government of India, Calcutta)

Received January 13, 1942
(Communicated by Prof. G. Sankaran)

IT is now generally recognized that the coagulation of blood fibrinogen is
brought about by thrombin produced from plasma prothrombin by the
action of thrombo-plastin and calcium. Prothrombin is present in the blood
stream in a soluble and inactive form. The exact nature of prothrombin
has not been elucidated, but its preparations contain globulin protein which
may be precipitated by dilution and acidification, or removed from plasma by
colloidal adsorption. Like other plasma proteins, prothrombin is formed
in the liver but with the help of vitamin K.

Mellanby 1 has prepared highly active prothrombin from beef blood and
described its properties. It has been found to give well marked protein
reactions. Being a protein in nature, it would be naturally of great interest
to study the action of proteolytic enzymes on prothrombin. Mellanby
however does not appear to have conducted any experiments to test the
action of proteolytic enzymes, since he found that prothrombin is rapidly
destroyed at 38 C. in concentrations of acid and alkali in which pepsin and
trypsin act.

Pope 2 has demonstrated the action of pepsin and trypsin on plasma
proteins at a pH, away from the optima of the action of these enzymes. The
enzymic degradation of the proteins may not take place but there is a possi-
bility of the disaggregation which can be demonstrated by a change in the
properties such as critical denaturation temperatures, etc. In the case of
prothrombin, since it is shown that its biological activity is very labile, any
slight action on the prothrombin protein may be reflected in its biological
activity. The biological activity referred to is its capacity to form
thrombin, in the presence of calcium and thrombo-kinase.

Douglas and Co-ebrook 3 reported that blood coagulation was accelerated
by the addition of trypsin. Wall-Schmidt-Leitz 4 ' 5 and his co-workers also
reported that trypsin accelerated blood coagulation. They came to the
conclusion that thrombin was a proteolytic enzyme either identical with, or
related to, trypsin and that coagulation was due to the enzymic hydrolysis
of fibrinogen to an insoluble modification. Tryspin is considered to have

123

124 N. K. lyengar

accelerated coagulation in so far as it hastened this hydrolysis. Other
proteolytic enzymes like papain were found to be active. Eagle and Harris 6
have made a detailed study of the action of trypsin on blood coagulation, in
order to throw some light on the mechanism of physiological coagulation.
Trypsin does not coagulate fibrinogen but apparently reacts with plasma
prothrombin to form the physiological coagulant thrombin. Papain
directly acts on fibrinogen to form an insoluble modification resembling
fibriil. It appears therefore that trypsin acts as a thrombo-plastic enzyme
in assisting blood-clotting. The possibility of trypsin being a thrombin can
be discounted in view of the complete inability of the former to clot fibri-
nogen meticulously free from prothrombin (Ferguson 7 ) even when calcium
and cephalin are also added. The possible role of trypsin in bringing about
the activation of prothrombin solutions has been brilliantly discussed by
Ferguson. 8 Prothrombin solutions used in his experiments were found to
contain 8 to 30 mg. per cent, of phospholipid. This amount of phospho-
lipid if supplied in the form of added cephalin solution would be powerfully
thrombo-plastic, while prothrombin solutions containing phospholipid in
firm combination with proteins require the mediation of trypsin for the
formation of thrombin. It is therefore concluded by Ferguson that trypsin
splits off cephalin from its inert protein combinations and make it ' available *
for the activation of prothrombin. In the words of Ferguson, the action of
trypsin consists in the " mobilization of cephalin and calcium at the colloidal
surface of the protein (prothrombin) substrate where the close juxta-posi-
tion of the three components permit of the formation of thrombin via an
intermediary prothrombin-calcium-cephalin complex or compound ".
According to this concept, the tryptic action is brought into line with the
classical processes of thrombin formation. Schmitz 16 and lyengar and
Scott 11 have demonstrated the presence of trypsin in blood plasma. One can
therefore consider plasma-trypsin as part of the normal physiological clotting
mechanisms. '*

A study of the action of plasma-trypsin on prothrombin at pH 7-2, the
physiological pH of blood, will therefore throw light on the formation of
thrombin on the one hand and also on the inactivation of prothrombin on
the other. The present work was undertaken to elucidate the role, if any,
of plasma-trypsin, in the physiological clotting mechanism. Incidentally,
it may also be possible to discuss in the light of the results obtained the
mechanism of the synthesis of prothrombin.

The experiments reported in this paper can be broadly divided into two
groups. In view of the thromboplastic activity of trypsin, the possibility
of the trypsin in plasma acting as the physiological thrombo-plastin circulating

Prothrombin and Plasma Trypsin 125

in blood can be visualised, and experiments have been designed to test this
hypothesis. In order to test this hypothesis, the clotting times of oxalated.
plasma on mere recalcification in a large number of samples of blood, from
different human subjects as well as from other species, have been determined. .
Simultaneously the trypsin content of each sample of plasma was also esti-
mated. Secondly, the action of plasma-trypsin on the prothrombin present
in plasma has been studied, to see whether any enzymic degradation of the
prothrombin protein is a necessary adjunct to the physiological inactivation
of prothrombin activity. Since prothrombin is rapidly destroyed at a tem-
perature of 36 in a solution of pH 8-4, the optimum for trypsin, specific
action of plasma-trypsin on the prothrombin protein could be evaluated,
only if the enzyme action was allowed to take place at pH 7-2, a reaction at
which prothrombin solutions are definitely known to be stable. The action
of high-grade commercial trypsin on purified prothrombin solutions has
also been studied.

Material and Methods

Preparation of purified prothrombin. This was prepared according to
Mellanby. 2 Litres of freshly bled ox blood is well shaken in a bottle con-
taining 20 c.c. of 20% neutral potassium oxalate, and the plasma separated by
high-speed centrifugalisation. The plasma is diluted with 10 vols. of distilled
water and brought to pH 5 3 (the iso-electric point of prothrombin) by the
addition of 1% acetic acid. The supernatant fluid is then poured off and tlfe
dilute suspension of globulin is then centrifuged. The precipitate is then
resuspendad in distilled water, equal to half the volume of the original
plasma. Dilute calcium bicarbonate solution is now added to an equal
volume of the globulin suspension, mixed by gentle shaking and allowed to
stand for about 10 minutes. The suspension is rapidly filtered through a
series of coarse filter-papers. The prothrombin is precipitated from the
rest of the solution by the addition of 1% acetic acid until the pH is approxi-
mately 5*3. The precipitate is then centrifuged, and the mass is quickly
dried by treatihent with acetone.

Preparation of fibrinogen This was prepared according to Eagle and
Harris. Repeated precipitations with 1-5 vols. of saturated NaCl yielded a
satisfactory product. This failed to coagulate on the addition of Ca and
tissue extracts but was promptly coagulated on the addition of thrombin.
The final product was brought to a concentration of 0*9% with respect to
NaCl by proper- dilution.

Trypsin. Digestive Ferments Cpmpany's trypsin ' Trypsin 1 : 110 '
was used in the experiments in%hich the proteolytic action of purified pro-
thrombin solutions was studied,

126 N, K* lyengar

Estimation of prothrombin. After many trials with Quick 9 and
Fullerton 10 techniques lyengar et al 17 have evolved an improved and con-
venient method for the determination of prothrombin time, this consists
in adding to 0-2 c.c. plasma maintained at 38 C., 0-2 c.c. of a solution of
1 in 20,000 Russel viper venom in 0-025M CaCl 2 solution. The interval
between the addition of the calcium thrombo-plastin solution and the first
appearance of the fibrin web is taken as the prothrombin time. The shorter
' this interval, the greater the prothrombin content.

Estimation of plasma trypsin. 2 c.c. plasma was precipitated with 8 c.c.
acetone, and centrifuged.- The precipitate was washed with acetone and
allowed to dry for a few minutes. The whole precipitate was then mixed up,
with 10 c.c. of phosphate buffer of pH 8-4, the optimum for plasma- trypsin
(lyengar 11 and Scott) in a glass pestle and mortar to get a uniform suspension.
3 c.c. of this suspension was pipetted to a test-tube containing 3 c.c. of 10%
trichlor-acetic acid. The contents are well shaken and filtered. The nitro-
gen in 3 c.c. filtrate is estimated by the micro-Kjheldal method. Few drops of
toluene are added to the remainder of the suspension and incubated at 37 C,
for 48 hours. At the end of this period, 3 c.c. of this suspension is mixed
with 3 c.c. of 10% trichlor-acetic acid, filtered and the nitrogen in 3 c.c. of
the filtrate estimated. The increase in non-protein nitrogen is taken as a
measure of tryptic activity of plasma. It is seen that by the above method,
the free trypsin, in plasma, is precipitated by acetone along with plasma
protein. If the solids are suspended in the buffer of pH 8-4, and incubated,
the autodigestion of the protein takes place giving rise to an increase in non-
protein nitrogen. Tryptic activity is expressed in terms of increase in
N.P.N. for 100 c.c. of blood plasma.

Although the measurement of tryptic activity is carried out at pH 8 4,
its action on prothrombin has been studied at pH 7-2 for the reasons enu-
merated in the early part of the paper. While we were engaged in a routine
determination of prothrombin levels in plasma of a large number of cases,
the clotting time of plasma on mere recalcification without the addition of
thrombo-kinase, was noted in every case. It was found that this varied over
a wide range while the prothrombin time itself with the addition of an opti-
mum amount of thrombo-kinase, was remarkably constant with very slight
variations. Can this variation be due to the varying amounts * of trypsin
present in blood plasma ? This has been put to test and the results are
given below ;

Prothrombin and Plasma Trypsin

127

Experimental
TABLE I

Relationship between * Clotting time on mere recalcification of plasma * and
the free trypsin content of plasma

Human subjects

Case No.

Prothrombin
time

Clotting time
on recalcifi-
cation of
plasma

Trypsin content
as measured by
increase in N.P.N.
calculated for
100 c.c. plasma

Human subjects

sees.

mg.

23-5

22-8

24-2

25-8

18-5

19-2

13-4

18-9

120 j

10-8

23*5

Rabbit

28-5

25-2

21-4

Monkey

22-8

21-4

Dog

18-3

It will be seen from the table that there is an inverse relationship
between the clotting time on mere recalcification and the trypsin content of
plasma. This can be noticed only when the clotting time is radically changed
as for instance from 45 sees, to 60 sees, or to 90 sees, or to 120 sees. In
these instances there is a significant drop in the trypsin content of plasma.
Minor changes in clotting time as for instance from 45 to 42 or 35, are not
reflected in the trypsin content. In fact, even the tendency may sometimes
be in the opposite direction. Such minor changes can only be ascribed to
experimental error in trypsin determination, as we are dealing with minute
quantities of trypsin from only 2 c.c. plasma. The figures in the table are
however magnified since they are calculated for 103 c.c. plasma.

The action of plasma-trypsin on the prothrombin also present in plasma
was next studied. The institution of blood banks in many of the larger
hospitals has made the clinician largely dependent on stored blood for
transfusion. It is now becoming increasingly apparent that such blood is not
equivalent in all respects to freshly drawn blood. The normal blood
contains a great excess of prothrombin beyond the amount necessary for
clotting. The work of Quick 12 ' 13 has proved a rational basis for the belief
that transfusion may diminish certain haemorrhagic tendencies, associated

128 N. K. lyengar

with prothrombin deficiency. Prothrombin content of blood stored in the
blood bank has been estimated by Rhoad and Panzer 14 at various intervals.
Their results have clearly shown that, although the blood is stored at 4 C. in
a sterile condition, the prothrombin content is gradually decreased. In a
week or more the blood would be practically useless in the treatment of the
acute prothrombin deficiency. The cause of this spontaneous deterioration
of prothrombin in blood is not known, Dilute solutions of purified pro-
thrombin appeared to maintain their biological activity quantitatively when
stored under identical conditions as in a blood bank. This has been indicated
by Mellanby in his experiments on dialysis of purified prothrombin solutions,
although the conditions of dialysis are not given. The complete stability of
such purified prothrombin solutions kept at a temperature of 4 under
sterile conditions, was confirmed by us. On account of the trypsin asso-
ciated with prothrombin in plasma, the possibility of tryptic action on the
prothrombin protein will have to be considered in this connection. It may
be pointed out that trypsin in such low concentrations as is present in plasma
and at such a low temperature as 7 C. and at a pH of 7-2, slightly removed
from the optimum pH of trypsin, cannot be expected to have any action on the
prothrombin protein. Pope has shown that even under conditions approach-
ing to the above, a disaggregation of the plasma proteins takes place. Minor
degrees of protein cleavage or perhaps mere intramolecular rearrange-
ment may take place even under these conditions. Mellanby has shown
that protein is an acid meta-protein or is associated with an acid meta-protein,
upon which the preservation of its properties depends. Prothrombic acti-
vity is such a delicate property of the protein that any slight change brought
about in the protein, may result in the deterioration of its potency. That
the trypsin in plasma may be responsible for the reduction of prothrombin
in stored blood, can therefore not be rejected summarily.

Prothrombin time of plasma was first determined immediately after the
blood was taken. The plasma was then kept in the frigidaire at a
temperature of 7 C. and the prothrombin time determined after different
intervals. Simultaneously with the prothrombin determination non-protein
nitrogen in the plasma was estimated in order to see whether destruction of
prothrombin is associated with an increase in N.P.N. as a result of tryptic
action. In some cases, the plasma was incubated at 37 C. and the
prothrombin destruction and changes in N.P.N. were followed at shorter
intervals. In order to ascertain if the rate of prothrombin destruction has
any relationship with the trypsin content of plasma, the trypsin was estimated
in plasma by the method given above, after autodigestion for 48 hours. The
results are given below.

Prothrombin and Plasma Trypsin

129

TABLE II (d)

Effect of incubation of plasma at 7 C. and 30 C.for different periods, on ' pro-

thrombin time * and corelat ion between changes in ' prothrombin time \

increase in N.P.N. and the free trypsin content of plasma

Prothrombin time after

Case

"Mrv

Prothrombin
time imme-

different intervals.
Plasma kept at 7 C.

Increase in
N.P.N

Trypsin
in 100 c.c.

diately

plasma

sees.

days

sees.

mg.

nil

23-2

nil

26-2

nil

18*5

nil

17-2

nil

38-4

TABLE II (b)

Prothrombin time after

Case

V T

Prothrombin
time imme-

different intervals. Plasma
incubated at 30 C.

Increase in

N.P.N

Trypsin
in 100 c.c.

No.

diately

plasma

sees.

hrs.

mgm.

sees.

nil

-37

-51

18-9

nil

-42

22-5

did not

nil

31-2

clot for 4

minutes

The results in Table II clearly show that plasma- trypsin slowly inactivates
the associated prothrombin even when kept at 7 C. There is no increase in
non-protein nitrogen even after a period of 3 days, which indicates that the
cleavage of the prothrombin protein has not taken place. Since prothrombin
activity has been affected, it should be assumed, that some change of the
prothrombin protein has been brought about by the trypsin, which cannot be
detected by the ordinary chemical methods employed in studying enzymic
degradations of proteins. The biological activity of prothrombin serves in
this case as an excellent method of detecting even the most superficial change
involving perhaps intramolecular rearrangement of the protein. When the
plasma is incubated at 30 C. the prothrombin destruction is considerably
accelerated. This is as should be expected since the tryptic activity is enhanc-
ed by the higher temperature. At the end of 24 hours, and 48 hours, there is

130

N. ft. tyengar

a definite increase in N.P.N. also and the quantity of prothrombin destroyed
is also greater. There is also a very rough lelationship between the rate of
destruction of prothrombin and the trypsin content of plasma.

In case (3) Table II (b) if purified prothrombin was added to the plasma
after incubation for 48 hours, the prothrombin time was immediately restored.
This clearly shows that in these experiments, it is the prothrombin that has
been affected and not any other clotting factor like fibrinogen.

Finally the action of commercial trypsin on purified prothrombin protein
has been studied.

200 mg. was dissolved in 250 c.c. water and brought to pH 7-2 by the
gradual addition of a dilute solution of sodium carbonate and the volume
was made up to 50 c.c. To 25 c.c. of this solution was added 5 c.c. of 10%
solution of trypsin and incubated at 37 C. The N.P.N. was determined '
immediately and at definite intervals. The prothrombin time of this solu-
tion was also determined both immediately and after stated intervals by
diluting Ic.c. of the digest to 100 c.c. with 0-9% saline, and activating
0*2 c.c. of this with 0-2 c.c. of a solution of 1 in 20,000 Russel viper venom
in 0-025 M CaCl 2 and then adding 0-2 c.c. of a solution of fibrinogen.

TABLE III
Action of commercial trypsin on purified prothrombin

Time allowed

Prothrombin time

Increase in N.P.N.
in the digest

sees.

mg.

1 5 minutes

1*8

2-4

Ihr.

3-5

2hrs.

Did not clot in

5-2

\ 5 minutes

The results reported in Table III confirm the conclusion that the pro-
throbmin property of the protein is so labile that even slight intramolecular
rearrangement can disturb the activity.

Summary and Conclusion

The work of Northrop, and Kunitz, 15 Eagle and Harris, and Ferguson
has elucidated the role of trypsin in blood coagulation. Experimental
evidence has been obtained to show that the trypsin in plasma may be the
physiological thrombo-plastin. The thrombo-plastic action of trypsin
depends on the amount present in plasma. Since the trypsin content of

Prothrombin and Plasma Trypsin 131

plasma is Very low, it is definitely below the optimum amount required to
have the maximum effect. Being present in sub-optimal amounts, the
clotting time on mere recalcification is found to vary with the trypsin content.
The prothrombin time is constant irrespective of the plasma trypsin content,
since during the determination of this factor, the optimum amount of
thrombo-plastin is added in the shape of Russel viper venom. Trypsin is
reported to have (Eagle and Harris) no direct coagulative action on puri-
fied fibrinogen. The coagulating action of trypsin was found to rest on the
fact vhat it reacts with prothrombin to form thrombin. This thromboplastic
action of trypsin is observed only within a comparatively narrow optimum
zone of trypsin concentration. The trypsin in plasma is so small in quantity
that it is highly probable that it is sub-optimal so far as its thrombo-plastic
action is concerned. Hence with varying concentrations of trypsin in plasma in
the sub-optimal range, the clotting time of recalcified plasma is found to vary.
The experiments on the incubation of plasma under sterile conditions at
a temperature of C. and 30 C. show that the gradual destruction of pro-
thrombin activity takes place. In the early stages, the destruction is not
accompanied by any increase in N.P.N. which is the earliest index of protein
cleavage. These results are suggestive of the possibility that mere intra-
molecular changes of the prothrombin protein or mere disaggregation of the
protein is enough to bring about a loss in prothrombin activity. This is
confirmed by experiments on the tryptic action of purified prothrombin

solutions.

Acknowledgment

My thanks are due to Sir R. N. Chopra and Dr. B. Mukerji for their keen

interest in this work.

REFERENCES

1. Mellanby - - Proc. Roy. Soc. t Sec. B, 1930, 107, 271.

2. Pope, B. J. Exptl Path-Bad., 1938, 19, 245.

3. Douglas and Colebrook . . Lancet, 1916, 2, 180.

4. Walschmidtz Leitz, et al. . . Nature Wissenshapten, 1928, 16, 1027.
5 __ . . z. sa. Physiol-Cheml, 1929, 183, 39.
6*. Eagle and Harris .- / Gen. Physiol, 1936-37, 20, 543.

7 Ferguson J H. - J- Lab. and Clin. Med. 9 1938, 24, 273.

* Am. J. PhysioL, 1939, 126, 661.

9! Quick, A. J. J - Am - Med - Asson-* 1938 110 1658t

lo! Fullcrton Lancet, Aug. 17, 1940, 195.

1 1 lyengar, N. K., and Scott, D. A. . . Trans. Roy. Soc. Canada, Sec. V, 1940, 34, 45.

12 Quick A J Am. J. Med. Science, 1935, 190, 501.

!3* Q ' .. J.A.M.A., 1938, 110, 1658.

14.' Rhoads and Panzer .. J6W, 1939, 112, 309.

15. Northrop and Kunitz - J ' Gen ^ hy ^ ^^

16. Schmitz - - Zt-schrift Physio l-Chemie, 1937, 2.0, 37.

17. lyengar, Sehra and Mukherjea . . Curr. Sci., 1941, 7, 326.

1659-41 Pnnted at The Bangalore Press, Bangalore City by G. Srinivasa Rao, Superinte i ent
and Published by The Indian Academy of Sciences, Bangalore.

THE GRAIN SORGHUMS OF THE DURRA GROUP

BY G. N. RANGASWAMI AYYANGAR, F.N.I., I.A.S.,

(Millets Specialist and Geneticist, and Principal, Agricultural College)

M. A. SANKARA AYYAR, B.A., B.Sc. AG.,
T. R. NARAYANAN, B.A. (CANTAB.), B.Sc. AG.,

AND
A. KUNHI KORAN NAMBIAR, B.Sc. AG.

(Assistants, Millets Breeding Station, Agricultural Research Institute, Coimbatore)
Received May 31, 1941

Introduction

AMONG the grain sorghums, the Durras form the most important group.
Snowderi 1 has recently made a systematic classification of the cultivated
sorghums. He has grouped the cultivated forms into six sub-series in a
descending order of affinity to the wild types. The sub-series Durra is the
last of these six and the most removed from the wild ones. It consists of
four species: Sorghum rigidum Snowden, Sorghum durra Stapf, Sorghum
cernuum Host, and Sorghum subglabrescem Schweinf. et Aschers. Of these,
the first named is a rare group and is reported to exist only in the Blue
ISfile district of the Anglo-Egyptian Sudan. Moreover, it is less closely
related to the other three species than they are among themselves. These
three, viz , S. durra, S. cernuum and S. subglabrescens form not only a
compact, well-defined group, but are also culturally the most important
among the grain sorghums. This article gives a brief review of the Durra
sub-series, with special reference to Indian and .particularly to Madras
varieties.

Origin, History and Nomenclature

The origin, history and nomenclature of the species included in this
sub-series have been fully reviewed by Snowden.

Chief Characteristics

The three important species of this sub-series are characterised by the
following features. The plants are medium stout to robust with a coat of
\vaxy bloom on the internode and leaf-sheath. The panicle is usually
compact, medium compact or medium loose (Fig. 1), and only very rarely
loose. The rachis is stout and grooved, and the branches and branchlets
are short, erect and hairy (Fig. 2). The peduncle is usually erect except in

133

134 G. N. Rangaswami Ayyangar and others

S. cernuum where it is mostly goose-necked (Fig. 3). The sessile spikelets
show a wide range in shape from ovate elliptic to rhomboid (Fig. 4). The
glumes are thick and spongy or thin and herbaceous. They are more or less
equal in length. When herbaceous,' there is often a transverse wrinkle
about the middle of the glume (Figs. 4 & 6). In the thickly coriaceous forms,
there is no wrinkling and the tip is strongly nerved. The lemmas are hyaline
and ciliate, and most often have a long awn. The mature grains exceed the
glumes in length and are as a rule readily separable. The embryo mark is
ovate to elliptic, flat or rarely concave. The lateral lines are prominent.
The endosperm is white in colour, mealy inside and hard towards the peri-
phery. Unlike most of the species of the earlier groups, e.g., Drummondii,
Guineensia and Caffra, the pedicelled spikelets in this are persistent (Fig. 4)
and have only short pedicels. They are lanceolate to elliptic, and antheri-
ferous or neuter. The stalk is generally sweet.

Sorghum durra Stapf.
(a) Characteristics:

The plants of this species are generally stout-stemmed and broad-leaved.
The height varies from 125 cm. in the Sudan and Sind varieties to 400 or
even 450 cm. in some of the Indian (Madras) varieties! The duration ranges
from 95-145 days. The stalks are usually pithy in the ripe stage, but are
mostly sweet. The internode (fourth from the top) is -9-2 -4 cm. thick.
The number of leaves varies from 7-16 in the mature plant. The panicle
is generally medium-loose to medium-compact, sometimes compact, and
rarely loose, and 9 -5-28 cm. long and 5-17 cm. broad. The peduncle is
erect as a rule, and is only rarely recurved. The heads are well emerged
from the boot in the majority of types with a clearance of 10-15 cm. The
branches are rather rigid at the bases. The racemes are somewhat crowded,
mostly, three to four noded and fully hairy. Sessile spikelets vary in shape
from obovate-elliptic to rhomboid, and are 4 -0-6 -5 mm. long and
2- 5-5-0 mm. broad. They maybe red, black, buff or straw coloured, when
dry. The glumes are 4-6-0 mm. long and 3-4-5 mm. broad, and may be
fully hairy or glabrate. The nodal bands may be fully hairy or glabrous
(Fig. 5). The glumes are thickly, coriaceous except at the tip, where it is
thinly coriaceous and unevenly hairy. The tip of the outer glume is broad,
triangular and strongly nerved. There are 12-16 primary and 6-8 secondary
neives on the lower glume. The upper glume also has 7-9 primary and 5-7
secondary nerves, and is ciliate on the margins. The glumes are never
wrinkled. The outer lemma is ovate to broadly elliptic, 4-0-5-5 mm. long,
3-4 mm. broad and 2-5 nerved, and the inner is ovate and shortly two-lobed,
with an awn up to 12 mm. in length. The anthers are 3-4 mm. long and

The Grain Sorghums of the Durra Group 135

1 mm. wide. Grains vary in shape from obcjvate to broadly ovate or sub-
rotund, and are 4-0-6-5 mm. long and 3-0-6 mm. broad, and have a broad
rounded and much exposed top. The majority of the types are yellow or
white grained ; red grained come next, while brown-grained types are some-
what rare. The pedicelled spikelets are lanceolate to linear oblong, 4-8 mm.
long and -75-2 mm. broad.

This species is characterised by the presence of obovate-elliptic to
rhomboid-sessile spikelet, thickly coriaceous glume with broad and strongly
nerved herbaceous tip, awned or rarely mucronate inner lemma and biconvex
grain with broad and round top and wedge-shaped base. It differs from
S. cernuum in having more thickly coriaceous glume which is not wrinkled
and which becomes more or less glabrous on the back at length. Moreover
the grain in S. cernuum is broadly elliptic to orbicular in shape and much
more flattened than in S. durra. S. durra can be distinguished from S. sub-
glabrcsctms in having thickly coriaceous and rather spongy glume which is
comparatively more hairy and not transversely wrinkled as in the latter.

(//) Distribution.

This species is now the most important grain sorghum in Egypt,
the Anglo-Egyptian Sudan, Eritrea, Arabia and India. In recent
times it has also been introduced into the United States of America and in
addition is also grown in parts of Middle 'and Western Asia, namely Iraq,
Mesopotamia and Palestine. In India Durra varieties are ' cultivated in
almost all the provinces where sorghum is grown, particularly in Madras,
Sind and Baluchistan. It forms the chief rain-fed variety in the Coimbatore,
Guntur, Cuddapah, Kurnool, Kistna, Godavari and Viz agapatam districts
and occupies about 46 per cent, of the total area (4-6 million acres) under
sorghum in the Madras Presidency, S. subglabrascens having 18 per cent, and
S. cernuum 17 per cent. Thus the species of the Durra sub-series occupy more
than four-fifth of the total sorghum area in the Madras Presidency.

Out of the 16 varieties into which Snowden classifies Sorghum
durra only nine, viz., mediocre, coimbatoricum. javanicum, fecundum, eois,
elongatum, fuscum, rivulare and maximum are found in India. The remain-
ing seven varieties, viz., aegyptiacum, Fiorii, rutilum, niloticum, melanoleucum,
erythrocarpum and lutecium are confined to Anglo-Egyptian Sudan, Egypt
and Eritrea. Among the varieties cultivated in India, vars. mediocre,
coimbatoricum and javanicum alone are grown in the Madras Presidency.
A short description of these is given below.

136 G. N. Rangaswami Ayyangar and others

(i) Var. mediocre (Burkill) Snowden. This variety is confined to the
districts of Northern Circars namely, Vizagapatam, Godavari and Kistna,
the Ceded districts of Cuddapab, Kurnool, Bellary and Anantapur and the
adjoining districts, viz., Guntur, Nellore and Chittoor. It is known in the
different districts under different local names such as Bommayi jonna,
Budda jonna, Desa jonna, Gidda jonna, Harasana jola, Mdllemari jonna,
Napa jonna, Pacha jonna, Pairu jonna, Pedda jonna and Zinkapuri jonna.
The Pacha jonna of the Circars and the Ceded districts is typical of this
variety.

Chief Characteristics. Types mostly rain-fed ; plants medium tall;
height 125-285 cm.; duration 100-130 days; stalk 0-9-1 -5 cm. thick, pithy ;
leaves 10-14 in number, 50-70 cm. long and 6-9 cm. broad ; sheath and
glumes mostly reddish purple; axil purple; midrib white; panicle medium
loose to medium compact, 10-25 cm. long and 5-1 2 cm. broad; awn long;
glumes glabrous and slightly or fully bleached; grains pearly yellow, invari-
ably with a characteristic brown wash, 4-5 -5 mm. long and 3-4 -5 mm.
broad; pedicelled spikelets 4-6 mm. long, and -75-1 -5 mm. broad and
turns brownish yellow on drying. This is the chief sorghum grown in
the Northern Circars. But being a paddy tract, rice forms the chief cereal
for food there, and sorghum is mostly exported. In the Ceded districts
and also in Guntur, Nellore and Chittoor this variety forms one of the chief
food grains. The yield ranges from 300-800 Ib. of grain and 900-1,500 Ib.
of straw per acre.

(ii) Var. coimbatoricum (Burkill) Snowden. As the name indicates, this
variety is confined to the district of Coimbatore. Recently it has spread
to the neighbouring districts mainly through the endeavour of the Agricul-
tural Department! This variety is commonly known by the name Peria-
manjal cholam and is rarely called Sadaimanjal The name Periamanjal
refers to the long duration, the great height and the yellow grain of this
variety.

Chief Characteristics. Plants are very tall (tallest among the Indian
sorghums), stout and long in duration. Height 300-450 cm. ; duration
130-40 days; stalk 1-2 cm. thick; leaves 12-16 in number, 60-70 cm. long
and 8-10 -5 cm. broad; leaf-sheath and glume reddish or blackish
purple; node glabrous; axil purple; midrib white; panicle large, medium
loose, 14-26 cm. long and 7-17 cm. broad; awn long; grain pearly yellow with
a characteristic brown wash and somewhat duller in colour than in var
mediocre; 4 -5-5 -5 mm. long and 3-5-4-5 mm. broad and tightly held by the
glumes. This variety is highly season-bound, the flowering period being

The Grain Sorghums of the Durra Group 137

confined to the months of October and November. The yields vary from
600-1,200 Ib. of grain and 2,000-6,000 Ib. of straw per acre. As a food grain
this is considered superior to the other varieties in the Coimbatore district,
(iii) Var. javanicum (Hack.) Snowden. This variety has a wider distri-
bution than the two described above being found in all the sorghum growing
provinces of India as well as in Afghanistan, Anglo-Egyptian Sudan, Egypt
and in Morocco. In the Madras Presidency it is grown chiefly in the districts
of Bellary, Anantapur, Chittoor and North Arcot. It is known under different
local names such as Hirajola, Konai cholam, Nagari cholam, Nettaijola, Nir
jola, Vellai cholam and Vibhuthivantha jonna. The Vellai cholam of Chittoor
and North Arcot is typical of this variety. This consists of types which are
grown both under irrigated and rain-fed conditions.

Chief Characteristics. Height 140-75 cm. in the irrigated and 165-225
cm. in the rain-fed ones; duration 110-15 days in the irrigated, and 120-35
iu the rain-fed types; stalk 0-9-1 -6 cm. thick and pithy; leaves 7-12 in num-
ber, 60-70 crn. long and 6 5-9 cm. broad ; panicles compact, ovate to conical,
10-20 cm. long and 6-5-10 cm. broad; glumes mostly reddish purple, always
fully hairy, 4-5-6 mm. long and 2 -5-4 mm. broad; awn long; grain mostly
white, 4-5-5 mm. long and 3-5 mm. broad. The yield varies from 400-1,000
Ib. of grain and 2,000-3,500 Ib. of straw per acre.

Of the three varieties of S. durra described above vars. coimbatoricum
and mediocre have yellow grains, and var. javanicum is mostly white grained.
Var. coimbatoricum differs from vars. mediocre and javanicum in having much
taller and more robust plants with larger panicles and bolder grains which
are rather tightly held by the glumes. Moreover in this variety both reddish
purple and blackish purple coloured leaf-sheaths are met with, while in
mediocre and javanicum blackish purple is very rare. In var. javanicum
the panicles are more compact than in the other two.

Sorghum cernuum Host.
(a) Characteristics :

Plants shorter than those of S. durra; height 110-300 cm.; duration
95-140 days; stalk 0-9-2-2 cm. thick, mostly juicy and sweet; leaves 7-15
in number, 45-75 cm. long and 5-10-5 cm. broad; node green and fully
hairy; axil of leaf-sheath purple; leaf-sheath and glume mostly reddish
purple, blackish purple being rare; awn long; panicle compact to medium
compact, 8-27 cm. long and 5 -5-19 cm. broad; peduncle mostly recurved
(Fig. 3) ; rachis fully hairy and branches short and sub-erect; sessile spikelets
broadly' ovate to obovate-oblong and fully hairy ; callus beard copious;
glumes fully hairy to villous, equal, thick and spongy below the middle and

138 G. N. Rangaswami Ayyangar and others

thin and papery above with a transverse wrinkle, creamy white to straw or
buff in colour, 4-6 mm. long and 3-4-5 mm. broad and tips breaking off at
maturity; grain rotundate or orbiculate, flattish, 4-6 mm. long and 3 5-5 mm.
broad, protruding beyond the glumes, mostly white, occasionally red and
rarely yellow in colour; pedicelled spikelets generally large, fully hairy and
mostly antheriferous, turning red in red grained and straw coloured in white
grained types.

The distinguishing characteristics of this species are the silky hairs on
the nodes and the glumes, completely bleached glumes which are somewhat
thick and spongy at the base, thin and herbaceous at the tip, transversely
wrinkled or depressed at the middle, and breaking off at the tips, and the
orbicular and flattened grains. This differs from S. durra in having more
hairy and transversely wrinkled glumes and more flattened grains ; and from
S. sub-glabrescens in having fully hairy and completely bleached spike-
lets which are invariably long awned.

(b) Distribution:

This species is less wide in its distribution than S. durra. It is found in
India, parts of Afghanistan, Persia, Arabia, Asia Minor, Egypt and British
Somaliland. At one time it was extensively cultivated in Egypt, although it is
now largely replaced by S. durra. This species figures to a slight .extent in
the United States of America having been first introduced in 1874 under the
name of white durra. In India it is limited chiefly to the uplands of the
Deccan. Varieties of this species are grown in Rajputana, Sind, Bombay,
Hyderabad, Central Provinces, Central Indian States, Mysore and Madras.
A few types are found in Bihar and Orissa also. The plants of these varieties
seem to do well only in areas with highly retentive clayey soils, low rainfall
during the growing period, and an absolutely rainless, cool weather during
the ripening stages. The ability of the plants of this species to resist drought
is shown by the fact that it is distributed over the driest areas of this Presi-
dency as well as of India in general. In the Madras Presidency S. cernuum
occupies a predominant place only in the Ceded districts. It is of minor
importance in the adjacent districts of Guntur and'Chittoor. Though iso-
lated areas occur here and there, where S. cernuum is grown under irrigation,
it is mostly a rain-fed species.

Snowden has divided this species into seven varieties, viz., truck-
menonm, yemense, agricolarum, globosum, orbiculatum, subcylindricum and
cernwm, all of which occur in India, and two are confined to India alone in

The Grain Sorghums of the Durra Group 139

their distribution. Of the above seven varieties only three namely, agrico-
larum, globosum and orbiculatwn are cultivated in the Madras Presidency
to a large extent, and a short description of these is given below.

(i) Var. agricolarum (Burkill) Snowden. Grown chiefly in the district
of Bellary and portions of Kurnool this is known locally as Yerrajonna and
Ycrrapusi jonna which refer to 'the red colour of the grain. It is usually
cultivated as a dry crop. Owing to the juicy stalk and the leafy nature of
the plants this is considered as a good fodder variety and yields 10,000-
15,000 Ib. of green fodder per acre. In extraction tests this gave 43-3
per cent, of juice with a Brix value of 12-6. This juice contained 6-6
per cent, of sucrose and 3-0 per cent, of glucose.

Chief Characteristics. Height 250-300 cm.; duration 120-40 days;
stalk 0-9-1 -4 cm. thick; leaves 12-15 in number, 50-70 cm. long and 6-8 cm.
broad with the margins turning red on drying; peduncle recurved in most
cases; panicle compact, 9- 15 cm. long and 6-8 cm. broad; grain red,
very bold, 5-5 mm. long and 4-5 mm. broad with a dimple in rare cases;
pcciicellcd spikclcts 4 ; ~5 mm. long and 1-2 mm. broad.

(ii) Var. globosum (Hack.) Snowden. The different forms of this variety
arc found chiefly in the districts of Cuddapah, Kurnool, Bellary, Anantapur
and Chittoor. They are known by the names Chitta jonna and Nallapiisi
jonna in Kurnool, Telia jonna in Bellary, Cuddapah and Chittoor, and Telia
Thota jonna in the district of Anantapur. They are mostly rain-fed types.

Chief Characteristics. Height 180-240 cm.; duration 100-130 days;
stalk 14 -4 cm. thick; leaves 10-14 in number, 50-75 cm. long and
6* 5-9 -5 cm. broad; panicle medium, to very compact, 10-20 cm. long and
7-10 cm. bfoad; grain very bold, mostly pearly white, 4-5-5 mm. long, and
3 '5-5 mm. broad; a few double grained due to the lower lemma being
fertile; endosperm mealy white; pedicelled spikelets 4-6 mm. long, and
1-2 mm. broad. This is the most important of the varieties of S. cernuum
cultivated in the Ceded districts. The yield is 300-600 Ib. of grain and
1,000-2,000 Ib. of straw per acre.

(iii) Var. orbiculatum Snowden. This variety has a much wider distri-
bution than either of the two described above, but in Madras it is grown
to a lesser extent and is mostly found in the districts of Bellary, Chittoor and
Gunlur. The types in this variety are known as Bdikalu jola -(white pearly
grained) in Bellary, Mudda (ball-like) jonna in Chittoor and Venna Mudda
(butter ball) jonna in Guntur. This is similar to var. globosum in height,
duration, and other plant characters, the chief point of difference being the
more ilattened nature of the grain in this variety.

140 G. N. Rangaswami Ayyangar and others

The three varieties of S. cernuum namely agricolarum, globosum and
orbiculatum, generally met with in the Madras Presidency, have been de-
scribed above. In these, var. agricolarum consists of red grained types only
and in this the spikelets and the margins of the leaves are reddish in colour,
when dry. In the varieties globosum and orbiculatum the majority of the types
are white grained. Variety orbiculatum can be distinguished from var-
globosum by the grain of the former being orbicular in shape and much mare
flattened than that of the latter.

Sorghum subglabrascens Schweinf. et Aschers.
(a) Characteristics:

Height 70-335 cm. ; duration 85-135 days ; stalks generally pithy, thinner
than those of S. durra and S. cernuum, range in thickness being 0-8-1-5 cm. ;
leaves 7-14 in number, 40-70 cm. long and 4-5-8 cm. broad; panicle
compact to very compact and sometimes loose, 8-25 cm. long and 5-12 cm.
broad; peduncle generally erect and rarely goose-necked; branches and
branchlets less hairy than in the other two species; sessile spikelets oblong
to hexagonal in shape, 4-6-5 mm. long, and 2-5-4 mm. broad, often hairy
when in flower, and ultimately glabrate; callus beard scanty; glumes
4-6 -5 mm. long and 2 -75-4 mm. broad, thick and papery except near the
base, wrinkled or depressed about the middle and with 12-14 primary and
5-8 secondary nerves; awn long in the majority of types; grain white, yellow
or red and rarely brown in colour, 3-5-5 mm. long and 3-4-5 mm. broad;
with a rounded tip and an abruptly compressed, wedge-shaped base; yellow
type invariably long awned ; pedicelled spikelet small, and reddish in red
and yellowish in yellow grained types.

The distinguishing characteristics of this species are the semi-membra-
neous, glabrate glumes which are usually transversely wrinkled about the
middle (Fig. 4) and broad topped grains with abruptly tapering wedge-
shaped bases. The plants are usually shorter and less robust than in
S. durra and somewhat less robust than in S. cernuum. S. subglabrescens can
be distinguished from S. cernuum by the glumes of the former being glabrous
or less hairy and the grains biconvex and broad topped with abruptly taper-
ing wedge-shaped bases. It differs from S. durra in having obovate-oblong to
hexagonal spikelets, less hairy to almost glabrous, thinner and transversely
wrinkled glumes, and abruptly tapering grain bases. Y

(b) Distribution:

This species is distributed in the Anglo-Egyptian Sudan Eritrea

taTStaf ^ ?*i II " a ' S bem to**"*

South Africa, Nyasaland and the United States of America.

TJie Grain Sorghums of the Durra Group 141

Of the 17 varieties into which this species is classified by Snowden,
10 are limited in their distribution to Africa, and of the remaining seven some
are found both in India and Africa, and the rest in India only. Of these
seven varieties, pabulare, rubidum, compactum, irungiforme and oviforme are
concentrated in the Madras Presidency, while paniculatella and rugulosum
are reported from Bombay and the Central Provinces. In Madras S. sub-
glabrescens is more abundant in the south than in the north where S. durra
and S. cernuum predominate. A short description of the varieties culti-
vated in the Madras Presidency is given below.

(i) Var. pabulare Snowden. The word pabulare indicates fodder, and
this variety is raised more as a fodder than a grain crop. Under the names of
Nilwajowar and Utavlijowar it is grown extensively in the Bombay Presi-
dency and the Central Provinces. In Madras it is not so prominent, and is
represented only in two districts Ramnad and Tinnevelly, where these are
known as Arisi cholam or Uppu cholam. These Arisi cholams have a great
affinity to the Irungu tholam (S. dochnd) in having reed-like stalks, narrow
leaves, large number of tillers, loose panicles and small grains which are
almost completely enclosed by the glumes. In these two districts the Irungu
cholam being the most common variety grown for fodder, and the white
grained forms of S. subglabrescens for grain, it is quite possible that the variety
pabulare, having the characteristics of both S. subglabrescens and S. dochna
might have originated as a product of hybridisation between the white-
grained forms of these two species. Similarly the Nilwa jowars of Bombay,
Bihar and the Central Provinces show some of the characteristics of both
S. cernuum and S. subglabrescens and seem to have been evolved through
hybridisation between certain white-grained types of these two species,

Chief Characteristics. Height 180-225 cm. ; duration 95-105 days; stalk
0-8-1 cm. thick; leaves rather stiff, 8-13 in number, 50-65 cm. long and
4 -5-5 -5 cm. broad; leaf-sheath and glumes either reddish or blackish
purple, node, junction and glume completely hairy; panicle loose conical,
18-25 cm. long and 8-12 cm. broad; awn 8-10 mm. long; grain chalky
white and small; glames extending nearly to the tip of the grain, slightly
bleached and transversely wrinkled; pedicelled spikelets small and sterile.
This variety is of minor importance in the Madras Presidency.

(ii) Var. rubidum (Burkill) Snowden. This is one of the four important
varieties of S. subglabrescens grown in the Madras Presidency. Its distri-
bution extends from Nellore in the North to as far as Madura in the South,
being cultivated in Nellore, Guntur, North Arcot, Ceded districts, Salem,

142 G. N. Rangaswami Ayyangar and others

Coimbatoie, Trichinopoly and Madura. Among those districts however,
its greatest prominence is in Salem and Trichinopoly where large areas are
grown under the name of Sen cholam. The name mbidwn has arisen from
the word rubidus meaning reddish, and the grain in this is generally red or
lidit red. This consists of both irrigated and rain-fed types the latter being
50-100 cm. greater in height and 20-30 days longer in duration than the
former. The most common names by which this variety is known are
Kunkuma jola, Makkattai cholam, Palapu jonna, Sakkaraguliga JQnna, Sen
cholam and Yerrajonna. The Sen cholam is the most typical of this variety.
Chief Characteristics Height 125-245 cm.; duration 100-135 days ;
stalk 1-1 -2 cm. thick; leaves 8-12 in number, 50-60 cm. long and 5-7-5 cm.
broad; leaf-sheath and glumes reddish purple; node and junction glabrous;
panicle medium compact in rain-fed and compact to very compact in irri-
gated types, 10-14 cm. long and 5-7 cm. broad; peduncle usually erect and
rarely recurved; glumes slightly bleached and wrinkled; awn long in most
of the rain-fed types, and absent in the irrigated ones; and grain bold, red,
light red or pink in colour. The yield is 1,5003,000 Ib. of grain and 3,000-
5,000 Ib. of straw per acre under irrigated condition while the rain-fed crop
gives 600-800 Ib. of grain and 2,000-3,000 Ib. of straw.

(iii) Var. compactum (Burkill) Snowden. This variety is found only
in India where it is confined to the Central Provinces and the Madras Presi-
dency. In the latter province it is grown chiefly in the districts of Coimbatore,
Trichinopoly and South Arcot, and to a small extent in Bellary and
Anantapur. It is known as Azhukku cholam, Chinna or Chitrai manjal
cholam., Dosakaya jonna, Kullamanjal or Kullanari cholam, Manja makkattai
and Sena jonnalu. The Chinna manjal and Manja makkattai are typical of
this variety. As in rubidum, irungiforme and oviforme the types in this
variety also fall into two groups of duration, the shorter (95-110 days)
grown under irrigation from March to June, and the longer (120-135 days)
grown rain-fed from July to December. The irrigated ones are 160-220 cm.
and the rain-fed ones 200-3 10 cm. tall. Other characteristics are: stalk
1-1 -4 cm. thick ; leaves 10-13 in number, 50-70 cm. long and 7-8 cm.
broad; leaf-sheath and glumes reddish purple-; nodal band, junction and
glumes glabrous; panicle medium compact to compact, ovate; awn long ;
glumes obovate, blunt tipped, and wrinkled with tips breaking off at matu-
rity, and grains pearly yellow with or without a brown wash. In the Chinna
manjal cholam the tissue at the nodal band develops a characteristic parple
colour which presents a checkered or cracked appearance at the ripe stage,
and this purple is linked with the sienna coloured dry anther and yellow grain
without the brown wash. The yields vary from 1,500-2,500 Ib. of grain and

The Grain Sorghums of the Durra Group 143

3,000-5,000 Ib. of straw per acre in the irrigated, and 500-800 Ih. of graift and
2,000-4,000 Ib. of straw in the rain-fed crops.

(iv& vj Var. ifungiforme (Burkill) Snowden and Var. oviform? Snowdcn.
These two varieties are similar in all morphological characters and cultural
features. The only difference is that the var. oviformc has a much denser
and shorter panicle than the var. irungijormc in which the panicle shape
varies from compact to medium compact. These two varieties are therefore
discussed together. As already recorded, both the varieties are pmely
Indian in their distribution and the majority of types are found in Madras
and a few reported from Bombay, Sind and the Central Provinces. Both
the varieties arc known by a number of local names, the most common
of these being Chinna vcllai, Ennai vcllai, Kattai vellai, Kokki vcltai, Kullanari
cholatn, Telia jonna, Uppam or Uppit cholam and Vcllai cholam. The last
named is perhaps the most typical of these. Variety inmglformc is more
widely distributed in Trichinopoly district while var. oviformc predominates
in Madura. In some cases both the varieties are known by the same local
name as Kullanari cholatn, Uppam cholam and Vcllai cholam,

Chief Characteristics. Height 150 230 cm. in the irrigated and 220-
350cm. in the rain-fed ones; duration 90-110 days in the irrigated and
125-35 days in the rain-fed ones; stalk 1-1*2 cm. thick; leaf-sheath and
glume reddish purple or blackish purple, brown sheath being rare ; leaves
10-14 in number, 50-60 cm. long and 5-7-5 cm. broad; node and junction
glabrous in the majority of types; panicle compact to medium compact,
15-20 cm. long and 8-10 cm. broad in irungiforme* and more compact and
8-14 cm, long and 5-8 cm. broad in oviforme; glumes ovate to obovate*
broad tipped, transversely wrinkled in the majority of types and 3-5-5 mm.
long and 3-4 mm. broad; and grains white, mostly pearly. Both the varieties
consist chiefly of irrigated types, the rain-fed ones being few and less impor-
tant. The irrigated crop is raised from January to June and gives 1,500-
3,000 Ib. of grain and 4,000-6,000 Ib. of straw per acre. The rain-fed is
grown from July to February and produces an acre yield of 500-800 Ib. of
grain and 2,000-4,000 Ib. of straw.

The five varieties of 5. subglabrescens, namely, pahulare, rubidum*
compactum, irungiforme and oviforme cultivated in the Madras Presidency
have been described above. Var. pabulare differs from the others In having
reed-like stalks, profuse tillering, loose conical-shaped, long panicles and
small grains which are almost completely enclosed by the glumes. Var.
rubidum is distinguished from the rest by its red or light red grains. Among
the remaining varieties compactum has pearly yellow grain invariably asso-*<

144 G. N. Rangaswami Ayyangar and others

dated with a long awn, and irungiforme and oviforme are white grained.
Var. irungiforme differs from var. oviforme in having more elongated and
less dense panicles.

Border Types

The three important species in the sub-series Durra have now been dis-
cussed. There occur however certain types that appear to be intermediate
between these species. They cannot be included under any one of these
three S. durra, S. cernuum or S. subglabrescens, and can only be classed as
intermediate or border types. Thus there are border types between S. durra
and S. cernuum i between 5. cernuum and S. subglabrescens, and also between
S. durra and S. subglabrescens. These border types are mostly found in
regions where all the three species are simultaneously grown as in the
Ceded districts. In the other districts where either one species alone is
grown or different species are raised in different seasons, these border types
are rare, probably due to lack of chances for intercrossing. The Telia kugu
jonna of Anantapur and Telia jonna of Cuddapali are considered as border
types between S. durra and S. cernuum, Manadanti and Palumadi jonna of
Bellary and Cherukku jonna of Kurnool as border types between S. cernuum
and S. subglabrescens and Kakimari jonna of Anantapur seems to be a border
type between S. durra and S. subglabrescens.

A Brief Review of Plant Characters met with in the Durra Group

Height and Duration. Most of the types in the Durra sub-series fall
within the medium duration group, and have the unimodal disposition in
their internodal lengths. 2 The relationship between height and duration was
studied in a mutant in a Pacha jonna type (S. durra var. mediocre) and the
short early was a simple dominant to the tall-late. 3

Root colour. In this sub-series there is a preponderance of the reddish
purple type, the blackish purple being few and the brown rare.

Root system. Most of the varieties of the Durra sub-series have very
well developed root systems. This is more evident in the rain-fed than in the
irrigated ones. Some, particularly when desheathed, have a tendency to
develop aerial roots. 4

u L f '* heath -~ ln this g r up the leaf-sheath covers more than the lower
half of the mternode, and the direction of the aestivation is normal, being
alternately clockwise and anti-clockwise.* Abeirations from this normal
condition have been met with in a Pacha jonna type called Edakula jonna

"**- re clockwise or

The Grain Sorghums of the Durra Group 145

Leaf-blade. In the majority of the varieties in this group the leaf-blade
is broad with smooth junctions and wavy margins. 6 In S. durra and
S. cernuum most of the types have hairy leaf tips.

Nodal band. This is generally green and mostly hairy in this group, the
glabrous types being in a majority in S. subglabrescens alone. Purple
coloured band is rare.

Axil of leaf-sheath. Most of the types in this sub-series have purple
coloured axil. Green axils are very few and deep purple ones rare.

Leaf-junction. In the majority of the varieties of this group the junc-
tion is generally green, hairy and smooth. It is worth noting that hairy
nodes and junctions are more numerous in the rain-fed than in the irrigated
types.

Midrib colour. In the Durra sub-series generally the midrib is white
in colour and the stalk pithy but sweet. In S. cernuum the majority have
juicy stalks. Apart from juiciness or pithiness, the midrib in some types in
all these species is yellow 7 and in a tare mutant in S. durra, it is brownish
purple. 8 The colour in these cases is confined to the mechanical tissue.
The yellow is dominant and the brownish purple recessive to the colourless
condition.

Peduncle. In this sub-series the peduncle rnay be erect or recurved, the
latter being in a majority in S. cernuum. In S. subglabrescens a half recurved
type is commonly met with in var. rubidum, compaction, irungiforme and
oviforme. Warty protuberances are also met with in certain types and these
are presumed to help in the liberation of the head from the boot.

Emergence of panicle. The heads are fairly well emerged in S. durra
and S. subglabrescens, but in S. cernuum the emergence is poor.

Panicle. In the Durra sub-series the panicle shape falls mostly in the

ovoid group 9 with variations in the degree of compactness. Within the
ovoid group the medium compact behaved as a simple dominant to the
compact type.

^ W2 ._The presence of awn is the most common feature in this group.
Of the three species, S. durra and S. cernuum-mostly rain-fed-consist almost
entirely of long awned types, while in S. subglabrescens both nil awned and
long awned types occur, with the latter in slight excess especially in the rain-
fed varieties. As already recorded, the yellow grain is almost invariably
associated with a long awn. The awn is greenish in the flowering stages
but the column takes on the tint of the glume on drying.

146 G. N. Rangaswami Ayyangar and others

Glume. In all the three species in this sub-series the glumes are thickly
coriaceous at the base and thinly coriaceous to herbaceous at the tip. In
S. cernuum and S. subglabrescens the herbaceous tip breaks and falls away
at maturity. Both the glumes (as in S. cernuum and the majority of
S. subglabrescens), or one only (as in the rest of S. subglabrescens), or neither
(as in S. durra) may be transversely wrinkled (Fig. 6). Glume wrinkling com-
mences in about 15 days after flowering, when the grains are in the dough
stage, and completes in about 10 days, by which time the grain becomes
fairly hard. Wrinkling tends to desiccate the upper half of the glume, with
the result that any colour present there gets bleached out, and hence in most
of the types of this group the glume is partially or completely bleached.'
Wrinkling has been recorded as dominant to its absence, 10 but it is not
stated whether one glume alone or both the glumes were wrinkled. In crosses
between types with both the glumes wrinkled and no wrinkling in any glume
the F! showed wrinkling on both the glumes, and the F 2 gave a 9 :7 ratio
of wrinkled to not-wrinkled. A heterozygous mutant with both the glumes
wrinkled occurred in' an irrigated Sen cholam (upper glume alone wrinkled)
and it gave a monogenic segregation between both glumes and upper alone
wrinkled. The relationship between the upper glume alone wrinkled and
the complete absence of wrinkling, and other aspects are under study. The
glumes in all these varieties are short nerved. In crosses with the long
nerved types of S. nerwsum the short nerved condition, behaved as a
simple dominant. 5

Grain. White, yellow and red are the most common grain colours met
with in this group; brown is rare. S. durra and S. subglabrescens contain
all the colour forms. In S. cernuum, on the other hand, white and red alone
are usually met; yellow and brown are very rare. The grain shape in this
sub-series varies from ovate to rotund or orbiculate with a wide range in the
size of the grain. The largest grain so far met with in sorghum is found in
var. niloticum and rivulare of S. durra and the most flat grain in the
Chaptijuar (S. cernuum) of the Central Provinces. A few types in this group
have dimpled grains. This character was first noted in a type of Sakkara-
guligajonna of Bellary classified under S. cernuum. Later on dimpled types
were found to occur in varieties of S. subglabrescens as well of this tract.

Pedicelled spikelets.-In this sub-series the pedicelled spikelets are
elliptic to lanceolate or linear-oblong in shape, sparsely to moderately
hairy, large, conspicuous and antheriferous, or small and neuter and
always persistent The pedicel is short. In red and yellow grafaSlS
the margins of the pedicelled spikelets turn red and brownish yellow

G. N. Rangaswami Ayyangar
and others

Proc. Ind. Acad. ScL, B, vol. A r K, PI. Ill

(a) $. durra

(b) S. cernuum
FIG. 1. Sorghum Panicles

FiG. 3. A Panicle showing
recurved (goose-necked)
peduncle

(a) Hairy (b) Glabrous

FIG. 5. Nodal bands

6-. 2V. K#ngasu<anii Ayyaiigar
and others

/W. Ind. Ac.id. Sci., /?, vol. XI-', PI.

FIG. 2. Panicle Branches

, . t ,

Sp.kelets and Grains (Front and Side views)

FIG. 6. Glume Wrinkling

wrinkled

The Grain Sorghums of the Durra Group

147

Chlorophyll colour grades. Most of the varieties of the Durra group
have green leaves. But certain types of S. subglabrescens particularly the
rain-fed Sen cholam have light green leaves. Dark green is the prevalent
colour in the African group Caffra. In crosses between the three grades
of green, dark green has proved a monogenic dominant to green, and
green a monogenic dominant to light green. A 9:6:1 ratio of dark
green, green and light green has been obtained connoting supplementary
factors.

Summary

A brief review of the three important species of the Durra group, v/z.,
5. duna, S. cernuum and S. subglabrescens, with -special reference to the
Indian and particularly to Madras varieties, 'is given. S. rigidwn, also
included in this group but which does not fit in with this, is omitted.
Detailed descriptions and distinguishing characteristics of the varieties of
these three species cultivated in the Madras Presidency are provided.
A short review of the important characters met with in this group has been
added at the end with special reference to panicle shape and glume wrinkl-
ing. Figs. 1-6 illustrate the different types of panicles, panicle branches,
spikelets, grain shapes, glume wrinkling and node hairiness in the different
species.

5.
6,

Snowden, J. D.
Ayyangar, G. N. R., Rao,

V. P.,andReddy,T. V.
___ , Ayyar, M. A. S,,

and Nambiar, A. K.
.__ and Rao, V. P.

Ayyangar, G. N. R.
, Rao, V. P., and
Nambiar, A. K.

_ and Ayyar, M. A. S.

and Nambiar, A. K.

10.

and Rajabhoosha-

nam, D. S.
Ramanathan, V.

LITERATURE CITED

The Cultivated Races of Sorghum, 1936.

" Studies in Sorghum Internodes and Leaf-sheaths," Proc.

hid. Acacl ScL, April 1938, 7, 4, 161-76.
ct The Inheritance of Height cum Duration in Sorghum," Mad.

Agri. Jour., April 1937, 25, 4, 107-18.

u Aerial Roots in Sorghum," Curr. Sci., April 1935, 3, 10, 485.
" Studies in Sorghum," Jour. Mad. Univ., 1939, 6, 2, 131-43.
" The Inheritance of some characters in crosses with the

Sorghums Milo and Kafir," Proc. Ind. Acad. Sci., Dec.

1935, 2, 6, 508-22.
" The Inheritance and Linkage affinities of Yellow coloured

Midrib in Sorghum," Curr. Sci., 1940, 9, 12, 542-43.

" Inheritance of characters in Sorghum- The Great Millet

VIII. A Brownish Purple Mutant," Ind. Jour. Agri. Sci.,

April 193,6, 6. 2, 481-83.
" A Preliminary analysis of Panicle structure in Sorghum,"

Proc. Ind. Acad. ScL, 1939, 9, 1, 29-38.
44 Some observations on Mendelian characters in Sorghum,"

Jour. Mad. Agri. Stud. Union, 1924, 12, 1-17.

AGE AND AFFINITIES OF THE BAGH FAUNA
BY DR. G. W. CHIPLONKER, D.Sc.

(From the Department of Geology, Benares Hindu University)

Received December 2, 1941
(Communicated by Prof. L. Rama Rao, M.A., F.G.S.)

IN spite of the varied interest stratigraphical, pateontological and pateo-
seographical, attaching to this series of strata, no adequate attention was paid
to their fauna until the present writer began his work. The author has in
a series of contributions to the palaeontology of these beds, 1 presented
the results of his study of the Echinoidea, Brachiopoda, Bryozoa, Lamelli-
branchia and the Ajnmonoidea, and discussed the age of these beds on the
basis of the affinities of each fossil group independently of the other ; so that
we possess satisfactory knowledge of all those members of the Bagh fauna
which could be studied with any exactness. Thus it is now that we are in a
position to discuss adequately the age of this series of strata and their alleged
faunal affinities with the Cretaceous deposits of the Trichinopoly District.

Among the earlier workers Carter 2 had assigned a Neocomian age to
these beds; while according to Bose 3 these strata stretched over a long
period extending from the Albian to the Senonian. The conclusions arrived
at by both these workers were, however, based on admittedly tentative identi-
fications of the fossils and as such their views carry little weight.

Duncan, 4 who for the first time studied in detail the echinoids from
these beds, because of the presence of

Nudeolites similis d'Orbigny

Salenia fraasi Cotteau

Cyphosoma cenomanense Cotteau

Hemiaster cenomanensis Cotteau
and H. similis d'Orbigny

of which the first species is from the Chloritic Marl and the remaining four
from the Cenomanian of Europe, and Lebanon thought it justified to assign
to the Bagh Beds an upper Green Sand horizon. This conclusion was
roughly corroborated by Vredenburg 5 from his study of the Bagh ammo-

i Chiplonker, 1937, pp. 60-71; 1938, pp. 300-16; 1939 a, pp. 236-46; 1939 b pp 98-109-

1939 c, pp. 255-74; 1941, pp. 271-76.
8 Carter, 1857, pp. 621-23.
8 Bose, 1884, pp. 37-44.
4 Duncan, 1865, pp. 349-63; 1887, pp. 81-92.
8 Vredenburg, 1907, pp. 109-25 ; 1908, pp. 23$

, pp. 239-40.

148

Age and Affinities of the Bagh Fauna 149

nites; while, according to Fourteau 6 both the echinoids and the ammonites
point to a lower Gault horizon.

More recently Mukerjee, 7 who was working on a small collection of
Mollusca from a few exposures of the Bagh Beds in the Jhabua and Ali
Rajpur States, has like Bose assigned to these beds a long period extending
from the Cenomanian to the Senonian, thus regarding them as approximate
equivalents of the Cretaceous Series of Southern India. Besides basing his
conclusions on extremely inadequate palaeontological evidence, as the present
writer has shown it to be in one of his earlier contributions to the palaeonto-
logy of the Bagh Beds, Mukerjee curiously enough thinks it reasonably
possible to compress the major portion of the Upper Cretaceous Period,
from the Cenomanian to the Senonian, in these poorly fossiliferous limestones
attaining a thickness of hardly forty feet as they do in Jhabua and Ali
Rajpur States.

To consider then from the present writer's work such of the more
important features of the palaeontological evidence as will help us to deter-
mine the age of the Bagh fauna as a whole, we find that out of the total
of forty-nine species which the author has recorded from these deposits,
we have only five species,

Neithea morrisi Pictet and Renevier
Plicatula batnensis Coquand
Hemiaster heberti (Coquand)
H. saadense Peron and Gauthier
and H. meslei Peron and Gauthier,

which are known to occur outside the Narbada valley. 8 Of these, the first
is a very common species in the various sections in the type area
around Chirakhan (lat. 22 IT 30" ; long. 75 7' 30"), and occurs in the
Albian and Aptian beds in England, Spain, Switzerland and Japan ; while,
the other four species are recorded from the Cenomanian beds in Algeria,
Tunis and Egypt.

Six, out of the seven species of Hemiaster represented in these beds 9
belong to the groups of Mecaster, Proraster and Integraster, all of which
make their first appearance in the Cenomanian. Further, we have in these
deposits the genus Diplopodia which is not known to survive the Cenomanian
age, while Hemiaster fourteaui Chiplonker, the commonest of the Bagh
species, has its nearest ally H. luynesi Cotteau in the Cenomanian of Palestine.

6 Fourteau, 1918, pp. 34-53.

* Mukerjee, 1935, p. 73 ; 1936, p. 81 ; 1938, pp. 193-98.

' Chiplonker, 1937, pp. 65, 67; 1939 a, pp. 241-42; 1939 c, pp. 258, 260.

Chiplonker, 1937, pp. 62-67 ; 1939 a, pp. 240-44.
Bla

150 G. W. Chiplonker

Among the remaining echinoids, while there is a mixture of lower and upper
Cretaceous affinities, the majority present an unquestionable lower Creta-
ceous aspect. Thus the echinoids on the whole point to a Cenomaman
(probably the lower portion, as is shown already) age. Among the ammo-
nites 10 which are represented by three species, Namadoceras scindice Vreden-
burg has probably Turonian affinities ; but Knemiceres mintoi (Vredenburg),
the commonest of the ammonites, has an aspect a little younger than the
Vraconian; while Namadoceras bosei Vredenburg has distinct middle Ceno-
manian affinities. The ammonites, therefore, on the whole point to a
middle to upper Cenomanian age. The brachiopod genus Malwirhynchia
which features very conspicuously in the type area for the Bagh Beds, has
unmistakable Upper Green Sand affinities^ The Lamellibranchia and the
Bryozoa, though, as is already remarked, show a mixture of affinities ranging
over a considerable part of the Cretaceous period, they, particularly the
Lamellibranchia, indicate middle Cretaceous as the predominant phase in
their affinities. 12

Thus, while discussing the age of the Bagh Beds on the basis of their
fauna as a whole, we are faced with a certain amount of diversity of evidence
as furnished by the various groups of fossils. It is, however, not an
uncommon occurrence ; because all groups of animals inhabiting a particular
basin of sedimentation do not necessarily, and often they do not, flourish nor
evolve at the same rate as those in the neighbouring basins. But each
group of animals is, however, bound to show more or less close affinities to
their allies in the adjoining basins, in accordance with the environments
as they affected them. Hence the inference of the age of the deposits con-
taining them, when based on the affinities of each group of animals sepa-
rately, is bound to be more or less different. Therefore, while fixing the age
of a formation we have to attach more weight, not to the whole range of affi-
nities shown by all the various forms, as was done by Mukerjee, 13 but to
the more predominant elements of the fauna and the general aspect as shown
by the assemblage of species.

In the present case we have recorded in these beds a few species of which
the age in other areas is definitely known ; while for the rest of the species we
have to rely upon their affinities towards species in other parts of the world.
Thus with the four Cenomanian species mentioned above and the definite
Cenomanian aspect of the echinoids, brachiopods and the ammonites which

10 Chiplonker, 1941, pp. 271-75.

11 Chiplonker, 1938, pp. 312-nl3.

12 Chiplonker, 1939 b, pp. 99-106; 1939 r, pp. 256-70.
18 Mukerjee, 1938, pp. 197-98.

Agt and Affinities of the Bagh Fauna 1 5 1

form the more dominant members of the Bagli fauna, we arc justified in
considering Cenomanian as the most appropriate age for these beds.

In the list of the Bagh fossils Bose mentions a number of South Indian
Cretaceous species. While admitting that they were only roug r ily identi-
fied, he endeavours to show with their help that the different members of the
Bagh Beds are approximately equivaUit to tho.se of ihe South Indian Creta-
ceous Series 14 ; and to explain what he considers as anomalous occurrences
of some of the South Indian species in the NLirbada valley, he u invokes
the idea of submergence of a land barrier intervening between the Narbida
valley and the Trichinopoiy District, duriig the "Nodular Limestone* 1
period, which could thus facilitate the intermigration of the faunas of these
two zoological provinces. Bose's work has already been sufficiently criti-
cised by Duncan, 10 and any further allusion to even the more important
features of his work would be nothing but repetition of Duncan's remarks.

The present author had an opportunity of seeing Makerjoe's moHuscan
collection from the Jhabua-AIi-Rajpur area; it is neither extensive nor well
preserved, and as mentioned on a previous occasion, 17 it needs a closer study
befoie his claim for the presence of Protocardiwn pondichcrricnsc cfOrbigny
and Cardiwn (Trachicardium) incomptwn Sowerby in the Bagh Beds could
be accepted. Turritclla (Zaria) multbtriata Reuss is another species in
Mukerjee's collection, which he lK mentions as a typical South Indian form,
U is neither a characteristic fossil in the Trichinopoiy deposits nor is it in any
way typical of South India. It is reported to be quite widely distributed in
Lybia and Central Europe. This species, however, as judged from the
numerous published figures and descriptions, appears to be a heterogeneous
group of, in all probability, related forms; and the specimens from the Bagh
Beds of Jhabua identified by Mukerjee as Zaria midtistriata Reuss, might be
found to belong to this stock from Central Europe. During the course of
the writer's study of the Bagh fossils Pitma mathuri C\\lp\Q\\k^i\ m is found to
be the only species in his collection which shows some distant relations to
P. arata from South India and P. vanh&peni from Pondoland, of which the
latter two species are again allied to European stock. These species there-
fore, cannot be considered as presenting a South Indian element in the Bagh
fauna; they rather add to the already abundant evidence which the present
author has brought forth from his study of all the different groups of fossils

" Bose, 1884, pp. 37-43, 48-50; Oldham, 1893, p. 250,
11 Bose, 1884, pp. 38-39.
l * Duncan, 1887, pp. 81-92.
17 Chiplonker, 1939 c, p. 271.
Mukerjee, 1938, pp. 197-98.
lf Chiplonker, 1939 c,. pp. 256, 270-71,
9*

152 G. W- Chiplonker

from the Bagh Beds, to show that, the fossil fauna of the Narbada valley
belongs to the Mediterranean zoological province and had no direct connec-
tion across the Indian Peninsula, with that V the Southern Ocean.

Acknowledgments

In conclusion, the author has to sincerely thank Dr. Raj Nath for the
keen interest he took in this work on the Bagh Beds. Thanks are also due
to the Director of the Geological Survey of India for the permission to work
in the Survey Museum and Library. The author takes this opportunity of
expressing his indebtedness also to the Dhar, Indore and Gwalior Durbars
for the kind permission to collect fossils from within their territories and for
the facilities accorded to me during the field work, but for which this work
on the Bagh Beds would not have been possible.

1. Base, P. N.

2. Carter, H.J.

3. Chiplonker, G. W.

4.
5.

8. -

9. Duncan, P. M.

10.

11. Fourteau, R.

12. Mukerjee, P.

13.
14.

15. Oldham, R. D.

16. Vredenburg, E.

17.

BIBLIOGRAPHY

"Geology of the lower Narbada valley between Nimawar and

Kawant," Mem. G.S.I., 1884, 21, pt. 1.
"Neocomian fossils from Bagh and its neighbourhood presented

by Lieut. R. H. Keatinge " in " On the contribution to the Geo-
logy of Central and Western India," Journ. Bom. Br. Hoy. As.

Soc. 9 1857, 5, 614-38.
"Echinoids from the Bagh Beds," Proc. Ind. Acad. Sci. (B), 1937,

6, No. 1, 60-71.

" Rhynchonellids from the Bagh Beds," ibid., 1938, 7, No. 6, 400-16.
"Echinoids from the Bagh Beds Part II," ibid., 1939 a, 9, No. 5

236-46.

44 Bryozoafrom the Bagh Beds," ibid., 19396, 10, No. 1, 98-109.
44 Lamellibranchs from the Bagh Beds," ibid., 1939 c, 10, No. 4

255-74.

44 Ammonites from the Bagh Beds," ibid., 1941, 14, No. 3, 271-76.
4 "Description of the Echinodermata fronv the strata on the South-

Eastern Coast of Arabia and at Bagh on the Narbada in the

collection of the Geological Society," Q.J.G.S., 1865, 21, 349-63.
"Notes on -Echinoidea of the Cretaceous Series of the Lower

Narbada valley with remarks upon their Geological age," Rec.

G.S.I., 1887, 20, 81-92.

4i Les fechinides des Bagh Beds," ibid., 1918, 49, pt. 1, 34-53.
In 4t General Report of the Geological Survey of India for the year

1933," ibid., 1935, 68, 71-73.
In "General Report of the Geological Survey of India for the year

1934," ibid., 1936, 69, 80-81.
In Gupta, B.C., and Mukerjee, P. N., "Geology of Gujarath and

Southern Rajputana," ibid., 1938, 73, pt. 2, 163-208.
A Manual of Geology of India, 1893, 2nd Edition.
"The Ammonites of the Bagh Beds," Rec. G.S.L, 1907, 36, pt. 2,

109-25.

"Additional note concerning a previous notice on The Ammonites of
the Bagh Beds," ibid., 1908, 36, pt. 3, 239-40.

ON THE INTERNAL BUNDLES IN THE STEM OF
RUM EX PATIENTIA L.

BY P. MAHESHWARI AND BALWANT SINGH

(Dacca University, Dacca)
(Received November 18, 1941)

/. Introduction and Previous Work

A FEW years ago, one of us (Muheshwari, 1929) investigated the anatomy
of Rumex crispits and found that the inflorescence axis of this plant possesses
a number of internal and inverted bundles in addition to the normal outer
ring of vascular bundles. A detailed study of their origin and development
revealed that they are not formed by the division of pith cells as was
previously supposed (Herail, 1885) but from the inner portions of the same
procambial strands which produce the normal bundles. It was also
demonstrated that the bundles are not merely inverted or obcollateral
but actually become concentric (amphivasal) due to the extension of
the cambium all round the phloem. The xylem in the internal bundles
is entirely secondary in origin, while the phloem is both primary and secon-
dary. A maximum of five internal bundles was found in association with a
single normal vascular bundle.

Since then Joshi (1930, in a brief note on the anatomy of Rumex den-
tatus, mentions that while the stem is normal in structure and devoid of any
internal bundles, there arises occasionally in the basal internodes a peri-
cyclic cambium, which forms an accessory ring of bundles between the cortex
and the piimary vascular ring.

Wiirke (1933), in a work dealing with the anatomy of the rhizome of
Rheum, also makes some casual remarks on the anatomy of a few species
of Rumex. R, patientia^ R, crispus and R. domesticus were found to have
bicollateral bundles, whose internal phloems later on give rise to inverted
bundles. Further details are not mentioned and after a general discussion
and confirmation of Maheshwari's work on R. crispus, the author passes
on to Rheum, which forms the main part of his contribution.

2. Observations

The material used in the present study was collected by one of us 2 in
1936, from the subutbs of Kiel, during an excursion with Prof. G. Tischler,
and consisted of a few pieces of the inflorescence axis preserved in fornialin-

* According to Herail the internal bundles of JR. patientia consist of phloem only.
a I am grateful to Drs. W. Gauger and H. D. Wulff of the Botanical Institute, Kiel, who
were present ia the excursion and helped me in the collection (P. Maheshwari).

100

154 P. Maheshwari and Balwant Singh

acetic-alcohol. Sections were cut freehand and stained in Safranin and Fast
Green.

A cross- section of an inter node shows a hollow pith and a single ring
of vascular bundles which at first appear to be quite normal. A closer exami-
nation reveals, however, the presence of internal phloem in many of the
bundles which are thus truly bicollateral. The external surface is provided
with ridges "and farrows, with collenchyma occurring underneath the former
(Fig. 1).

Fio. 1 . Diagram of a portion of a t.s. of the inflorescence axis of Rumex patientia. In this and
the subsequent figures the sclerenchymatous sheath is represented by cross lines, phloem
by dots, cambium by a single layer of cells, xylem parenchyma in black, and xylem vessels
by empty spaces, x 33.

Sections of the older internodes presented a still larger number of bi-
collateral bundles, the internal phloem lying completely within the sclerenchy-
matous sheath of the outer bundle (Fig. 2) and not scattered in patches at the
periphery of the pith as is the case in the Convolvulacese, Solanaceae, Apocy-
naceae, Asclepiadacese, etc. The older of the phloem groups showed a
distinct cambium on their outer side, just facing the protoxylem (Fig. 5).
This cambium has a clear tendency towards lateral extension and later
completely surrounds the phloem (cf. Rumex crispus). Most of its activity
is however confined to the outer side only, resulting in the formation of

On the Internal Bundles in the Stem of Rumex patientia L. 155

obcollateral or inverted bundles. The xylem in this bundle is obviously all
secondary but the phloem is partly primary and partly secondary. The
central region of the phloem, which is primary, is particularly rich in sieve
tubes. In a few cases the internal bundles were ob-bicollateral with phloem
in the centre and xylem on both sides (Fig. 4) or amphivasal (Fig. 3) with
xylem vessels or xylem parenchyma completely surrounding the phloem.

FIGS. 2-8. Figs. 2-6. Vascular bundles of the inflorescence axis of Rumex patientia showing
various stages in the development of internal bundles, x 173. Fig. 7. A normal bundle
of Rumex crispus with two adjacent internal bundles, x 173. Fig. 8. A normal and
an internal bundle of Rheum rhaponticum L. x 173.

J56 P. Maheshwari and Balwant Singh

The oldest pieces of the stem, available to us, showed conspicuous
internal bundles. In some sections, 3 such bundles were found to be associated
with a single outer bundle, all enclosed in a common sheath of sclerenchyma
and forming a distinct unit (Fig. 6). In rare cases a single internal bundle
lies in association with 2 partially anastomosed normal bundles (Fig. 5).

For comparison, we examined some previously prepared slides of Rumex
crispus and found that there is no essential difference in the stem anatomy of
the two species. In one case, in R. crispus, we found two internal bundles
detached from the sheath and lying at some distance inward in the pith
(Fig. 7). Their deeper position does not mean, however, that such bundles
actually arise through a division of pith cells. It is more likely that a
sclerification of the cells between the outer and inner bundles was probably
delayed for some time and when it did take place, each side formed its own
sheath, leaving some unlignified parenchyma between.

Such a separation of the internal bundles which is rare in Rumex, is
common in the allied genus Rheum (Fig. 8). According to Wiirke the
conditions in the two genera are closely similar in the earlier stages but later
on the internal bundles of Rheum get detached from the normal vascular
ring due to a division of the intervening cells.

We do not deny the possibility of the formation of internal bundles
from pith cells in other plants. Indeed, this has been conclusively
demonstrated in the case of tobacco, (Esau, 1938), where pith cells resume a
meristematic character and give rise to groups of sieve tubes irregularly
arranged in the perimedullary zone.

3. Conclusion

The phylogenetic or physiological significance of the internal bundle
has been discussed by several authors. Worsdell (1915, 1919) considers
the medullary phloem to represent a vestigial structure, the remnant of a
former system of medullary vascular bundles, in which the xylern has dis-
appeared and adds that " the morphological origin of this internal phloem
bundle is from an amphivasal bundle, for the latter is the typical and more
primitive form of the medullary phloem bundles, wherever they occur *\
The inversely oriented internal bundles are explained by supposing that only
the outer portion of the originally amphivasal bundle is retained.

Maheshwari (1929), in his study of Rumex, adduced evidence to show
that the presence of internal bundles is an advanced character, the species
with higher chromosomes being generally found to possess them and those
with the lower numbers lacking them. Wiirke (1933) is in complete agree-
ment with this view. Alexandrov and Alexandrova (1926) and Hartwich

On the Internal Bundles in the Stem of Rumex patientia L. 157

(1936), working on the internal bundles of the inflorescence axis of Ricinus
communis, also regard their presence as derived.

In order to test the hypothesis that the species of Rumex with higher
chromosomes are likely to be provided with internal bundles while those
with lower numbers are likely to be without them, it is necessary to examine
more material belonging to a variety of forms. With an instrument like
colchicine in hand, it may also be possible to induce polyploidy either in the
same species or its hybrids and then examine them anatomically to see if this
change is associated with the appearance of the internal bundles. This
would open up a new field of experimental anatomy, that would probably
lead to the solution of other problems.

4. Summary

The inflorescence axis of Rumex patientia has bicollateral bundles
whose internal phloems give rise to obcollateral, ob-bicollateral and amphi-
vasal bundles. Sometimes two or three internal bundles lie in association
with a single outer bundle. It is concluded that the presence of such
bundles is an advanced character. Further, the condition in Rheum, where
the internal bundles lie detached from the outer ones, is easily derived from
that in Rumex.

5. LITERATURE CITED

1 . Alexandrov, W. G., and

Alexandrova, O. G.

2. Esau, K.

3. Hartwich, W.

4. H<5rail,H.J.

5. Joshi,A.C.

6. Maheshwari, P.

7. Worsdell,W.C.

^ Wiirke,H.

" t5"ber Koncentrische Gef&ssbundel in Stengel von Ricinu

communis" Bot. Arch., 1926, 14, 455-60.
*' Ontogeny and structure of the phloem of tobacco," Hilgardia*

1938, 11, 343-99.
, '* Uber die Entstehung der leptozentrischen Leitbfindel in der

Inflorescenz-Achse von Ricinus communis" Diss. Berlin,

1936, 40.
. " Recherches sur Tanatomie compare de la tige des Dico-

tyledones," Ann. Sci. Nat. Bot., 1885, VII, 2, 203-314.
"Anomalous secondary thickening in the stem of Rumes

dentatus L.," Jour. Tnd. Bot. Soc., 1931, 10, 209-12.
" Origin and development of internal bundles in the stem of

Rumex crispus," ibid., 1929, 7, 89-177.
"The origin and meaning of medullary (intraxylary) phloem

in the stems of Dicotyledons. I. Cucurbitaceae," Ann.

Bot., 1915, 29, 567-90.
" The origin and meaning of medullary (intraxylary) phloem

in the stems of Dicotyledons. II. Composite," ibid.,

1919, 33, 412-58.
" Untersuchungen iiber die Maserbildung beim Rhabarber-

rhizom," Diss. Berlin, 1933, 40.

THE EMBRYO-SAC OF EUPHORBIA
HETEROPHYLLA L.-A REINVESTIGATION

BY P. MAHESHWARI

(Dacca University, Dacca)
Received November 18, 1941
/. Introduction and Previous Work

FROM the point of view of floral morphology and embryology, the family
Euphorbiacese has been of great interest for a long time. No less ^ than 8
different types of embryo-sacs have been reported which are shown diagram-
matically in Fig. 1. Most of the plants exhibit the Normal-type with a
monosporic, 8-nucIeate gametophyte. Modilewski 1 (1909, 1910, 1911)
found a tetrasporic, 16-nucleate type in Euphorbia procera and E. palustris,
which is now designated . as the Penaa-form and has since been reported in
Acalypha australis (Tateishi, 1927). In Mallotus japonicus (Ventura, 1934)
also, 16 nuclei are present but they are organised differently an egg appa-
ratus, two polar nuclei and eleven antipodal cells (Drusa-form). Arnold!
(1912) thought that he had found 4-nucleate embryo-sacs developing from a
single megaspore (Oenothera-type) in Glochidion, Codiceum and Ceramanthus
(Phyllanthus). This claim has been disproved in the case of the last two
genera by Lundberg (1931) and Maheshwari and Chowdry (1936) respec-
tively and it seems that Glochldlon will also yield, similar results on reinvesti-
gation. It is certain that Arnold! missed the antipodal cells in his prepara-
tions and was thus led to a wrong interpretation (for a detailed discussion
see Maheshwaii, 1937).

1 In his "Introduction" Sanchez (1938) quotes Modilewski's work (1910) on Euphorbia
procera as follows:

44 In this species, instead of only one surviving megaspore dividing to form the normal
8-nucleate embryo-sac, all the four megaspores divide simultaneously without exception
so that each of them after two successive divisions gives rise to a 4-nucleate embryo-sac.
At this stage, one of the 4-nucleate embryo-sacs divides twice to form a mature
16-nucleate embryo-sac, while the other three degenerate."

This is obviously a misunderstanding of the original in German. What Modilewski really
says t is that there are several megaspore-mother cells, all of which may undergo reduction
division and become 4-nucleate. Only one divides further, however, and forms a 16-nucleate
embryo-sac. He summarises the situation as follows (p. 417):

"Eine von den vierkernigen Embryosackmutterzellen entwickelt sich zu einem reifen

sechzehnkernigen Embryosack, wahrend die ubrigen degenerieren "
158

Embryo-sac of Euphorbia heterophylla L> A Reinvestigation 159

Remarks

Occurs in by far the
largest number of investi-
gated genera and species.

Reported in Codixum.
Ceramamhus and Glochi-
dion by Arnoldi H912).
First two cases already
disproved (sec M aheshwari,
J937) ; the third is also
extremely doubtful.

Reported in: Euphorbia
matiritanica (Ventura, 1933).

Reported in :
Eurphorbia procera

OT.odilewski. 1909)

E.paluftris (Modi!ewsl:i,
1911)

Acalypha indica
(See -i aheshwari and
John, 1940)

Probably occurs in
Euphorbia dulcis
(Carano, 1926)

Reported in WaWotus
/apon/cus (Ventura, 1934)

Reported in Euphorbia
heterophylla (Sanchez,
1938). Dealt with in the
present paper and shown
to be incorrect.

FIG. 1. Diagram showing the different types of embryo-sac development
reported in the Euphorbiaceae

160 P. Maheshwari

Carano (1926) noted that in Euphorbia dulcis three of the four nuclei,
formed after the first two divisions, pass down to the chalazal end of embryo-
sac and during the next division the spindles of these 3 chalazal nuclei fuse
together resulting in a secondary 4-nucleate stage. This by a further division
gives rise to an 8-nucleate embryo-sac organised in the normal fashion.
A comparison with Bambacioni's figures of Frit illaria persica (1928) seems to
indicate that the development is identical.

Yet another variation was noted by Ventura (1933), who reports an
Allium-type of embryo-sac in E. mauritanica, and D'Amato 2 (1939), in a
work dealing with several spp. of Euphorbia, reports that in most cases the
Normal-type was observed, but some spp. occasionally or regularly show the
AHium-type.

More recently Maheshwari and Johri (1940) have published a prelimi-
nary note on the embryo-sac of Acalypha indica, in which 16 nuclei, formed
by 2 divisions of the megaspores, are sometimes organised as in Euphorbia
virgata and Acalypha australis, but more frequently form four pairs of 2 cells
each leaving 8 nuclei to fuse in the centre. Several other irregularities have
been noted and described in the full paper which is in the press. 3

The embryo-sac of Euphorbia heterophylla, which forms the subject of
the present paper, was first investigated by Modilewski (1910), who reported
a Normal-type of embryo-sac in this and several other species of the same
genus. His description of this species being very brief and unillustrated,
Sanchez (1938) reinvestigated it, and found an Adoxa-typz of embryo-sac!
From his figures and descriptions, however, this appeared so doubtful that
a reinvestigation was undertaken.

2. Observations

The material was collected from some plants growing in the Government
Nursery, Dacca, and in the writer's private garden. Nawaschin's fluid and
formalin-acetic-alcohol were used for fixation. The sections were cut at
10 /i and stained in iron-haematoxylin.

A description of the ovary and ovules has already been given by Sanchez
and my observations agree with his. The growth of the integuments is very
tardy but the nucellus is well developed. The inner integument starts first
at about the time the megaspore-mother cell is already well formed but is
soon surpassed by the outer. '

* * **

Embryo-sac of Euphorbia heterophylla L. A Reinvestigation 161

With regard to the early development of the embryo-sac Sanchez writes
as follows :

" Following the synezetic contraction the macrospore-mother cell
proceeds to usual heterotypic division, resulting in the formation of two
daughter nuclei. These nuclei move and lie opposite each other at the ends
of the enlarged macrospore-mother cell (Fig, 3 B) 4 one toward the micro-
pylar end and the other toward the chalazal end. A large vacuole is formed
between them while the two daughter nuclei become surrounded with dense
protoplasm. From this stage the macrospore-mother cell grows rapidly
especially in width (Fig. 3 B).

The two daughter nuclei divide simultaneously in the usual manner
into 4-nucleL These nuclei are not separated by walls, but remain in pairs
separated by the large central vacuole. All the four macrospores become
functional because they all participate in the formation of the female gameto-
phyte or embryo-sac. Thus none of them degenerated or disintegrated as
observed in the normal type of embryo-sac development, where only one

surviving macrospore becomes functional The third division seems to

take place immediately after the four nuclei are fully developed, for the
4-nucleate embryo-sac stages are comparatively few.'*

A critical study of the figures presented by Sanchez, the more important
of which are reproduced here (Fig. 2) reveal a great gap between the mega-
spore-mother cell and the 2~nucleate stage (compare also his diagrams of the
ovules at this stage, Fig. 2 a-c). The beginning of vacuolation even before
the reduction divisions are over is most unlikely. As pointed out by Rutgers
(1923), Fagerlincl (1938) and Maheshwari (1941), polarity in the embryo-sac
begins only after the formation of the megaspores. There is no case where it
is known to commence immediately after the first reduction division as
supposed by Sanchez.

Indeed it was this discrepancy in the drawings of Sanchez that suggested
this reinvestigation.

My observations show that the megasp ore-mother cell stage is identical
with that figured by Sanchez (Figs. 3 and 4). The first reduction division
is followed by wall formation and the second division also proceeds quite
normally in the lower dyad but is frequently delayed in the upper. A row of
four cells is formed in most cases but sometimes the division is incomplete
in the upper dyad and only 3 cells are then observed (Figs. 5-8).

4 This is reproduced here as Fig. 2 e.

162

P. Maheshwari

FIG. 2. Drawings of megaspore-mother cell and 2-nucleate and 4-nucleate embryo-sacs (copied
from Sanchez, 1938). Figs, a, b and c show diagrams of ovules at the stages shown in
d, e and /.

The lowest cell of the tetrad enlarges and functions. I was able to see
the 3 degenerating megaspores quite distinctly in several preparations and can
therefore state definitely that the development is not of the Adox&-type but of
the

Vacuolation begins first only after megaspore formation has been com-
pleted and the chalazal cell has enlarged appreciably. The subsequent stages
showing 2, 4 and 8 nuclei are passed through normally and are identical with
those figured by Sanchez. It is therefore unnecessary to duplicate them
here.

3. Discussion

The above observations remind one of exactly similar errors committed
by some other authors.. Perhaps, Typha latifolia presents a more or less

it
I

Embryo-sac of Euphorbia heterophylla L.A Reinvestigation 163

identical case. Schaffner (1897) studied the embryo-sac of this plant and
mentions having taken extreme care in tracing out the development step by
step. A row of megaspores was " never seen " and the development was
stated to be of the Adoxa-typt. Nevertheless, Dahlgren (1918;, twenty
years later, proved that the 4 cells are formed as usual and it is the chalazal
megaspore which functions to give rise to the embryo-sac.

FIG. 3. L.S. ovule at megaspore~motbcr cell stage, x 300. FIG. 4. Nucellus with megaspore-
mothercelL x 1,050. FIGS. 5,7. L.S. ovule with stages in megasporogenesis. x 300.
FIGS. 6, 8. NuccIU of ovules in Figs. 5 and 7 respectively, x t,050.

It is a great pity that several authors have not drawn any detailed
drawings but merely given diagrams showing the number of nuclei. It is
impossible to discuss these cases, but other errois of interpretation, where
at least the illustrations were executed with sufficient care, have often been

164

P, Maheshwari

rectified without much difficulty. The chief traps for workers on embryo-
sac development are either in connection with megasporogenesis or the
organisation of the mature embryo-sac. With regard to the former it may be
stated that as a rule vacuolation and polarity in the embryo-sac follow the forma-
tion of megaspores and are never seen before the reduction divisions are over.
Thus, the megaspore-mother cell and dyad stages are free from any conspi-
cuous vacuoles. The early tetrad stage also does not show appreciable
vacuolation, which starts first only after the four megaspores (or megaspore
nuclei) have been formed and the next stage is about to commence. This
rule applies to all embryo-sacs, whether monosporic, bisporic or tetrasporic.
Thus, the difference between the four-nucleate stage of a monosporic embryo-
sac and a tetrasporic one is very well marked (Fig. 9). The former shows

Megaapore) Megatporog'enesis

Type

mother cell |M e i os j s j | MeiosJs II

Vacuolation
and Polarity

Development of the embryo sac

Mature
embryo sac

Monbsporic
8-nucleate

Bisporic

8-nucleate

',A///um-type]

Tetrasporic
8-nucleate
Adoxa-typTe)

FIG. 9. Diagram to show the stage at which vacuolation begins in the development of
monosporic, trisporic and tetrasporic embryo-sacs

a large central vacuole with the nuclei and cytoplasm limited to the periphery,
while the latter begins to show a central vacuolation only at a later stage,
just preparatory to the next division, and even then this is far less appreciable
than in the monospbric and bisporic embryo-sacs. Note also the difference
between the 2-nucleate stage of a monosporic and a tetrasporic embryo-sac
(Fig. 9). The well-marked polarity and vacuolation in Figs. 3 b and 4 a
of Sanchez ( reproduced here as Figs. 2eand2/) entirely go against the
interpretation that they are pre-reduction stages.

Embryo-sac of Euphorbia heteroptiylla L. A Reinvesti gallon 165

I should like to state here that I do not consider vacuolation and
polarity to be an infallible means of judging whether the enibryo-sac is niono-
sporic or tetrasporic, but I agree with Rutgers (1923) that it serves as a good
and fairly reliable guide, which merits proper consideration.

The other error, concerning the stage immediately preceding the organisa-
tion of the mature embryo-sac, is also well exemplified in the Euphorbiaceae.
Arnoldi (1921) mistook the normal 8-nucleate embryo-sacs of Codimim and
Phyllanthus for 4-nucleate because of the early fusion of the polar nuclei
and the disappearance of the antipodals. Sometimes the antipodal cells
(or nuclei) may be in a narrow pouch that is easily missed in thin sections.
In still other cases they become laterally placed due to a downward
extension of the main body of the embryo-sac (see Kajale, 1940, and the litera-
ture quoted therein).

4. Summary

A reinvestigation of the embryo-sac of Euphorbia heterophylla shows
that the development is not of the^afoxa-type as reported by Sanchez (1938)
but of the Normal-type. The megasporc-mother cell gives rise to a tetrad
of megaspores of which the chalazal cell functions to produce a normal
8-nucleate embryo-sac.

It is pointed out that as a rule vacuolation and polarity are not seen in
embryo-sacs until after the reduction divisions are over. They form such
reliable guides that all cases, where the reported observations are not in
accordance with the above, deserve a fresh study.

5. Acknowledgements

In conclusion, I wish to express my gratefulness to my wife and sister
for the assistance they gave in the preparation and staining of the slides
and to my colleague and friend Mr. Reayat Khan for going through the
typescript.

LITERATURE CITED

Arno | di w _ * 2ur Embryologie einiger Euphorbiaceen," Trav. Mus. hot.

' * Acad., St. paersbourg , 1912, 9, 136-54.

Bambacioni V. .. "Ricerche sulla ecologia e sulla embriologia di Fritillaria

' * persicaL." Ann. Bot. (Roma), 1928, 18, 7-37.

Cararto, E. "Ulterior! osservazioni su Euphorbia dulcisL. in rapporto

col suo comportamonto apomittico," ibid., 1926, 17, 50-79.

Dublin K V O ** Die jiingeren Entwicklungsstadien der Samenanlagen von

* " ' * Typha latifolia L.," Svensk. hot. Tidskr., 1918, 12, 207-11.

D'Amato F ** Ricerche embryologiche e caryologiche sul genere Euphor*

' ' * biar Nitovo Gior. Bot. Ital., 1939, 46, 470-509.

166

P. Maheshwari

Fagerlind, F.

Kajale, L. B.
Lundberg, F.
Maheshwari, P.

- and Chowdry, O. R.

and John, B. M.

Modilewski, J.

Rutgers, F. L.

Sanchez, S. T.
Schaffner, J. H.
Tateishi, S.

Ventura, M.

"Wo kommen tetrasporiscjie durch drei Teilungsschritte

vollentwickelte Embryosiicke unter den Angiospermen

vor?" Sot. Notiser, 1938, 461-98.
"A contribution to the embryology of the Amarantacecc,"

Proc. Nat. Inst. ScL, India, 1940, 6, 597-625.
*' Bemerkungen iiber die Embryosackentwiklung bei Codiceum"

Bot. Notiser, 1931, 346-49.
* 4 A critical review of the types of embryo-sacs in Angio-

sperms," New PkytoL, 1937, 36, 359-417.
** Recent work on the types of embryo-sacs in Angiosperms

A critical review," Jour. Ind. Rot. Soc., 1941, 20, 229-61.
"A note on the development of the embryo-sac in Phyllan-

tkus niniri Linn.," Curt: Sci., 1937, 5, 535-36.
"A note on the embryo-sac of Acalypha indica L. s " ibid., 1940,

9, 322-23.
"Zur Embryobildung von Euphorbia procera" Ber. dtsch.

hot. Ges., 1909, 27, 21-26.
"Weitere Beitrsge zur Embryobildung einiger Euphorbiaceen",

ibid., 1910, 28, 413-18.
"Uber die abnormale Embryosackentwiclclung bei Euphorbia

palustris L. and anderen Euphorbiaceen," ibid. 9 1911, 29,

430-36.
" Embryo-sac and embryo of Moringa oleifera. The female

gametophy te of Angiosperms," Ann. Jard. hot. Suitenzorg,

1923, 33, 1-66.
41 Embryo-sac development in Euphorbia heterophylla Linn.,"

Univ. Philippines Nat. & Appl. Sci. Bull., 1938, 6, 59-75.
" The development of the stamens and carpels of Typka lati-

folia," Bot. Gaz.> 1897, 24, 93-102.
44 On the development of the embryo-sac and fertilisation of

Acalypka austraHs L. (Preliminary note)," Bot. Mag. Tokyo,

1927, 51, 477-85.

" Sviluppo del gametofito femminile di Euphorbia mauritanica

L.," Ann. Bot. (Roma), 1933, 20, 267-73.
" Sulla poliembrionia di Mallotus japonicus Muell. Arg.,"

Ibid., 1934, 20, 568-78.

175M1 Pnnted t The bangalore Press, Bangalore City, by G. SnnivAsa Rao, Superintendent,
and Published by The Indian Academy of Sciences, Bangalore.

A LEAF SPOT DISEASE OP ZINGIBER OPFICINALE
CAUSED BY PHYLLOSTICTA ZINGIBERI N.SP.

BY T. S. RAMAKRISHNAN, M.A. *

(Agricultural Research Institute, Coimbatore)

Received October 17, 1941
(Communicated by Rao Bahadur V. Ramanatha Ayyar)

IN 1938 and succeeding years a leaf spot disease has been reported on ginger
from Godavari and Malabar districts. The disease is common in the months
of August, September and October. The spots vary in size. Some are small
and roundish being a millimeter in length and half in breadth. Others are
oval or elongated having a size of 9-10 x 3-4 mm. (Fig. 1). The spots
are almost white in the centre and have a dark brown margin; Just surround-
ing the spot is a halo of yellowish colour. The central portion is thin and
papery and more often torn up. In this portion are also seen a number of
minute blackish pycnidia. The pycnidia are formed immersed in the tissues
of the leaf under the epidermis. But later they become erumpent and can be
seen distinctly on the surface as the mesophyll tissue collapses and the leaf
becomes thin in the affected areas. The spots are usually isolated but they
may also become confluent resulting in big patches. Sometimes a large
number of spots develop on a leaf and in consequence the entire leaf turns
brown and dries up.

Microscopic examination revealed that the pycnidia are those of Phyllo-
sticta. Each pycnidium measures 78-150^ in diameter and has a definite
ostiole. When mounted on slide the characteristic worm-like mass of spores
coming out of the ostiole can be seen under the microscope. The spores are
hyaline, oblong and measure on an average 4-3x 1-6 p, the range being
3 .7_7.4x 1-2-2-5/a.

Other leaf spot diseases have been recorded on ginger. Sundararaman
(1922) has described Colletotrichum zingiberece as the cause of a leaf spot
disease in Godavari district of the Madras presidency. Stevens and Atienza
(1932) have reported from the Philippines a leaf spot of ginger caused by
Coniothyrium zingiberi. In the description of the fungus they have stated
that it may be mistaken for a Phyllosticta. The same has been observed
in Hawaii (1937).

Examination of the specimens from Godavari and Malabar districts
showed only Phyllosticta on the spots. The fungus was readily brought into

167

T' S. Ramaknshnati

culture by transferring bits of affected portions of leaves to french bean agat
plates after having previously sterilised them by immersion in mercuric
chloride solution (1/1000 strength) for 2 minutes and washing in sterile
water. In a week's time pure growths developed and began to form pycmdia
on agar. JFrom these further isolations were made.

The fungus grows readily on agar media. The following statement
gives the growth characters of the fungus on the different media tried.

TABLE I

Growths on Different Media

Medium

Nature of growth

French bean agar
Quaker oats agar

Potato dextrose agar . .
Richards' agar
Sterilised ginger leaves

Thin, greyish white aerial growth, pycmdia in plenty on the

medium or slightly immersed, zones faintly visible.
Thick growth, aerial growth white, but submerged portion

dark olive, pycnidia in plenty but hidden by the aerial

mycelium. *
Thick growth, aerial growth smoke grey, submerged growth

dark-olive, zones visible, pycnidia numerous.
Thick growth, serial mycelium creamy white, submerged

growth dark, margin irregular, pycnidia formed.
No aerial growth but entire leaves studded with numerous

pycnidia.

On culture media the pycnidial formation starts on the 4th or 5tii day. The
pycnidia are light in colour in the beginning but with age the colour deepens
and finally they turn light to deep brown. They are isolated or in groups.
Each, pycnidium has an ostiole and a very short neck. Sometimes a pycni-
dium shows two ostioles (Fig. 4) in all probability formed by the fusion of
two pycnidia. The wall of the pycnidium is thin. The pycnidia formed in
cultures are much bigger than those in nature. Some are spherical but in most
cases they are only subglobose. They measure on an average 177 - 6 JLC (range
100-270^). The ratio of the two diameters is 1:1-1.

The hyphae are hyaline or coloured. Sometimes several hyphae unite
to form strands. Coloured hyphae are common on potato dextrose, quaker
oats and Richards' agars. On french bean agar and sterilised ginger leaves
coloured hyphae are rare. The hyphae very often form swollen cells of various
shape's. Round glistening bodies are found inside these and some coloured
hyphse (Fig. 6).

The spores are hyaline and oblong with rounded ends. They are often
biguttulate (Fig. 5). Even after keeping in culture for over three years no
Qploured spore was ever noticed in any of the cultures.

Leaf Spot Disease of Zingiber Officinate caused by P. zingiberi n.sp. 169

The fungus grows well over a wide range of H-ion concentration. It
was grown on Richards' agar of different pH values and the diameters of
growths are represented below :

TABLE II
Growth on Media of Different pH Values

pH value of media

3-5

4-3

4.9

5-8

6-7

Diameter in mm. in 7 days ..

63-5

62-5

The best growth is formed between 4-3 and 5-8 with a falling off on both
sides of this range.

Pathogenicity. The parasitism of this fungus was tested by inocula-
tions on the leaves of ginger plants. When bits of culture and spore suspen-
sions from growths on agar media, were used, successful infections were
obtained only when the leaves were previously wounded. The controls and
inoculations made on unwounded surfaces remained healthy without deve-
loping any pathological symptoms. But inoculations on wounded leaves
produced small water-soaked spots on the third day. Later there was an
increase in size of the spots. The central portions of these spots became
yellowish and thin and still later dried into white membranous patches
showing tearing of the tissues (Fig. 2). Pycnidia developed in the central
portions in 8 days.

Wound infections were successful on the leaves of turmeric- (Curcuma
longd). Spots with white thin membranous centres developed and in these
pycnidia were formed.

Inoculations were made on ginger leaves using cultures grown on
sterilised ginger leaves. Two series of experiments were conducted, one within
6 months of the isolation of the fungus and another after two years. In the
earlier inoculation experiments successful infection was obtained on un-
wounded ginger leaves but it took a longer time for the spots to develop.
On wounded leaves evidences of infection were noticed in 60 hours but on
unwounded leaves these were visible only after 6 days. In the second series
of experiments an isolate which had been for two years in culture was grown
on sterilised ginger leaves and spore suspensions from this were used
for inoculation purposes. But no successful infection was obtained when the
leaf surface was free from wounds. The parasitism of the old culture was
not improved by one passage through sterilised portions of the host tissue.
In nature injuries caused by insects might help in easy infection of leaves.

170 T. S. Ramakrishnan

The symptoms of the disease agree with those described by Stevens and
Atienza (1932). But the fungus under study is undoubtedly a Phyllosticta
whereas the fungus responsible for the disease in the Philippines is described
as a Coniothyrium though the authors state that it may be mistaken for
Phyllosticta. The local isolate did not show any coloured spores though it
has been under observation for over three years. Stevens and Atienza give
the range of spore size as 3-5-4 x 7-10 p but the average is not given. The
average of 200 measurements of the spores of the local isolate is 1 -6 x4-3/z
the range being 1-2-2 -5x3 -7-7 -4ft. This is decidedly much less than that
recorded for the Philippine organism. For these reasons and since no
Phyllosticta has been recorded on ginger till now the local organism is named
Phyllosticta zingiberi.

The diseased plants were obtained from the same village where Sundara-
raman (1922) had first noticed leaf spot caused by Colletotrichum zingiberece.
But this fungus was not noticed on any of the specimens.

Control At present this is not a very serious disease of ginger.' But
it has been observed to be common in Godavari and Malabar districts and
in some years causes a reduction in yield of rhizomes due to the destruction
of large areas of chlorophyllous tissue. Preventive measures have been
carried out against this disease with success in Godavari district. The plants
are sprayed with 1% Bordeaux mixture before the outbreak of the disease
and ones again if necessary and these operations are reported to have given
good protection against infection.

Phyllosticta zingiberi. Spots oval or elongated, centre whitish, pycnidia
on both sides of the spot, subglobose, dark brown in colour, ostiolate,
pycnidia from infected plants 78-150/4 in diameter; spores hyaline one-celled,
oblong, 4-3xl-6/L, (3 7-7 4 x 1 2-2 5) biguttulate. On culture media
pycnidia generally larger.

Habitat. In spots on the leaves of Zingiber officinale.

Phyllosticta zingiberi. Maculse ovales vel elongate, centro subalbidas;
pycnidiis in utraque superficie maculae, subglobosis, colore fuscis, ostiolatis ;
pycnidiis plantarum infectarum diametro 78-1 50/x; (pycnidiis autem medif
culture generatim latioribus); sporis hyalinis, unicellularibus, oblongis
4-3 x 1-6/A (3-7-7-4X 1-2-2-5) biguttulatis.

Habitat. Maculae foliorum Zingiberi officinalis.

The typs specimen is deposited in the herbarium of the Government
Mycologist, Agricultural Research Institute, Coimbatore, S. India.

S. Ramakrishnan

Proc. Ind. A cad. Sci., /!, vol. XY\ PI. V

Phylhsticta zingiheri n.sp.

Diseased leaves from nature, FIG. 4. Group of pycnidia (diagrammatic).

Spots formed on inoculated leaves. FIG. 5. Spores. x600.

A pycnidium from culture. x200. FIG. 6. Irregularly swollen hyphse from culture.

> 600.

Leaf Spot Disease of Zin giber Officinal* caused by P. zingiberi n.sp. 171

I am thankful to Mr. K. ML Thomas the Government Mycologist who
has helped me in various ways during this investigation. I am indebted
to Rev. Fr. Balan, s.J., of St. Joseph's College, Jrichinopoly, for the latin
translation of diagnosis.

Summary

A leaf spot disease caused by Phyllosticta zingiberi is common in Goda-
vari and Malabar districts. Spots with whitish centres develop on the
leaves and in these pycnidia of the fungus are formed. Wound inoculations
were successful on ginger and turmeric. Soon after isolation, cultures on
ginger leaves are able to infect unwounded ginger leaves.

This fungus does not agree with the description of Coniothyrium zingi-
beri. The spores are smaller and never coloured. Hence it is given the name
of Phyllosticta zingiberi.

LITERATURE CITED

1 .' Sundararaman, S. . . Mem. Dep. Ag. Ind., 1922, 11, 209-17.

2. Stevens, F. L., and Aitenza, Phil. Agriculturist, 1931-32, 20, 171 .

J.D.

3. . . Report Haw. Ag. Exp. Sta., 1937, 44.

THE ORIGIN OF SIPHONOSTELE IN THREE
SPECIES OF SELAGINELLA SPR.*

BY GIRIJA P. MAJUMDAR

(Department of Botany, Presidency College, Calcutta)

Received October 29, 1941
(Communicated by Dr. H. Chaudhuri)

Introduction

SEVERAL types of steles are found in the vascular plants, but in the earliest
of these plants, such as, the Psilophytales, the Psilotales, several fossil and
living Lycopodiales and a few of the living ferns, particularly in their seedling
stages, the axes are characterised by a solid hadrocentric stele. This solid
xylocentric stele is, therefore, taken to be the primitive stele or the protostele
from which all the other types of steles are believed to have been derived in
the course of phylogenetic specialisation.

One of such derivatives is the siphonostele with pith or medulla in the
centre. Two types of siphonostele are met with, the ectophloic siphonostele
and the amphiphloic siphonostele. The former has phloem only on the out-
side of the tubular xylem. This is by far the most common type found in
the axes of Gymnosperms and Angiosperms 5 and in ferns, like Osmunda.
The amphiphloic siphonostele, on the other hand, has phloem on both
sides of the xylem cylinder and is represented in the living ferns, like Adiantum,
Pteris, Osmunda cinnamomea, Todea hymenophylloides? Ophioglossum vul~
gatum, Botrychium Lunaria, etc., and also in some families of herbaceous
Angiosperms. 5

So far three possible ways of pith formation have been suggested :
(1) The pith is the included cortex or the fundamental tissue. Hence its origin
is extrasteler. (2) The pith represents the undifferentiated xylem elements.
Hence the origin is intrastelar. (3) The pith is partly extrastelar and partly
intrastelar in origin, as in the Osmundaceae and Ophioglossacege. 2

According to Van Tieghem (1890), Jeffrey, 8 Gwynne-Vaughan, 2 Tansley, 14
and Boodle, 1 the pith is extrastelar in origin in the rhizomatous solenostelic
(= siphonostelic) ferns. Jeffrey 9 further stated that in all cases the pith must
be regarded as derivatives of the cortex, i.e., the origin of pith in all cases is
extrastelar.

* Read before the monthly meeting of the Botanical Society of Bengal 1941
172

The Origin of Siphonostele in Three Species of Selaginella Spr. 173

From his extensive studies of the anatomy of Pteridophytes, both fossil
and living, Bower 3 generalises that the origin of the medullation is determined
by two factors, viz., the position of axes and insertion of the appendages.
He concludes that (1) in all upright columner microphyllous stems the pith
is tracheary in origin, i.e., intrastelar, and he cites Lepidodendreae and the
cone of living Selaginella spinulosa as examples; (2) in all creeping mega-
phyllous shoots, e.g., the rhizomatous ferns, the pith is extrastelar in origin
(p. 562). Eames and MacDaniels 5 , however, think that it has been estab-
lished beyond doubt that in seed plants at least the pith is morphologically
extrastelar; in most of the Pteridophytes the same condition obtains, prob-
ably in a few the pith is intrastelar in nature (p. 114).

Thus there is still a divergence of opinion 'as to the origin of pith in the
microphyllous upright stem of the Pteridophytes. A great difference of
opinion also prevails with regard to the origin of the internal phloem and
internal endodermis of the amphiphloic siphonostele. It will, therefore,
be interesting to follow the origin of pith in three of the most specialised
species of Selaginella with upright microphyllous axes.

These are: S. incequalifolia Spr., S. Wallichii Spr., and S. canaliculata
Baker. Specimens of S. incequalifolia were procured from the Orchid House
of the Royal Botanic Gardens, Calcutta, and those of S. Wallichii and
S. canaliculata were supplied by a student from his garden house at Dumdum,
a suburb of Calcutta, All the three species were kindly identified by the
Curator of the Herbarium, Royal Botanic Garden, Calcutta. The observa-
tions are based upon freehand sections cut serially from base upwards of
materials preserved in formol-acetic-alcohol and stained in safranin and fast

green,

Observations

S. inwqualifolia (Figs. 1-3) is tri-stelic with an accessory stele in the
upright stem. The accessory stele gradually enlarges and unites with one
of the lateral steles to form the siphonostele in the upper region of the stem,
and in so doing encloses a mass of extrastelar ground tissue which forms the
pith. In this case, therefore, the pith is distinctly extrastelar in origin.

S. Wallichii (Figs. 4-7) is also tri-stelic with an accessory stele in the
lower region of the upright stem. Here also the accessory stele enlarges
and unites with a lateral stele to form the amphiphloic, siphonostele. In this
species almost all the stages in the origin of the siphonostele from separate
steles can be traced. The pith is clearly extrastelar in origin, and the character
of the pith cells are strikingly similar to those of the cortical cells. The
similarity in this case is certainly not " merely physiological " as is assumed
by the opponents of the theory of the extrastelar origin of pith,

174 Girija P. Majumdar

5. canaliculata (Figs. 8-12) resembles the other two species in having
almost the same structure in the upright stem. Here also the siphonostele
in the upper region of the stem has been formed by the union of a lateral and
the accessory steles, and more or less a complete series in its origin can be
followed. The stage shown in Fig. 12 appears to be different from any
described above. It seems that a single stele has enlarged and bent in such
a way that the two free ends have come to meet with the result that a mass of
extrastelar ground tissue has been enclosed. A siphonostele with pith has
thus been formed.

Discussion

So far as the writer is aware only two cases of medullations in living
Selaginella have been reported : one in the creeping rhizome of S. Icevigata
Baker var. Lyallii Spr., reported by Gibson 6 where the stelar arrangement
very closely resembles that of a typical Filicinean amphiphloic siphonostele
with ramular gaps. Bower 3 admitted with Jeffrey 9 that the pith in this case
is extrastelar in origin and cites the case as an illustration of his hypothesis
that the origin of pith in creeping microphyllous form may be extrastelar by
adjustment to resemble that in rhizomatous ferns.

The other case is the axis of the strobilus of S. spinulosa A. Br. reported
by Bower, 3 who found pith in all stages of development in the stele in serial
sections from base upwards. He regarded this to be a clear case of intra-
stelar origin of pith in a stem where there were no ramular or foliar gaps
" to provide that continuity with the cortex without which cortical intrusion
cannot take place'*. But a doubt certainly arose in his mind as to the real
nature of this pith when a longitudinal section through the strobilus revealed
the tracheidal nature of its cells (elongated), and the presence of nucleus and
protoplasm in them. Prof. Bower anticipated that " it may be argued that
the softer central tissues are merely the result of imperfect development of
tracheids and that they would mature into tracheids later," but stated " that
the tissue have the appearance of maturity while the condition of the sporangia
and of the strobilus as a whole shews that further development is not to be
expected/* Convinced thus of the intrastelar origin of pith in the strobilus of
S. spinulosa he cited this case as an illustration of his general hypothesis that
the origin of pith in the upright microphyllous stock is always intrastelar.

Mitchell, 11 who also worked out the anatomy of the strobilus in S. spinu-
losa, on the other hand, reported that the vascular system of this organ is
essentially simpler than it is in the vegetative axis. It has a single vascular
chord with typically 8 marginal protoxylem groups. The metaxylem con-
sists of small elements which are frequently not thickened towards the centre

The Origin of Siptionostele in Three Species of Selaginella Spr. 175

and there may be more or less a well-marked procambial area extending
from the tip downwards.

It may, therefore, be suggested that the so-called pith which Prof. Bower
observed in the strobiius of this species, but the presence of which has not
been corroborated by other workers in the young or mature, upright or
creeping stem, is a collection of undeveloped tracheids or ill-differentiated
metaxylem. A comparison of the figures given by Bower (Figs. 1 and 5
PL XLVI1), Gibson (Fig. 3, PL IX) and Mitchell (Fig. 9, PL VIII), strengthens
the suggestion made here. Other examples of this type are not rare. Prof.
Gwynnc-Vaughan in the sporeling of Osmunda regalis noticed the presence
of parcnchymatous cells surrounding xylem tracheids, which he thought
" may be undiffcrcntiated cells in the process of differentiation as they still
have well-developed nuclei." Bower's figure of the t.s. of the rhizome of
Ophioglossum rcticulatum points to the possibility of the few parenchymatous
cells noticed in the otherwise solid xylem core as being still undifferentiated
tracheids. The present writer's observation on the structure of the creeping
stolons that arc annually given off from the base of the erect rhizome of
Nephrolepis cxaltata Schott. shews that the central procambium takes a very
long time to differentiate into metaxylem proper.

When the above facts arc taken into account with the mode of the origin
of the modulation in the upright sterns of the three species of Selaginella
described In this paper Prof. Bower's general hypothesis of the intrastelar
origin of pith in the upright microphyllous forms seems to need revision. It
is seen that the medullation here has nothing to do with the ramular or foliar
gaps, and the pith is definitely of extrastelar origin. The presence of internal
phloem and internal cndodermis is satisfactorily explained as due to the
origin of the arnphiphloic siphonostele from the polystelic condition and as
a result of fusion of more than one steles. One need not conceive of their
origin (or presence) as 4i entirely de now ", or by " decurrency through the
branch gaps into the pith ", or by " involution at the leaf gap," or by " gradual
encroachment upon the pith and then by imagination," or that in their
origin they " crept round the edges of the branch gaps," or " have subsided
into them *\

Selaginella belongs to the class Lycopsida where the steles are not
characterised by leaf gaps, and there is no provision in their living represen-
atives for secondary growth in their axes. The axes of the species under
investigation grow erect and sometimes attain a great length. To meet the
increasing demand for conducting and mechanical tissues the vascular
cylinder here enlarges not by the activity of a cambium which is absent, but

176

Girija P. Majumdar

by Increasing the number of steles which results in the polystelic condition
observed in these species. A similar conclusion was previously reached by
Scott 12 working on the origin of polystele in dicotyledons. He stated that the
need for the enlargement of the vascular system is met by increasing the
number of steles rather than the size of a single central cylinder. Bower 4
also developed the same idea of the expansion of the conducting and mecha-
nical tissues in his Size and Form in Plants.

Conclusion

The observations recorded in the foregoing pages thus warrant the
following general conclusion with regard to the origin of the amphiphloic
siphonostele in Selaginella :

The origin of the siphonostele in Selaginella is correlated with the poly-
stelic condition and the amphiphloic siphonostele originated as a result of
a fusion of a number of separate steles. The polystelic condition probably
originated in response to the necessity of increasing the amount of conduct-
ing and mechanical tissues in the absence of provision for secondary growth.
The pith is extrastelar in origin and the presence of internal phloem and
internal endodermis is directly due to the origin of the siphonostele from the
polystelic condition.

My thanks are due to Dr. A. C. Joshi for his kindly going through the
MSS. and many helpful suggestions which have considerably enhanced the
value of the paper.

1. Boodle, L. A.

2. Bower, P.O.

5. Eames, A. J., and
MacDaniels, L. H.

6. Gibson, R.J.H.

7. Gwynne-Vaughan, D. T.

8. Jeffrey, B.C.

REFERENCES

" Comparative anatomy of the Hymenophyllacea?, Schizseacese .
and Gleicheniaceas. IV. Further observations on Schizsea "
Ann. Sot., 1903, 17, 511.

" On the primary xylem and the origin of medullation in the

Ophioglossaceae," ibid., 1911,25, 537.

" On the medullation in the Pteridophyta," ibid., 1911, 25, 555,
Size and Form in Plants, 1930.

An Introduction to Plant Anatomy, 1925.
"Contributions towards a knowledge of the anatomy of the
genus Selagmella. L The stem," XJIH. Sot., 1894, 8, 133

f the

o fi h * central ^ler in vascular plants,"

Rep. Bnt. Assocn., Toronto, 1897, 869; London, 1898,
The Anatomy of Woody Plants, 1917, 283-91.
" Tbe Pteropsida," Sot. Gaz., 1910 ? 401 ,

The Origin of Siphonostele in Three Species of Selaginella Spr. 177

11. Mitchell, G. . . " Contribution towards a knowledge of the anatomy of the

genus Selaginella Spr. V. The Strobilus," Ann. Bot.,
1910, 24, 19.

12. Scott, D. H. . . " Origin of polystele in dicotyledons," ibid., 1891, 5, 514.

13. Tansley, A. G. . . " Lectures on the evolution of the Filicinean vascular system.

Lecture V. The evolution of the solenostele," New Phyt.,
1907, 6, 148.

14. t f "Lectures on the evolution of the Filicinean vascular system.

Lecture IX. The leaf-trace. Ontogeny," //</., 1908, 7, 1.

EXPLANATION OF FIGURES IN PLATES VI AND VII

All figures are photomicrographic representations

FIGS. 1-3, Plate VI. S. inaequalifolia Spr. Transverse sections at different levels of the upright

stem. Stages shewing the union and coalescence of the ventral lateral and accessory steles

to form the amphiphloic siphonostele with extrastelar pith.
FIGS. 4-6, Plate VI and FIG. 7, Plate VII. S. Wallichii Spr. Same as above. The stages are more

complete.
FIGS. 8-12, Plate VII. S. canaliculata Baker. Same as above. Fig. 12 shews a stage where the

lateral stele alone appears to have formed the siphonostele.

THE GENUS CEPHALOGONIMUS IN INDIA AND

BURMA

BY G. D. BAHLERAO, D.Sc., PH.D.

(From the Imperial Veterinary Research Institute, Izatnagar)
Received February 26, 1942

BETWEEN 1930 and 1937 five species of the genus Cephalogonimus, have been
recorded from India: C. emydalis Moghe, 1930, C, magnus Sinha, 1932,
C. gangeticus Pande, 1932, C. mehri, Pande, 1932 and C. minutum Mehra,
1937. Chatterji (1936) recorded C. burmanica from a tortoise in Burma.
Several writers in the past have utilised the relative position of the testes
(tandem or oblique), the comparative size of the suckers, the position of the
genital pore, the presence or absence of the oesophagus, the length of the
intestinal caeca, the extent of the vitellaria and the cirrus sac, the position of
the ventral sucker, the nature of the receptaculum seminis and the position
of the ovary as criteria for distinguishing one species of the genus from the
other. Sinha (1932) has also included the terminal part of the excretory
bladder in distinguishing the species C. gangeticus from C. lenoiri. The
writer has found in his studies of these characters that the relative position of
the testes (tandem or oblique), the shape of the ovary (almost rounded or
transversely oval) and the comparative size of the two testes are variable
in different individuals of the same species. The comparative sizes of the
oral and the ventral sucker appear to be a constant feature : in all the speci-
mens from Lissemys punctata the oral sucker was invariably larger than the
ventral sucker. The position of the genital pore and the terminations of the
intestinal caeca were also quite constant. Unnecessary importance has been
attached to the presence or absence of the oesophagus. As a matter of fact,
a very short oesophagus at least is always present. In certain species, how-
ever, its presence cannot be detected in toto preparations. In such cases,
this organ can only be seen in a sectionised specimen. On examination of
some individuals of the same species the extent of the vitellaria was found to
be extremely variable. This has already been pointed out by the writer
in 1936. Usually the left vitelline gland is the longer but, in rare instances,
the right side gland was found to be the longer. The location of the ventral
sucker in the body varies from the anterior fourth to the anterior third.
The position of the ovary and the termination of the cirrus sac with respect
to the ventral sucker are also somewhat variable. The shape of the

178

The Genus Cephalogonimus in India and Burma 1/9

receptaculum seminis among the Treniatoda is dependent on the number of
spermatozoa it contains. In regard to the terminal part of the excretory
bladder, it may be remarked that this is a generic character and not a
specific one. The omission of a reference to this point is not to be regarded
as indicating its absnece in the species concerned.

In the light of the remarks offered above, it is found that the species
C. mehri and C. minutum are quite distinct and can be separated from all
the known species of the genus. In regard to C. burmanica, Chatterji (1936,
85) mentions that the intestinal caeca terminate " a little anterior to the
posterior end of body". In the text-figure 3, on p. 85, however, the caeca
are shown to terminate a little behind the testes and at a distance much in
front of the posterior end of the body. If the figure, which is very distinct,
is taken to be correct representation of the parasite, it more nearly approxi-
mates to the species C. europceus Blazoit, 1910. From this species it can,
however, be distinguished by the extent of the vitellaria, the position of the
genital pore and by the length or the oesophagus.

It has already been pointed out by the writer (Bhalerao, 1936) that the
species C. magnus and C. gangeticus are identical and that the latter is a
synonym of the former. It was further pointed out that both these species
are synonymous with C. amphiumce Chandler, 1923. A recent examination
of some material at the disposal of the writer has convinced him that C. amphi-
uma is quite distinct from C. magnus as in the former species the intestinal
caeca terminate very close to the posterior end of the body, while in the latter
they terminate midway between the hinder border of the posterior testis and
the posterior end of the body. It has previously been remarked that this
character is not subject to variations as are some other organs. Further,
on comparing C. magnus with the description of C. emydalis as given by
Moghe (1930) and from some material at the disposal of the writer, it is
found that in regard to the internal anatomy the two species are quite
identical. Pande (1932) states that C. gangeticus (syn. of C. magnus) differs
from C. emydalis in the subterminal position of the oral sucker, the aceta-
bulum being situated more towards the posterior end, in the presence of an
oesophagus, in the cirrus sac not being coiled on itself, in the position of the
ovary, the form of the receptaculum seminis and in the location of the uterus
and the vitellaria. Examination of some material at the disposal of the
writer revealed that, in C. emydalis also, the oral sucker is subterminal, the
ventral sucker is situated between the anterior third and fourth of the body,
the oesophagus is very small, the cirrus sac usually coils during contraction,
the position of the ovary and the extent and disposition of the vitelline

180 G. D. BahleraO

follicles are always variable and the uterus completely fills up the post-testi-
cular region of the body. The only respect in which C. magnus differs from
C. emydalis is the relatively large size of its body and organs. It is therefore
considered that C. magnus is merely a large variety of C. emydalis and not
a species distinct from the latter.

REFERENCES

Bhalerao, G. D. . . " Studies on the Helminths of India. Trematoda II, "

/. Helminth, 1936, 2, 181-206.
Chatterji, R. C. .. "The Helminths parasitic in the Fresh-water Turtle of

Rangoon," Rec. 2nd. Mus., 1936, 38, 81-94.
Moghe, M.A. . . "A new species of Trematode from an Indian Tortoise, "

Ann. Mag. Nat. Hist., 1930, Ser. 10, 6, 677-81.
Pande, B. P. . , " On two new species of the genus Cephalogonimus Porier

from water tortoises of Allahabad with remarks on the

family Cephalogonimida Nicoll," Bull. Acad. Sci., Allahabad,

1932, 2, 85-100.
Sinha,B.B. .. "On the morphology and systematic position of Cephah-

gonimus magnus, sp, n. (Trematoda) from Trionyx gange-

ticus," Ann. Mag. Nat. Hist., 1932, Ser. 10, 10A, 419-28.

BIONOMICS AND CONTROL OF AEOLESTHES
HOLOSERICEA F. (CERAMBYCID^E: COLEOPTERA)

BY KHAN A. RAHMAN, B.Sc. Ao. (EDIN.), PH.D. (CANTAB.)
P.A.S., CLASS I

AND

ABDUL WAHID KHAN, M.Sc. Ac. (Pb.)

(Punjab Agricultural College and Research Institute, Lyallpur)

Received January 13, 1942
(Communicated by Dr. Hamid Khan Bhatti, F.A.SC.)

Aeolexthes hohsericea F. was collected for the first time by the Director, Im-
perial Forest School, Dchra Dun, in 1889 from Sal and Terminalia tomentosa.
Stcbbing (1914) made some observations on the life-history of this pest be-
tween 1901 09 and also described the adult. Beeson (1941) recently discussed
its life-history, economic importance, food-plants and control. Nevertheless,
information as to the duration of its various stages, seasonal history and con-
trol (when the attack is in progress) has remained singularly meagre and in
the present paper an attempt is made to throw light on these points.

Distribution

Stebbing (1914) collected it from U.P., Oudh, Hyderabad, C.P., D.I.
Khan and Ganjam; Gahan from N.W. India, Bombay, Nilgiris, Ceylon,

Assam, Andaman, Nicobar, Siam and Malay Peninsula and Lefroy secured
it from Bengal. According to Beeson (1914) A. hohsericea is distributed
throughout the greater part of the forest of India; " it extends up the sub-
montane valleys of the Himalayas to considerable elevation, occurs in the
Indus plains and in thcSundarbans, in moist forests, and in dry, and in Ceylon,
Burma, the Andamans and Nicobars". In -the Punjab we have found it
between the altitudes of 3,500 to 8,000 ft. above sea-level in Kulu, Bandrol,
Raison, Katrain, Naggar and Manali in the Kulu Valley, Kotgrah and
Simla in the Simla Hills,

Food-Plants

Stebbing (1914) found it on the following plants : Sal (Shorea robustd),
Terminalia tomentosa, Hardwickia binata, Chloroxylon swietenia, Tamarix
afticulata, Acacia arabica, Guava (Psidium guava), Mango (Mangifera indicd).
Beeson (1941) mentions the following as its food-plants : Aegle marmelos,
Alnus nitida, Anogeissus latifolia, Bauhinia acwninata, Bauhinia retusa,

181

182 Khan A. Rahman and Abdul Wahid Khan

Bauhinia variegata, Bombax malabaricum, Bridelia retusa/ Butea frondosa,
Careya arborea, Cedrela toona, Cynometra ramiflora, Duabanga sonnera-
tioides Eucalyptus robusta, Excacaria agallocha, Ficus bengalensi^ Grewia
oppositifolia, Kydia calycina, Largerstramia parviflora, Lannea grandis,
Mallotus philippinensis, Miliusa velutina, Morus alba, Myristica andamamca,
Ougeinia dalbergioides, Pentacme suavis, Pinus longifolia, Prunus commum^
Pterocarpus marsupium, Pyrus communis, Quercus incana, Sapium sebiferum,
Shorea assamica, Soymida febrifuga, Ttctona grandis, Terminalia balerica,
Terminalia myriocarpa. We collected it from Kosh (Alnus nitida\ Cherry
(Prunus avium), Apple (Pyrus malus,) Crab apple (P. baccatd), Apricots
(Prunus armeniaca}, Walaut (Juglans regia,} Plum (Prunus domzsticd), Peach
(Prunus persica) and Mulberry (Moms alba).

Description of Various Stages

^ag.Egg 2 -25 mm. long, 1-0 mm. broad, elliptical, tapering towards
either extremity, micropylar end slightly broader with a small petiole.

L arV a. Larva yellow with dark brown head. When full-grown it
measures 75mm. in length and 13 -5 mm. in breadth. Its body is clothed
in fine bristles which are abundant on the thorax and the last abdominal
segment. Antenna 4-segmented, 1st segment thick, rest minute with sen-
soria. Prothorax larger than meso-thorax. Thoracic legs persent. Abdo-
minal segments possess series of tubercles on their dorsal and ventral
aspects. Spiracles, pit-like, elliptical, brownish.

Pupa. Pupa yellow, 42mm. long, 35mm. broad, Head small, slightly
deflected. Thoracic sterna and 2nd and 7th abdominal segments covered
with bristles. Last abdominal segment bifurcated, curved dorsally.

Female. Female measures 32 mm. in length, 10 mm. in breadth. It is
dark brown in colour with silvery or golden reflections on the elytra. Antenna
11-segmented, basal segment small, rounded, ,2nd segment with wrinkled
surface, 3rd to 10th segments sfender but thickened at their distal ends, llth
segment very thin and tapering. Prothorax wrinkled and furrowed. Elytra
with well-developed shoulders inner edge of each of which terminates in a
small sharp spine.

Male. Male resembles the female but is smaller with smaller antennae,
the last segment of each of which is flattened dorsally.

Life-History

Females lay eggs from May to October, They select injured areas on
the bark, more often previously attacked parts of the stem, for egg-laying.

Bionomics &

0/Aeolesthes holosericea F.

183

They make minute incisions on the injured edges of the bark into which
th^y push their eggs singly and longitudinally with their ovipositors. 1-5
eggs are laid daily. In confinement a female laid a maximum of 92 eggs in
its life-time.

Eggs hatch out in 7-12 days. The youn^ larva bites irregular holes in
the egg-shell most of which it usually eats. Larva on hatching feeds upon
the inner layers of the bark in shallow and zig-zag galleries, but when it is a
few days old it starts feeding on the inner layers of the bark and outer layers
of the sapwood making shallow, wide, zig-zag and long galleries. When
almost grown up the larva enters the main wood through a self-prepared
kidney-shaped entrance hole which later on serves as an emergence hole for
the beetle. The larval stage occupies from 2 years 3 months and 5 days to
2 years and 8 months. If the larva is full-grown by October it pupates after
a rest of 3 to 25 days only, but when it becomes full-grown in November, the
pre-pupal period may be prolonged to 4J-5 months. Such larvae pupate in
the middle of April

The grub constructs a pupal chamber the distal end of which it plugs
with a brownish white matter. The pupa lies naked in this chamber. The
pupal stage occupies from 1 month 10 days to 3 months 10 days.

If pupation occurs in October the beetle is formed within the chamber

in December to February, This beetle remains quiescent in the stem
throughout the winter and spring, its resting period varying from 3 months
11 days to 5 months 2 days. But when the larva enters the resting stage in
winter and pupates in April, the beetle has a shorter resting period of
1 month 10 days to 1 month 17 days.

TABLE I

Eggs
laid

Eggs
hatched

Adults

emerged

15- 6-38
17- 6-38

22- 6-38
25- 6-38

26-4-41
26-4-41

3- 6-38

12 738

264~4I

13- 6-38

22- 8-38

25-5-41

15- -6-38

24- 9-38

25-5-41

4-10-38

16-10-38

30-5-41

14-10-38

26-10-38

2-6-41

DURATION OF STAGES

Life-cycle

completed

Egg

Larva

Pupa

days

rn. d.

m. .

months; d.=* days

184

Khan A. Rahman and Abdul Wahid Khan

From the date of oviposition to the date of emergence of the beetle its
life-cycle is completed in 2 years 7 months to about 3 years.

Seasonal History

Beetles begin to appear on the wing towards the end of April and con-
tinue to do so upto the middle of July. They live upto the end of October.
The annual calendar of activities of the pest is as follows :

Months

Stages present

April

May- June . .
July

August-September
October
November . .
December-March

One-year old grubs and 2-year old resting grubs present;

beetles start emerging
Beetles keep on emerging and laying eggs
Beetles continue to emerge; and oviposit-grubs present. Pupae

scare

Beetles, eggs and grubs present
Beetles, eggs and grubs present ; old grubs pupate
Grubs present, old grubs start resting ; few pupate
Active and resting grubs and immature beetles present

Damage

The attacked tree is recognised by the fibrous and faecal matter which
falls from the larval tunnel to the ground below. The bark around the
attacked area splits up. Attacked sherry trees suffer from gummoses also.

Grubs only do damage. When young they feed upon the inner parts of
the bark and the outer parts of the main wood, each grub eating out as much
as half a square foot of it Older grubs bore into the main wood and injure
the inside of the stem. The damage done to the bark is more serious and when
more than one grub feed in the stem the tree dies ; young apple plants, how-
ever, are killed by a single grub.

The damage done by the beetle itself is negligible. It feeds upon the
damaged area of the stem and interior of the tunnels made by the grubs.
The beetle spends the day hiding in the tunnel or beneath the injured
bark; it flies at night. It has never been seen feeding upon leaves.

Control
I. Prevention of oviposition

1. Painting solignum on injured areas on the stem. The females lay
eggs in the edges of the injured bark. Therefore, when such areas were painted
with solignum, the beetles refrained from laying eggs. Young apple
or cherry trees should be painted every summer because young trees when
attacked succumb within a month or two. Such trees are usually attacked
on the stem quite adjacent to the soil. This part, therefore, must receive
an occasional examination and treatment.

han A. Rahman

and Abdul II "ah id Khan

Proc. I ml. A cad. Sci., B, vol. XV, PL VII

Fir,, 1. Adult, ti. female; /, male, IMC,. 2. Kgg. FIG. 7. Egg slit.

I ; i; *i. I,arva. n, clorsal view; /, ventral view.

FIG. 4. Pupa.

Fir.. 5. Pupal ctmmher. Km, H. Ilok* (/) thn

a, chamber frtms which adult which fsccal matter (F)
has emerged; /;, chamber is pushed out.

from which bcetkt has not

FIG. 6. Emergence holes.

bionomics & Control of Aeolesthes holosericea F.

//. Killing grubs in their tunnels

The tunnel is cleaned to some extent with an iron wire or knife and
cotton wool soaked in kerosene oil is introduced into it. This is finally
plastered with clay mud. The fumes of kerosene oil penetrate into the
tunnel, reach the grub or pupa and kill it. Others have suggested potassium
cyanide crystal or a mixture of petrol and chloroform and though these
chemicals are also successful they are expensive and not so readily available
as kerosene oil which is also the cheapest. During 18th June-28th September
1940, 109 trees were treated with kerosene oil and cent, per cent, mortality
of the pest was obtained.

LITERATURE

Stebbing, E. P. . . Indian Forest Insects, 1914, 301.

Beeson .. Forest Insects, 1914, 136.

PHRAGMOTELIUM MYSORENSIS, A NEW RUST
ON INDIAN RASPBERRY

By*M. J. THIRUMALACHAR

(Department of Botany, Central College, Bangalore)

Received January 13, 1942
(Communicated by Prof. T. S. Raghavan)

THE genus Phragmidium founded by Link in 1824 contains several hetero-
genous elements and four species that were originally placed in it, have been
made the types of four new genera: Hamaspora Kornicke, Phragmotelium
Sydow, Frommea Arthur and Earlea Arthur. Of these the first three genera
were segregated from Phragmidium because of easily distinguishable morpho-
logical characters and they are at present accepted by urediniologists. The
genus Earlea erected by Arthur (1906) to provide for those species of Phrag-
midium which lacked uredia in their life-cycle, is not accepted by Sydow (1915)
or Dietel (1928), and Arthur himself now states that emphasis which was
formerly placed upon the succession of spore-forms, should be transferred,
because of a better understanding of the development of the rusts, to the
vegetative states. The presence or absence of uredia "which according to
Arthur, belong to the same state as telia cannot therefore be used to set off
Earlea as a separate genus.

Of the first three, the genus Phragmotelium is characterised by telio-
spores which have smooth walls and which germinate soon after ripening
without any rest period. Their pedicels are not well developed and they do
not swell in water. Aecia are further more lacking and their place is taken
by aparaphysate primary uredia which form the first spore-form. As
against this, the teliospores in Phragmidium are warty or verrucose, and they
germinate only after a long rest period. Their pedicels are better developed
and they swell when placed in water. Ttie aecium which is the first spore-
form in this genus is of the caeoma type.

Sydow (1921) transferred seven species of Phragmidium to Phragmo-
telium and three more species are mentioned by Hiratsuka (1935) whose
logical position is in the genus Phragmotelium. He does not recognise the
genus Phragmotelium, which according to him is a section of Phragmidium
a view with which the present author does not agree. The three species
involved are Phragmidium formosanum Hirats P. Rubi-fraxinifolii Syd. (tenta-
tively placed by Sydow in this genus as he had not seen the teliospores which
186

Phragmotelium mysorensis, a New Rust on Indian Raspberry 187

were later discovered by Hiratsuka), and P. Okianum Kara., and it is clear
that they should be transferred to Sydow's genus as Phragmotelium formo-
sanum (Hirats) comb. nov. Phragmotelium Okianum (Kara.) comb. nov.
and Phragmotelium Rubi-fraxinifolii (Syd.) comb. nov.

Yet another species which is new has been recently found by the author
on Rubus lasiocarpus Smith. It distinguishes itself from Phragmotelium
burmanicum (Syd.) Syd., recorded on the same host, by its much larger and
many septate teliospores. Indeed comparison of descriptions shows that
this new species is easily the largest teliospored form for the genus. It

FIG. 1
Leaf of Rubus lasiocarpus showing infection, x nat

188 M. J. Thirumalachar

resembles Phragmotelium griseum (Diet.) Syd. But this species has telio-
spores with three to four germ pores, a thicker epispore, fewer septa, slightly
smaller size, and above all there are no paraphyses associated with its telia,
which are characteristic of the new species. The name Phragmotelium
mysorensis Thirumalachar and Mundkur is proposed for the new species.

The fungus attacks only leaves (Fig. 1) causing severe blotches. The life-
cycle of the rust includes all the spore-forms, viz., pycnia, aecia, uredia and
telia. Uredia and telia are formed during the months of August and Septem-
ber and pycnia and aecia in the months of October and .November.

Pycnia

Pycnia are sub-cuticular (Fig. 2) and manifest themselves macroscopi-
cally as yellow specks. They are mostly formed on the midrib and the secon-
dary veins (Fig. 5). The infected areas show slight swellings. The pycnial
mycelia and pycnosporophores are uni-nucleate.

Aecia

Aecia are of the caeoma type, covered with cylindric paraphyses, which
are slightly recurved (Fig. 10). The aecial initials are formed in the hypo-
dermal position by the concentration of hyphae in the intercellular spaces
(Fig. 9). The topmost cells of the hyphal knot which are uni-nucleate
elongate and form a palisade layer of cells (Fig. 3). Each of these cells cuts
off one or two sterile cells which degenerate after a short period. Plasmogamy
takes place between any two fertile cells, the connection being established by
the dissolution of the septa (Fig. 4). In many cases the fusion of superposed
cells takes place (Fig. 6). In any case the fusion cell elongates (Fig. 7), and
by repeated cell divisions develops chains of aeciospore-mother-cells, which
in turn form the intercalary cells and the aeciospores. Aeciospores are
spherical and minutely verrucose, with three germ pores which become dis-
tinct during germination (Fig. 8). The spores measure 16 x!0-6ju.

Uredia

Uredia are formed associated with telia. They are yellow and are
surrounded by cylindric incurved paraphyses (Fig. 17). Urediospores are
also yellow, echinulate (Fig. 14), with a single indistinct germ pore. The
spores are stipitate, binucleate and measure 16 x llju. When germinated on
slides by the method suggested by the writer (1940) the germ tube with two
nuclei can be observed (Fig. 15). Formation of appresorium at the tip of
the germ tube is also of common occurrence.

Phragmotelium mysorensis, a New Rust on Indian Raspberry 189

FIGS. 2-9

Fig. 2. Camera lucida drawing of a pycnium. X1800. Fig. 3. Fertile cells of the aecium.
X1800. Figs. 4 and 6. Fusion of fertile cells. X1800. Fig. 5. Showing the development of
pycnia and secia on the midrib of the leaf. x400. Fig. 7. Development of aeciosp ores-mother-
cells. X1800. Fig. 8. ^Eciospore germination showing three germ tubes, X56Q. Fig. 9,
Section through an aecial initial. X900,

M. J. Thirumalachar

minating teliospore. X400. Fig
Fig. 15. Showing urediospore
1*17. Section though a

cyli f ric paraphyses - x9 - Fi *-

iUm ' X4 ' Fig ' "' Ger '
eXOSp re - Xl260 '

Phragmotelium mysorensis, a New Rust on Indian Raspberry 191

Telia

Telia are hypophyllous, chestnut-brown. In colour and are minutely
distributed on the leaf surface. They are associated with paraphyses (Fig. 12).
The teliosporcs are stipitate, and mature spores are five to six-septate. Telial
initials are hypodermal from which cylindrical hyphsc with two conspicuous
nuclei are differentiated. Each of these divides into a stalk cell and a spore
initial. The latter by a series of conjugate mitosis and periclinal wall
formation forms five to six-septate teliospores. Seven septate teliospores
are also developed in rare eases.

Mature teliospores are yellowish-brown, stipitate, smooth-walled with
two germ pores in each cell The cells of the teliospores are superposed,
and are slightly constricted along the septa. Teliospores germinate soon
after they are mature (Fig. 13). In sections through the telia with spores
of different maturity promycelia with basidiospores were observed in all the
mature spores. It is evident that the teliospores are not resting spores.
Following germination and formation basidiospores the promycelium breaks
down, and the empty spore coat shrivels up and turns black in colour.
Material collected for spore germination studies from old infected leaves
had spores without cell contents,

Teliosporc germination indicated that the promycelium emerges
through the germ pore and at one time more than one cell of the teliospore
germinates simultaneously. The yellowish-brown contents of the teliospores
migrate into the promycelium. A four-celled promycelium is formed giving
rise to basidiospores. The basidiopores arc thin-walled, spherical and uni-
nucicatc (Fig, 16), They measure 11 x 10 -5f*.

Leaves of Ruhus lasiocarpus with freshly erupted teiia were taken and
suspended over young plants raised from cutting under rust.free conditions.
The leaves of host plant were kept moist by spraying them with water. The
plants were enclosed under bell-jars. After twenty days small discolourised
spots were observed indicating the first sign of infection. Eight days after
the pycnia were observed appearing macroscopically as orange yellow
specks. Further development could not be followed owing to the death of

the host plant, ^ t ^

Description of the Rust

Phragmotelium mysorensis Thirumalachar and Mundkur, Spec. nov.

Pycnia epiphyllous, sub-euticular, without paraphyses. Aecia of the
coma type, covered with long cylindric paraphyses; aeciospores yellow,
oval or spherical minutely verrucose, with three germ pores, measuring
16 x 10-6/A. Uredia hypophyilous, associated with telia, minute, sparsely

192 M. J. Thirumalachar

distributed, surrounded by incurved cylindric paraphyses; urediospores
yellow, spherical, echinulate measuring 16 x 11/x, with an indistinct germ
pore. Telia erumpent, chest-nut brown, sparsely distributed on the leaf
surface, provided with paraphyses ; teliospores stipitate, five to seven septate
5 -celled spore measuring 64-5 to 73 5 x 23 -'2 /A, 6-celled spore 77- 4-85 x
23 -2ft, and 7-celled spore 82-88 x23-4^, yellowish-brown smooth thin-
walled, with two indistinct germ pores in each cell; pedicel hyaline, not
swelling in water measuring 40 to 48/*, spores germinating in situ immediately.
Basidiospores thin- walled, spherical, measuring llxlO-5fi.

Hab. on leaves of Rubus lasiocarpus Smith, leg., Thirumalachar,
Nandi Hills, Mysore State, 16-2-1941. Type deposited in Herb. Crypt ,
Ind. Orient, of the Imperial Agricultural Research Institute, New Delhi.
Phragmotelium mysorensis Thirumalachar et Mundkur Spec. nov.

Pycnia epiphylla, sub-cutkrularia, aparaphysata, Aecia caemoidea,
paraphysibus, longis, cylindricis tecta; aeciosporae flavidae, ovatae vel
sphericae, tenuiter echinulate, tribus germinatioins sporis instructae, magni-
tudinis 16xlO-6fi. Uredia hypophylla, mixta cum teliis, minuta, sparse-
disposita, cincta paraphysibus curvatis, cylindricis; urediospora flavidae,
sphericae, echinulatae, magnitudinis 16 x lift, prsedite uno germinations
poro indistincto. Telia erumpentia castaneo-brunnea, sparse distributa per
foliorum faciem, paraphysata; teliosporae pedicellatae, 5-7 septatae; sporae
5-cellulatae magnitudinis 64 5-73 5 x 23 2 ft ; Sporae 6-cellulatae, magni-
tudinis 77 -4-85 -Ox 23 -2 ft. et sporae 7-cellulatae, 82-88 x 23 -4ft; sporae
omnes flavidobrunneae, tersis et tenuibus parietibus ornatae, et singular sporae
duobus germinationis pores indistinctis instructs; pedicellus hyalinus est
neque tumescit in aqua ; magnitudinis 40-48/x. Sporae statim germinantes ;
basidisoporae tenuibus parietibus ornatae, sphericae, magnitudinis 11 x 10 -5ft.

Hab. in foliis Rubi lasiocarpi smith, leg. Thirumalachar, Nandi Hills,
Mysore State, India, 16-2-1941.

Conclusions

The absence of the caeoma type of aecia was one of the chief reasons that
led Sydow (1921) to establish the genus Phragmotelium, a primary aparaphy-
sate uredium taking its place as the first spore-form. But Phragmotelium
mysorensis, it will be noted, is characterised by the presence of an aecium of
the caeoma type and by the absence of primary uredia. The teliospores with
smooth spore walls and with a capacity to germinate immediately ; and with
pedicels that cannot swell in water, leave however no doubt as to the position
of this rust in that genus.

Phragmotelium mysorensis, a New Rust on Indian Raspberry 193

- That there would be transition forms between Phragmidium and Phrag-
motelium cannot be doubted, and there is nothing surprising in coming
across species with overlapping characters. For instance species of Phrag-
midium formerly placed in the genus Earlea have teliospores with smooth walls,
though they germinate after a long rest period. Paralleling this we have
Phragmotelium mysorensis in which the primary uredia are replaced by an
aecium, though the other characteristics distinguishing the genus Phragmo-
telium are all present in the species.

The author desires to express his gratitude to Dr. B. B. Mundkur,
Imperial Agricultural Research Institute, New Delhi, for help in writing this
paper, in identifying the fungus, and in obtaining the latin diagnosis, and to
Dr. M. A. Sampathkumaran, Professor of Botany, University of Mysore,
for encouragement and guidance.

Summary

1. Phragmotelium mysorensis is a new species of rust attacking the
leaves of Rubus lasiocarpus Smith.

2. All the four spore-forms, viz., 0, 1, II and III occur on the same host.

3. Pycnia are sub-cuticular, and aecia are of the caeoma type with
paraphyses. Development of caeoma and the initiation of the dicaryon
phase has been studied.

4. Uredia are hypophyllous, pulverulent, covered with incurved para-
physes.

5. Telia are hypophyllous, associated with the uredia and covered
with paraphyses. Teliospores are five to six septate. Teliospores are smooth,
thin-walled, with two indistinct germ pores in each cell. Pedicels are hyaline
and do not swell in water. Teliospores germinate soon after maturity, and
form sporidia which are uni-nucleate.

6. Sporidial infections indicate that the rust is autoecious.

REFERENCES

1. Arthur, J. C. . . "Eine auf diestruktur und Entwicklungsgeschichte begrundete Klassi-

fication der Uredineen," Res. Sci. Bot. Congr. Vienne, 1906, 331-48
(Original not seen).

2. Dietel, P. .. Uredinales in die naturlichen Pflanzenfamilien, 1928, 2Aufl. 6, 24-98.

3. Hiratsuka, N. . . " Phragmidium of Japan," Jap. J. Bot., 1935, 7, 227-99.

4. Sydow, H. and P. Monographia Uredinearum, Leipzig, 1915, 3.

5. Sydow, H. . . " Die Verwertung der verwandt schafts verhaltnisse und des gegewarti-

gen entwicklungsganges zen Umgrenzung der gattungen bei den
Uredineen," Ann. Mycol. Berlin, 1921, 19, 161-75.

6. Thirumalachar, .. "A method for germinating and staining teleutospores," Jour.

M. J. Ind. Bot. Soc., 1940, 19, 71-75.

FEMALE GAMETOPHYTE AND EMBRYOGENY IN
*CYMBIDIUM BICOLOR LINDL.

BY B. G. L. SWAMY

Received January 10, 1942
(Communicated by Rao Bahadur G. N. Rangaswami Ayyangar)

Introduction

AN account of microsporogenesis and development of the male gamete
in Cymbidium bicolor was recently published by the author (1941). The
present work deals with the megasporogenesis and embryogeny.

The material was fixed in formalin-acetic-alcohol and embedded in
paraffin after the usual methods of dehydration and infiltration. Sections
wer$ cut between 8 and 20 microns and stained in Heidenhain's iron-alum
hematoxylin followed by a counterstain of either eosin or light green in

clove oil.

Megasporogenesis

. Sections through even the fully opened flowers rarely showed the nucellar
protuberances, in which the differentiation of the archesporiumandits further
development is initiated only after pollination. The sign of successful
pollination is the enlargement of the gynostegium and material was collected
at various stages for microscopic examination.

The archesporial cell is hypodermal in origin and is easily differentiated
by its large size, conspicuous nucleus and rich contents. Further develop-
ment is seen only in material collected ten days after pollination. Even at
the archesporial cell stage pollen tubes can be observed along the placental
ridges.

The archesporial cell functions directly as the megaspore mother cell
(Fig. 1). The nucleus passes through the usual stages of the meiotic divi-
sion (Fig. 2), and a dyad is formed (Fig. 3). The chalazal cell enlarges
(Fig. 4) and gives rise to an 8-nucleate embryo-sac (Figs. 5-9). The deve-
lopment of the embryo-sac thus conforms to the Allium-type.

In some cases the nuclear divisions are suppressed at the antipodal end
or they show a belated development, so that occasionally only 6-nucleate

* The name of Cymbidium bicolor has been revised in Gamble's Flora as
Alolfolium (Gamble: Fl. Mad. Pres., Part VHI, p. 1436).

194

Female Gametophyie & P^mbryogeny in Cymbidium bicolor Lindl.

FIGS, 1-10
ation. Magnification of Fig. I, X1260, of the rest X 560.

196 B. G. L. Swamy

embryo-sacs are seen. Usually the antipodal nuclei are smaller in size than
the micropylar ones. This difference is often noticeable even at the 4-nucleate
stage (Figs. 7-9).

Fertilization and Endosperm Nucleus

The germination of the pollinium and the formation of the tube nucleus
and the male nuclei has already been described by the wrter in a previous
paper (Swamy, loc. c/r.). Fertilization is porogamous (Fig. 10). The male
nuclei which are bean-shaped during their passage through the pollen tube
become spherical after being discharged into the embryo-sac.

One of the male nuclei fuses with the egg nucleus and forms the zygote.
The other enters into triple fusion with the partially fused polar nuclei
(Fig. 10). Even before they complete fusion the three nuclei show slight
hypertrophy and sometimes become five to six times larger than before
(Figs. 11, 12, 29 and 30). But gradual degeneration sets in, though the
remains of the nuclei persist even up to late stages of embryogeny.

Embryo

The fertilized egg divides after a long rest, sometimes extending up to
nearly two months after fertilization. The first wall is transverse (Fig. 12)
and very often oblique (Fig. 28) and even vertical (Fig. 29). The
next three to six divisions are very irregular, the walls being laid down with-
out any definite order (Figs. 13, 14 and 15). One of the cells at the chalazal
end of this resultant irregular mass continues to divide further and forms a
linear row of 6-10 cells (Figs. 16, 17 and 18). The chalazal two or three cells
of this proembryo undergo vertical divisions (Figs. 19 and 20) followed
by more anticlinal and periclinal divisons (Figs. 21 and 22) and form the
embryo (Figs. 23, 24, 25 and 26).

Some of the suspensor cells at the micropylar end begin to elongate and
present a fluffy appearance (Figs. 16, 17, 18, 22, 23, 24, 25, 26 and 35).
Some of them grow into the micropyle, and some others surround the deve-
loping embryo. They do not extend in any case out of the outer integument
but occupy the space available in it (Fig. 25 a). These cannot be considered
strictly haustorial as they do not reach any nutritive region even during
the late stages of embryogeny nor do they digest the surrounding tissue
during their elongation. This type of elongation is restricted to only
5 to 7 cells at the top. In a mature seed at the time of opening of the
fruit, these elongated cells are degenerated and only their dwindled remains
are seen.

P~aU

bicoior ,W/. 19?

FIGS. 11-22

Fig. 11. Zygote and the hypertrophied endosperm nucleus. X560. Fig. 12- Two-celled
embryo. X560. Figs. 13, 14 and 15. Early stages of embryo development. X560 *Figs 16 and
17. Later stages; note the enlargement of the suspensor cells towards the micropyiar end
X400. Figs. 18, 19, 20, 21 and 22. Stages depicting the subsequent development of the embrvo*
Fig. 18, X560. Fig. 19, X400. Fig. 20, X200. Fig. 21, x!60. Fig. 22 X160

198

6. G. L. Swamy

X200. Fig. 25a. An optical
showing the

FIGS. 23-26

n^ S1X ^ eIon ^ ted <=" towards the micropylar end.
3* * rou ^ the longitudinal plane of the ovule
^ xl. Fig. 26.

Female Gamcto^yte & E,n[>ry eny / Cymbidium bicolor Lindl. 199

Fios. 27-36

Figs. 27 and 29, Vertical division of the zygote, x 560. Fig. 28. Zygote divided by an
oblique wall. X560. Fig. 30, One of the two cells dividing. X560. Figs. 31 and 32. Further
stages illustrating the development of the plural embryos. X560. Figs. 33 and 34. Later stages
of twin embryos. x50. Fig, 35., Double embryos at the time of dispersal. X200. Fig 36
Mature seed showing the reticulate thickenings of the cells of the seed coat and the position
of the twm embryos,

200 & G. L. Swamy

Polyembryony

Occasionally two embryos are seen in a single ovule. Their develop-
ment has been traced in detail and is shown in Figs. 27 to 36. In some
cases where the first division of the zygote is vertical (Fig. 27) or oblique
(Fig. 28), the two resulting cells get separated (Figs. 27, 29) and each develops
independently into an embryo (Figs. 30, 31, 32, 33, 34, 35 and 36).

Usually one of the two embryos is slightly smaller than the other
(Figs. 35 and 36), probably due to want of space or nutrition and its tardy
development as compared with its fellow. Out of 2,000 mature seeds examm-
ed under the microscope 27 contained double embryos, i.e., about 2%.

The mature seed (Fig. 36) has a single layer of the integument composed
of translucent cells with deep brown reticulations. The seed measures about

13x52 A*.

Conclusions

The AlUum-typQ of development in terrestrial orchids was first reported
by Vermoesen (1911) and subsequently by others. Stenar (1937) has recently
shown the same type of development in Acroanthesmonophyllus. Among
strictly epiphytic orchids this type of development has now been recorded
in Cymbidium bicolor.

The vertical division of the zygote is rare among angiosperms and has
been recorded only in a few plants belonging to the Amentiferce. It has been
reported in Leitneria floribunda (PfeifFer, 1912), in Sassafras (Coy, 1928)
and in a few members of Loranthaceae (Schnarf, 1931). Vertical divisions
of the embryo upto 6-celled and 8-celled stages have been recorded in
Balanophora dloica (Ekambaram and Panje, 1935) and in B. abbreviate* and
B. indica (Zweifel, 1939). As mentioned before, in Cymbidium bicolor the
vertical division may lead to the origin of two embryos and results in
cleavage polyembryony.

Summary

1. The hypodermal archesporial cell directly functions as the mega-
spore mother cell.

2. The mode of development of the embryo-sac conforms to the Allium
type. Sometimes only 6-nucleate embryo-sacs are seen due to a reduction
of the number of divisions at the chalazal end.

3. The pollen tube enters through the micropyle. Double fertiliza-
tion has been observed.

Female Gametophyte & Embryo geny in Cymbidium bicolor LindL 201

4. A long suspensor is formed during the development of the embryo.
The terminal cell forms a filamentous proembryo which gives rise to the
embryo.

5. Some of the suspensor cells at the micropylar end elongate and
enlarge to enormous proportions. They are however not haustorial and

degenerate in later stages.

6. Cleavage polyembryony is reported and its mode of origin and deve-
lopment traced. The plural embryos are monozygotic.

The author takes this opportunity to express his sincere gratitude to
Dr. P. Maheshwari, D.sc,, for his valuable help.

1. Buchholz, John T.

2. Coy, Georgia V.

3. Kkambaram and Panjc

4. Pfeiffer, Wanda M.

5. Schnarf, K.

6. Stenar,H.

7. Swamy, B* G, L,

8. Vcrmocsen, C.

9. Zweifel, Rudolf.

LITERATURE CITED

41 Determinate cleavage polyembryony with special reference

to Dacrydium," Bot. Gai., 1933, 94, 579-88.
** Morphology of Sassafras in relation to phylogeny of angio-

sperms," ibid., 1928, 86, 149-71.

M Contributions to our knowledge of Bulanophora. II. Life-
history of B. dlolca," Proc. fnd. Acad. Set., 1935, B 1,

522-534.
"The morphology of Leitneria floribunda" ibid., 1912, 53,

Ig9"203.

** Vergkh'htndc Embryologlc der Angiospermcn," Berlin, 193 L
"Om Aeroanthes monophyllus (L) Greene* dess Geografiska

utbredning oeh cmbryologi," Sartryck tir Fcxtskrift till

FiL der Erik Modiun> Heimbygdas Tidskrlft, Formvarderen,

1937-38, 1 77-22 1.
**The development of the male gamete in Cymbidium bicolor

Lindl.," Prdc. fad. Acad. Scl.> 1941, B 14, 454-60.
** Contribution a Fetude de Fovule, du sac embryonnaire et

de la Fecandation dans les Angiospermes," La Cellule;

1911, 27 ? 113-62 (original was not available).
** Cytologisch-ernbryologische Untersuchungen an Balanophora

ahbreviata Blume und B. indica Wall./* Sonderabdruck .

Vtert. Natur. Gasellt. Zurich., 1939, 84, 245-306.

A STUDY OF THE LIFE-HISTORY AND CONTROL

OF BATOCERA HORSFIELDI HOPE (LAMIID^:
COLEOPTERA) A BORER PEST OF WALNUT TREE

IN THE PUNJAB

BY KHAN A. RAHMAN, B.Sc. AGRI. (EDIN.), PH.D. (CANTAB.),

P.A.S., CLASS I

AND

ABDUL WAHID KHAN, M.Sc. AGRI. (PB.)

(Entomological Laboratory, Punjab Agricultural College, Lyallpur)

Received December 9, 1941
(Communicated by Dr. Hamid Khan Bhatti, F.A.SC.)

Introduction

A LONG-HORNED beetle, Batocera horsfieldi Hope, is a serious pest of walnut
in the Kulu Valley and Simla Hills in the Punjab and of " walnut and alder
in the Darjeeling Himalayas and of oak in the Kumaon Himalayas "
(Beeson, 1941). In addition to these plants Beeson (1941) also found it on
Salix tetrasperma, Trema amboinensis and mentions its occurrence on Parlo-
wina tomentosa in Japan. In the Kulu Valley and Simla Hills, though
present between the altitudes of 3,500 ft. to 8,000 ft. above sea level, we
have found it to be more destructive between the altitudes of 5,000 to

8,000 ft.

Description of Stages

Adult (Fig. 1 a, 6). Beeson's (1941) description of the adult is
below:

" Beetle black with fine ashy or yellowish grey pubescence, pronotum
with 2 elongate white or yellowish spots, elytra with numerous shining black
tubercles of the base, and several rounded or broken elongate white marks
extending to the truncate apex, scutellum white or yellowish." Males and
females can be separated as follows:

Female (Fig. 1 a)

Male (Fig. 1 6)

(1) 60 mm. long and 21 mm. broad
(2) Antennae 70-76 mm. long ; as long as body
(3) Anal extremity narrower and without any
hairs

53 mm. long and 17-5 mm. broad
Antennae 76-86 mm. long ; longer than body
Anal extremity blunt and clothed in brown-
ish hairs

202

Study of Life-History & Control of Batocera horsfieldi Hope 203

Egg (Fig. 2). Egg is oval in shape and brownish in colour with a thick
and leathery chorion ; it measures 11 mm. in length and 4-5 mm. in breadth.

Larva (Fig. 30, 6). The larva is pale yellow in colour and when full-grown
it measures 90 mm. in length. The head is small in size, triangular in shape
and dark brown in colour. Antennae are minute, two segmented, and are
embedded in oval pits. Prothorax is the broadest segment, measuring 19 mm.
in width ; from here the body tapers gradually to the anal segment, which is
the narrowest measuring 13 pun. in breadth. Prosternum is broad; dark
yellow and studded with numerous tubercles. Thoracic legs absent. The
newly hatched larva resembles the full-grown one but is smaller; its length
being 10 -5 mm. and its greatest width (across the prothorax) 4 -Omm.

Pupa (Fig. 4). The pupa is creamy white to pale yellow in colour and
is 55 mm. in length and 20 mm. in breadth. It lies in a specially prepared
pupal chamber (Fig. 5).

Life-History and Habits

Under field conditions this pest completes its life-cycle in 22-32
months. Emergence of the adults from the pupal chambers commences in
the early parts of June and continues till about the end of July ; at the end of
October or early November these adults die after copulation and oviposition :
the adults emerge by cutting out a 4-5" long, curved passage from the
chamber through the stem which terminates (on the stem) in a circular hole
of <75" to 1 -0" diameter (Figs. 5 a, 6). The adults rest on their food-plant
and when disturbed they stridulate. They feed on the bark of young twigs,
but the damage done by them is negligible.

A female, lays 55 to 60 eggs singly in an upright position in specially
made transverse slits (Fig. 7) in the bark of the trunk or main limbs of the
food-plant. Eggs hatch within a period of 8-15 days, depending upon the
season. . As soon as the head of the larva comes out of the egg-shell, it begins
to bore into the tree; the larva never leads an exposed life. Frass issuing
from the egg-slit always indicates the hatching of the egg. The young larva
feeds on the inner side of the bark in shallow, narrow and zig-zag tunnels.
After a few days it feeds both upon the inner side of the bark and outer
regions of the sap wood making shallow, wide, and zig-zag galleries. It *
makes irregular tunnels and thorws out quantities of chewed up plant tissue
mixed with fecal matter (Fig. 8/) through holes (Fig. 8 h). In heavily
infested trees heaps of chewed up fibre mixed with fecal matter are found
beneath the hole; large quantities of frass also remain in the tunnels, and
under the bark.

204

Khan A. Rahman and Abdul Wahid Khan

The larval stage lasts for 20-25 months. The full-grown larvae do not
show any activity during winter months, Le. 9 from October-March. The
pest has a distinct prepupal stage which lasts 50-182 days depending upon
the season. Prior to pupation the larva constructs a lunar-shaped chamber
in the main stem (Fig. 5 b\ In this chamber it spends its prepupal and
pupal stages. The pupal stage occupies 46 to 90 days. The beetle does not
leave the pupal chamber at once on emergence, but rests in it for a varying
period of 5-6 months. It ultimately emerges out through a 4-5" long
curved tunnel which terminates, as pointed out above, in a circular hole of
75-1-0" diameter. The plant heals up the tunnel and the hole in 2-3 years.

Grubs less than one year old continue feeding throughout winter ;
they hibernate as full-grown larvae in the pupal chamber.

Table below gives the complete life-cycle of the pest :
m.= months; d. days

Eggs

Adults

DURATION OF STAGES

Life-cycle
completed

laid

hatched

emerged

Egg

Larva

Pupa

___ jt

days

m. d.

rn. d.

cu. u.

18- 7-36

26-7-36

1-6-38

20 19

1 16

22 13

10- 9-37

24-9-37

5-6-40

24 24

32 25

3-11-37

18-9-37

17-7-40

23 24

32 13

13- 6-39

21-6-39

3-6-41

21 24

1 18

23 21

13- 6-39

21-6-39

5-6-41

21 24

1 20

23 23

13- 6-39

21-6-39

7-6-41

21 24

1 22

23 25

Methods of Control

The control measures against this pest are directed against the beetles,
eggs and the larvae and consist in: (1) Catching and destroying the adults,
(2) destroying eggs and young larva! in egg-slits, (3) treating of holes in the
stem and main branches with potassium cyanide or kerosene oil.

Catching and destroying the adults. The adult beetle (Fig. 1 a, b) is a
large-sized, stoutly built insect. It rests on tree trunks during June-October
when it should be searched out, captured in a handnet and destroyed.

Destroying eggs and young larva in egg-slits. -They are laid on the main
stem usually upto 15 ft. from the ground; they may also be laid in thick
branches. The position of the egg-slit is revealed when the young larva*

S^S K T "? PUSh Ut fraSS " The e ^ s and b should be
destroyed by probing the egg-slit with a knife or an iron wire

Study of Life-History <& Control of Batocera horsfieldi Hope 205

Treating of holes in the stem and main branches with potassium
cyanide or kerosene oil. -This method is best suited for the control of the
older grubs. Before injecting either of the poisons, frass should first be
removed from the entrance of the hole and a pointed wire should be pushed
into the hole to clear the tunnel Two grams of potassium cyanide or a
plug of cotton wool soaked in kerosene oil should be inserted into the hole
and the hole closed with rnud. After each treatment heaps of frass should
be removed from under the treated plant.

LITERATURE CITED
Bccson, G P. C. ., Forest Insects, DehraDun, 1941,146-47.

INDIAN WATER MOULDS III
BY ABDUL HAMID, M.Sc.

(From the Botany Department, Panjab University. Lahore)

Received November 7, 1941
(Communicated by Dr. H. Chaudhuri, D.SC,, Ph.D.)

Introduction

WATER moulds are the saprophytic inhabitants of water which is calm or
slowly flowing. They flourish well in sewer waters where organic matter
is plenty and there is less of external disturbances.

The author collected some interesting forms from the tanks adjoining
the Mandirs and Gurdwaras; here, when it was breezy, the infected
insects would come to rest near the edge of the tank, thus making
collection easy.

Small ditches near the " Rajbahas " (a small canal distributary) where
green organic matter is abundant are the favourite resorts of the forms like
Pythiogeton and others. They are mostly found growing on submerged
plants, twigs and fruits in springs, creeks, troughs, ditches and moats,
puddles and pools.

Months of October and November are doubtlessly favourable for their
growth but the reproductive phase extends upto the middle of December
and after that the severity of cold and frost brings them to their resting
stage. The other favourable period ranges from the middle of February to
May when the rising spring temperature spurs them into activity again.
During rains, they get disturbed and it becomes difficult to collect
specimens, but rains are helpful in the distribution and migration of
the species.

Two papers on Indian Water Moulds have been published by Chaudhuri
and Kochhar (1935) 1 and Chaudhuri and Lotus (1936). 2 In the present
paper, the author has described only those forms that have not been previ-
ously reported in India.

1 " Indian Water MouldsI," Proc. Ind. Acad. Sci. 9 August 1935, 2, No. 2.
* " Indian Water MouldsI!," ibid., April 1936, 3, No. 4. ' -

206

Indian Water Moulds III 207

Considerable difficulty was felt in following the spore discharge as in
some forms it took place late at night and continued for 7-8 hours.
Similarly discharge of the eggs had sometimes to be followed during night.

Culture. The usual water culture methods of cultivation were adopted
with slight modifications. A boiled house-fly was placed for about 36 hours
in the specimens of water collected from different places. When a white
halo appeared round the fly, it was taken out and washed in several changes
of sterilized water. The first infection invariably consists of a mixture
of species, hence it is necessary to isolate them and grow them in pure
culture. Pure culture from single sporangium was made by taking out
a mature sporangium which was washed in acidulated water and finally
inoculating it on to the bait. In a couple of days the hyphas radiating from
the bait were seen. After giving two or three washings in sterilised
water, the culture was transferred to another pair of sterilised petri-dishes
containing sterilised water. This process was repeated after every four days
and culture transferred to a fresh pair of dishes.

Various baits were tried but boiled maize seed proved to be the
most favourite bait. The maize seed is freshly autoclaved to such an extent
that it bursts exposing fresh milk-white endosperm. Grapes have been
tried as baits but did not prove very successful. The hyphse grew out but
soon died forming a gelatinous mass. Egg yolk was successful and occa-
sionally very useful in the production of oogonia. Fly and other insects
were equally useful. Uncooked potato pieces served a useful purpose in
the cultivation of the present forms. This was found to be the favourite
medium for the new form described in this paper.

Staining. The staining process is preceded by killing and fixing opera-
tions. The fixing and killing was done in acetic formalin. The fixative
contained 5 c.c. of acetic acid, 10 c.c. of formalin and 85 c.c. of water.

Various staining methods have been tried. Eosin alone gave very faint
colour; erythrosin did not give satisfactory results. Consequently the use
of mordants was thought advisable. Various iron salts 'were tried but
without any satisfactory results. Eventually -05% solution of aluminium
* sulphate was prepared and the fixed material was put in it for 2-6 hours.
The material was taken out, washed twice or thrice in water and put in
a strong solution of erythrosin, to which a few drops of glycerine had been
added. The material after four hours was removed and thickened in
glycerine and mounted in jelly or glycerine and sealed with Canada balsam.
This was found to be quite satisfactory. Cotton blue-lactic acid stain has
also been tried and gave good results.

208

Abdul Hamid

PLATE I. Achlya obfangata

1-8. Fig. 1. Hypha and arrangement of oogonia. xl!2. Figs. 2-3. Zoosporangia
lytlospores. x!05. Fig. 4. Oogonium with diclinous antheridia. xl!2. Figs 5-6
i with diclinous antheridia. x300. Fig. 7. Oogonium with bulbous base. x300.
ogonium with large number of eggs. X300.

Indian Water Moulds III 209

Description

I. Family Saprolegniacece
7. Achlya Nees v. Essenbeck

A. oblongata de Bary, Bot. ZeiL, 1888, 46, 646, P. 10, Figs. 7-9.
(Plate I.)

Growth somewhat dense, reaching upto 2cm. on maize in distilled
water. Hyphas branching, tapering towards the apex (Fig. 1). The branches
are smaller in diameter. The diameter of the branches ranges from
2-77~22*2ju, at times upto 27-7/z; generally it is 11 -I/*. Zoosporangia
thicker than the vegetative hyphae, mostly club-shaped; 88 -8-1 36 -I//- long
(Fig. 2) chlamydospores few but with thick and dense contents and various
shapes (Figs. 2, 3). Zoospores coming out in groups stick at the tip of
the sporangium.

Oogonia somewhat rounded, plentiful and on short lateral stalks. The
stalks not thicker than oogonia and not equal in length to the diameter
of the oogonia. The diameter of the oogonia varies from 55- 5-111 -1^,
and generally it is from 83- 3-108 /*. Wall smooth and thin. Egg 1-16
(Figs. 4-8), diameter 20-27 /A. The contents of the eggs are thick and
granular. Antheridia always diclinous (Figs. 4-8), mostly covering the
whole oogonium. The oogonia are borne alternately and in racemose
manner.

Growth in culture. On house-fly in distilled water hyphae abundant;
club-shaped sporangia many. Spores discharged through an apical pore.
On egg yellow in distilled water vegetative growth luxuriant. On potato in
leucine (0-1% in water) gemmae (chlamydospores) formed, growth mode-
rate. On maize growth vigorous, when put in leucine water (0-1%) hyphae
develop zoosporangia ; oogonia not formed. Growth on fly in leucine
water (0-1%) was vigorous, oogonia produced after 2 weeks, but with
0-1% potassium phosphate and potassium nitrate growth was scanty.

The specimen differed from the description of de Bary's specimen in the
presence of certain clavate sporangia and less number of gemmae and
eggs, but these differences are not striking enough as to justify its position
as a new species.

Collected from Lahore in April, 1936.
2. Achlya androcomposita sp. nov. (Plate II)

Growth not delicate, hyphae 13 -8-41- 6 p, branched, zoosporangia
400-750 n long, cymosely branched (Figs. 2-4). Zoospores 8 3 ft in diameter.

Abdul Hamid

PLATE IL Achlya androcomposita

ia. X98. Fig. 2. Arrangement of

and
x487. Pig 7

showing diclinous

Indian. Water Moulds 111 211

Oogonia globular, borne racemosely <f% 1), diameter 6 1-75 jit, walls
smooth. Stalks almost equal in length to the diameter of the oogonia. The
number of eggs varies from 4-11, generally 6-8 (Figs. 5-7). Diameter of
eggs 20- 8-30-5/4, mostly 2S*27/i, centric, antheridia androgynous as well

as diclinous (Figs, 5-7); diclinous condition more abundant Antheridia
compound supplying more than one oogonia. Sometimes antheridia arise

from the base of the stalk of oogonia,

A chlya androcomposita

Hyph robustiorcs, 13-8-41-6/t diametro, rarnosx. Zoosporangia
400 -750 /A longa, cymosc ramificata. Zoosporae 8-3/n diametro, Oogonia
globosa* in raeemis, diametro 6 1-75 ft, pariete polito. Stipites longitudine
diametro ovomm fere aequalcs. Numcrus ovorum variabilis, 4-11, gene-
ratlin 6-8. Ova diamctro 20'8-30-5/i, plerurnque 25 -27 /A. Antheridia
androgyna aut diclina; diclina conditio frequentior. Antheridia composita,
pluribus oogoniis suppeditantia. Interdum antheridia ab imo oogonii stipite
orientsa.

Growth in culture. --On insect in watergrowth meagre but stout. On

potato and water slight growth but numerous oogonia. On egg yolk-
growth dense, Zoosporangia abundant. On maize in water slight growth.
On maize in leucinc water (0*1%) vigorous growth and oogonia formed

after seven days, Nitrate solution retarded the growth.

Achlya androcomposita has diverse affinities. The presence of (a) un-
pittcd wall of oogonia; (b) compound antheridia; (c) diclinous as well as

androgynous antheridia; (d) measurements of eggs (20-30- 5 /x) and oogonia
61-75 ft; (e) the number of (4-11); and (/) the length of the zoospo-
rangia, mark it to be a new species of Achlya. It approaches A. dubia
and A. in unpittcd oogonia, but differs from, these in having

both diclinous and androgynous compound antheridia and larger number
of It ha% nearly the same number of eggs as A. Klebsiana but the

position of anthcrtdia is strikingly different.

Collected from Amrhsar, Lahore and Hoshiarpur.

IL Family Pythiacea

1, Pythiogeton Von Mindcn (1916)

Habitat. -The fungus was isolated from samples of water containing

decaying twigs, obtained from sewers and ponds, in the vicinity of Lahore.

Morphological characters of the genus. Hyphae delicate, generally
non-septate, sporangia of different ages occurring in clusters. Sporangia

212 Abdul Hamid

asymmetrical, club-shaped or curved, attached to delicate hyphae at right
angles to the axis. At first a tubular structure is sent out with a mucilage
plug at the tip. The plug gradually dissolves and the contents (plasma)
are poured out forming a cloudy mass in water. Undifferentiated plasma
later on breaks into swarm spores. Sporangia proliferate frequently.
Re-innovation occurring in some cases twice or thrice. Each sporangium
having a tube of its own. Although sexual organs have been described in
the three species of Pythiogeton, viz., P. utriforme, P. transversum,
P. ramosum but the sexual organs have not been observed in the specimen
described here.

Pythiogeton sterilis sp. nov. (Plate III)

Characters as above. Delicate hyphae, break on slightest disturbance,
non-septate; 2-5-3-8^ in diameter. Sporangia in swarming numbers,
attached to the hyphas at right angles to the axis, 1 00-313 /^ x 30-60 fc
(Figs. 1, 4-6), spore mass which is still in undifferentiated condition is
poured and during an interval of 7-8 hours (Figs. 4-6). The spore mass
is sent out through a tubular structure by the dissolution of the mucilage
plug at the tip of the tube (Figs. 7, 9-10). This discharge usually occurs
at night. Occasionally plasma of the sporangium is discharged into a vesi-
cle (Fig. 8). Internal proliferation frequent and a number of times (Figs.
10-12). Occasionally a portion of the spore mass was delimited inside the
sporangium (Figs. 14-15). No sexual organs observed.

Pythiogeton sterilis

Characters ut supra. Hyphs tenera, minima conturbatione contain-
gentes, non septate, diametro 2 5-3 8 /x. Sporangia cumulata hyphis angulo
recto ab axi orientibus affixa, 100-313 x 30-60^. Sporarum massa non-
differentiatarum intervallo 7-8 horarum effunditur. Ejectio sporarum
masse fit habitualiter nocte per tubulum aliquem dissolutione obturamenti
mucilaginei in extremo tubulo. Quandoque sporarum plasma vesiculo
aliquo injicitur. Interna proliferatio frequens pluribusque vicibus (in eodem
sporangio). Interdum portio sporarum massse in sporangio delimitatur.
Organa sexualia non visa.

Growth in culture. Isolated from decaying twigs in dirty sewer and
pond water with potato pieces (fresh). Later on developed favourably on
maize in water. Attempts to cultivate it on fly and leucine were not futile,
though the growth was scanty, dwarf but abundant fruit bodies were formed.
It offered great resistance to its cultivation on other media like insect, eeg
yellow, potassium nitrate and potassium hydrogen phosphate.

Indian Water Moulds ///

213

PLATE III. Pythiogeton stenlis

FIGS. 1-7. Fig. 1. Mature sporangium showing lateral stalk. x487. Fig. 2. Sporangium

borne directly between hyphal continuity. X487. Fig. 3. A developmental state of sporangium.

X 337. Figs. 4-5. Mature sporangium bursting at the tip. x 187. Fig. 6. Cluster of sporangia.

X112. Fig. 7. An empty sporangium showing the discharge tube and a mature sporangium

just be/ore the tube formation. Xl87

214

Abdul Hamid

PLATE III. Pythiogeton sterilis (continued)

FIGS. 8-16.' Fig. 8. Sporangium discharging its contents into a compact vesicle. x326.
Fig. 9. Long stout discharge tube pouring out spore mass. X487. Figs. 10-12. Sporangia
showing internal proliferation. x300. Fig. 13. Empty sporangium with vesicle and discharge
tube, x 300. Figs. 14-15. Some of the spores delimited Insicc the sporangium, x 300. Fig. 16.
Abnormal gemma like sporangium, x 300.

Indian Water Moulds /// 215

Hyphal growth without these sporangia! bodies was delicate and silky.
Best temperature for growth was 22-25 C. Leucine 0*1% invigorated the
growth in culture. Best period of vegetative activity was found to be
January-March. Although it can resist high summer temperature but the
bacterial growth kills the fungus. Daily baths with fresh water made the
fungus rather sturdy and bacteria-free.

P. sterilis differs from the other three varieties, viz., P. utriforme,
P. ramosum and P. transversum in the absence of sexual organs for which
reason it has been placed 'under a new species.

Collected from Lahore.

Summary

This is the third contribution in the series " Indian Water Moulds ".
Two species of Achlya, viz., Achlya oblongata and A. androcomposita nov.
sp. and one species of Pythiogeton, viz., P. sterilis nov. sp., have been
described.

The author takes this opportunity in expressing his gratitude to Dr. H.
Chaudhuri under whose guidance this work has been done and to
Rev. Father Rapinat and Prof. H. Santapau for the Latin diagnosis of the
two new species.

INDIAN WATER MOULDS IV

BY H. CHAUDHURI AND M. L. BANERJEE

(From the Botany Department, Panjab University, Lahore)
Received November 7, 1941

Fam. Saprolegniacea
Protoachlya Coker

THIS genus was established on a species collected at Chapel Hill (America)
and previously described as Achfya. Coker had first named it as Achlya
paradoxa, but later changed this to Isoachlya paradoxa. Finally in 1923
he established it as a new genus and named it Protoachlya paradoxa.

Generic characters. Hyphas more delicate than in Achlya, sporangia
subcylindrical to clavate or flask-shaped, blunt and usually thickest beyond
the middle, proliferating like a cyme as in Achlya, and also, less frequently,
by growth through the empty sporangia as in Saprolegnia. Spores diplane-
tic on emerging ciliate and all or some showing sluggish or less often active
motion, some remaining attached in an irregular clump to the tip of the
sporangium. Oogonia borne singly, the great majority on short lateral
stalks from the main hyphse and with or without a few pits, eggs usually
few, centric. Antheridia androgynous or diclinous, typically pyriform with
their tips applied to the oogonium. Gemmae spherical to pyriform or elong-
ated. Vegetative behaviour not noticeably different from the other genera.

Habitat. The soil samples along with water were collected from a
drain in Lahore and from Hiran Minar tank, Sheikhupura, during the month
of November, 1938.

P. paradoxa Coker (Plate I)

Hyphae slender, spirally twisted, little branched, largest 60 /-t at the base,
others smaller generally between 29 -84-44- 76 ju,, zoosporangia mostly club-
shaped, 508 -2-28 1ft x 62 -68-38 -5 /*, rounded at the top with a distinct
short papilla (Figs. 1-4), secondary zoosporangia usually formed by internal
proliferation through the older ones, new zoosporangia formed entirely
outside the primary zoosporangia (Figs. 2-4), rarely formed by cymose
branching, zoospores biciliate, developed in several rows and all of the
same zoosporangium behaving in the same manner, diplanetic. Chlamydo-
spores pyriform or elongated with thick walls. Chlamydospores are deve-
loped mostly on the tips of hypha^ either in chains or terminal (Figs. 8, 9).
216

lutiian Water Moulds IV

217

PLATE L Protaachlya paradoxa
FKJS. 1-9. Fig, 1. Cymose branching of zoosporangia. X105. Fig. 2. Zoosporangium

wing raptured xS2S, Fig* 3, Secondary zoosporangium formed through the primary

x>oiporang2um. x!09. Fig, 4* S^ondary zoosporangium with mature zoospores. x!09.
Fig. 5. Germinating zootpores. x 487, Fife, 6. A portion of zoosporangium showing encysted

res, X4I7* Fig, 7. Encysted zoospores. X525. Fig. 8. Three chlamydospores in

x4S7. Fig, 9 . Tw chltmydospores, x487.

.218 H. Chaudhuri and M. L. Banerjee

They are mostly elongated and rarely globular. They are thick-walled which
measure 3 -2^. A few oogonia and antheridia developed.

Cultural characters. In pure culture the authors failed to get any
oogonial and antheridial formation, but in some contaminated dishes,
a few oogonia and antheridia were formed, but these were more or less
disorganised.

On egg albumen in sterilised water growth extensive, reaching a length
of 1^ inches from the margins of the bait, plenty of zoosporangia formed ;
on egg albumen in tap water growth vigorous, zoosporangia and chlamy-
dospores formed. Later a few disorganised oogonia and antheridia appeared;
on egg albumen in 1% potassium phosphate growth very vigorous with
pLuty of zoosporangia and chlamydospores; on egg albumen in 1% potas-
sium nitrate similar growth as above; on egg albumen, in 1% asparagin
very little vegetative growth ; on boiled house-fly in sterilised water growth
not vigorous, zoosporangia, however, formed; on grams in sterilised water
growth not vigorous, zoosporangia and chlamydospores formed.

When a drop of zoospore-suspension was placed on a slid 2 and
covered over with a cover slip in an incubator having a temperature of
35 C, the zoospores germinated after 24 hours (Fig. 5).

Discussion. This species has already been described by Chaudhuri
and Kochhar (1935) 1 when certain variations were observed from the
characters given by Coker. 2 In the present specimen further variations have
been observed which may be summarised as follows:

Highest diameter of the hyphae recorded by Coker is about 37 ^ when
grown on mushroom-grub, and by Chaudhuri and Kochhar (1935) 18*6^
and rarely upto 28 /x. But in the present case it was found to be upto
60 \L in rare cases, though usually when grown on egg albumen in water
it varied between 29-84-44-76^. Zoosporangia are furnished in most
cases by an apical papilla. They are club-shaped to globular with all
intermediate forms (Figs. 1-4). The size is again variable 508 -2-281 //>
x 62-68~38-5fi. Coker (1923) gives the diameter as 20-30 /^ and Chaudhuri
as 27 -6-33 -8 JLC.

The secondary zoosporangia are formed by internal proliferation
through the primary ones (Figs. 3-4). This point has been noted by
Coker as a characteristic feature in Protoachlya, and thus differing from

1 Proc. Jnd. Acad. Sci., 1935, 2, No. 2.

2 Coker, 1923*, The Saprolegniaceas.

Indian Water Moulds IV 219

Saprolegnia. This character has been found to be the most common feature
in the present specimen. Cymose branching which has been so amply
shown by Coker and Chaudhuri and Kochhar has been found to be of rare
occurrence in the present case.

The zoospores are diplanetic, formed in several rows, showing great
variation in behavioiir. Coker dealing with the behaviour of the zoospores
says, " The behaviour of the spores on emerging is remarkable and very
variable. The usual behaviour is for some of the spores, perh'aps half
or a third to swim slowly away on emerging, "the other remaining attached
to the sporangium mouth and encysting there." The observations made
in the present case are quite different. All the zoospores of the same
zoosporangium behave alike. Either all are very active and swim far away
from the zoosporangium or they show very slow movement settling down
away from the mouth of the zoosporangium. In three cases it was noted
that the zoospores collected to form irregular clumps at the mouth of the
zoosporangium, thus simulating Achlya to a certain extent. The normal
behaviour was, however, of active movement, and the zoospores charged
out with great rapidity and each one dashed rapidly away. All the zoo-
spores were distinctly seen to possess cilia.. The zoospores are oblong to
oval inside the zoosporangium but become spherical outside (Fig. 7). The
size of the zoospore as given by Chaudhuri and Kochhar is 12 -5-13 -2 /*
while in the present specimen it was 10-0-12-2^.

Pythiopsis de'Bary, 1888

Morphological characters of the genus as given by Coker and noted
by the authors are:

Hyphze slender, much or little branched. Sporangia typically short and
plump, spherical, oval, pyriform with a distinct apical papilla, or varying
to elongated and irregular, primarily borne at the tips of the hyphae and
multiplied from lateral stalks below the older ones to form more or less
dense clusters. Spores emerging and swimming as in Saprolegnia, pip-shaped
with two apical cilia, sprouting after the first encystment-monoplanetic.
Gemmae resembling the sporangia or oogonia, formed plentifully, often in
chains, producing zoospores after a rest. Oogonia borne like sporangia and
gemmae and resembling them, in youth, typically spherical, oval or pyri-
form with unpitted walls, 'smooth or with a few blunt processes. Antheridia
short and thick, typically androgynous from the close neighbourhood of the
oogonia. Eggs one or few (eccentric with a lunate cap of droplets on one
side in Pythiopsis cymosa\ structure doubtful in Pythiopsis Humphrey ana).

220

r
*

PLATE II. Pythiopsis intermedia

Proliferating zoosporangium. Fig. 2. Zoosporangium

z<Prangia. 3 a. Cymosely borne zoo-

fZOOSPOrangium - Fig ' 5 - Abnormal internal proli-
mternal P rol *ration. Fig. 7. Zoosporangium half a
mass coming out in semi-

Indian Water Moulds IV 221

P. intermedia sp. nov. (Plate II)

Hyphae slender, 4- 8-5 -5^ in diameter at base, much branched. Zoo-
sporangia globular or clavate, usually proliferating internally (Figs. 1, 4-6)
and borne in a cymose manner. A zoosporangium with two apical papillae
has been seen (Fig. 2). The secondary zoosporangium has been seen to be
growing partly inside and partly outside the empty primary zoosporangium
(Fig. 5). In cymose branching 'the secondary zoosporangium is formed
either very near the primary zoosporangium or at a fairly long distance
away (Figs. 3 and 3 a). Zoospores usually 9-5/x, biciliate, monoplanetic.
Oogonia plentifully formed in old cultures, spherical, unpitted, 25-6-35-2^
in diameter (Figs. 4-5). Eggs 22- 4-28 -8^ in diameter, single, eccentric.
Antheridial branches long arising far away from the oogonium, one to each
oogonium, androgynous (Figs. 9-10), clavate, later curving along the oogonial
wall. Gemmas resembling zoosporangia formed at the tips of the hyphae
but have a long drawn out apex. Gemmae formed if the mould is kept in
stagnant water for a long time or if the acid in the medium is slightly
increased.

Pythiopsis intermedia sp. nov.

Hyphae tenues, basi 4-8-5-5/I diametro, ramosissimas. Zoosporingia
globosa vel clavata, habitualiter interne proliferentia, in cymis disposita.
Zoosporangia cum duabus papillis apicalibus. Aliquando sporangium
secundarium ex parte extra et ex parte intra zoosporangium evacuatum
crescens. Sporangium secundarium vel proxime a primario formatur vel
remote. Zoosporae habitualiter 9 5 /i, biflagellatae, monoplaneticae. Oogonia
rotunda efoveolata, diametro 25-6-35-2^, in veteribus culturis abunde
occurrentia. Ova 22 -4-28- 8 /* diametro, solitaria,, excentrica. Kami
antheridiales longi, clavati, procul ab oogonio orientes, unus pro unoquoque
oogonio, androgyni, post aliquod tempus jexta oogonii parietem incurvantes.
Gemmae zoosporangiis similes, in summis hyphis um apicibus longe evectis
formatae. Gemmae habitualiter occurrentes sive in aqua stagnante qua longo
tempore hyphse continentur, sive in medio cujus aciditas leviter augetur.

Cultural characters. On egg albumen in tap water growth vigorous
and zoosporangia formed in plenty; in eggs albumen on acidulated water
vegetative growth only; very few zoosporangia formed; on egg albumen
in 1% potassium phosphates abundant zoosporangia formed; on egg albu-
men in 1% potassium nitrate vigorous vegetative growth; on egg albumen
in 0-1% asparagin very little vegetative growth; on boiled house-fly in
1% potassium phosphate vegetative growth vigorous, plenty of zoospo-
rangia, oogonia and antheridia also formed; on boiled house-fly in tap

222 H. Chaudhuri and M. L. Banerjee

water-growth sparse, a few zoosporangia formed; on corn grains in tap
waS Sktod water and 1% potassium phosphate-not much growth
Tnty case; on pea grains in tap water, acidulated water and 1% potassium
phosphate results same as above.

Zoosporangia make their appearance 30 hours after the placing of new
baits After 6 hours all the hypha bear zoosporangia. A zoosporangmm
takes 30-45 minutes to discharge the zoospores. Soon after liberation ot
the zoospores, multiplication takes place by either cymose branching or
internal proliferation. The zoospores swim for 20-25 minutes and then
settb down.

Cultured from a soil sample taken from a drain in Lahore in November,
1938.

Discussion. In this species of Pythiopsis the zoosporangia are mostly
globular with a dimension of 32 -0-41 -6^ (Figs. 2, 7). Some are elongated
(Figs. 1 , 4, 5) and their dimensions are usually 3 1 2 x 23 2 p or 28 8 x 22 4 ^
The hyphse are much thinner, being 4-8-5-5^ while in the other two
species (P. cymosa and P. Humphreyana) the thickness is much greater.

The zoospores come out of the zoosporangium in a semi-differentiated
condition by the breaking up of the apical papilla (Figs. 1-6). Outside,
in the medium, the zoospores are delimited (Fig. 8) and each swims away.

The size of a zoospore is usually 9 -6/1 while in Pythiopsis cymosa the
zoospores are 8-6-10-8^ mostly 9ft and in Pythiopsis Humphreyana they
are 8 -6 ft. Zoosporangia multiply by cymose branching (Figs. 3, 3 a) as in
other two species and also by internal proliferation (Figs. 1, 4, 6) which
is not found in the other species of Pythiopsis.

The oogonium measures 25 -6-35 -2 ft. In Pythiopsis cymosa the oogo-
nium measures 18-30 ft, while in Pythiopsis Humphreyana 3 3-89 ft, average
being 43 p. The oogonium in this new species contains a single egg measur-
ing 22 -4-28 -8 ft (Figs. 8-9). The egg in Pythiopsis cymosa measures 14-8-
18 -5 ft and in Pythiopsis Humphreyana 24-40/1. (average 30 ^). The egg
is eccentric as in Pythiopsis intermedia and contains a single drop of oil
whereas in the egg of Pythiopsis cymosa there is a lunate cap of oil droplets.
The wall of the oogonium is unpitted as in the other two species but the
thickness is 3 -2ft in contrast to 1*4-2 ft of Pythiopsis Humphreyana.

As this species differs in above-mentioned characters from the only
two species of Pythiopsis the authors have described it as a new species.

The most important point is the internal proliferation of the zoospo-
rangia, a character not to be found in either Pythiopsis cymosa or Pythiopsis

Indian Wafer Moulds

223

PLATE III. Saprokgnia rhcetica

FIGS. 1-7. Fig. 1. Internal proliferation of zoosporangium. X105. Fig, 2. Secondary
zoosporangium branching. x!05. Fig. 3. A portion of mature zoosporangium. x 487. Fig. 4
Complicated gemma. X105. Figs, 5-6. Oogonia with 12 eggs. X487. Fig. 7. Oogonium
with 9 eggs. x487. . ' - '

224 H. Chaudhuri and M. L. Banerjee

Humphreyana. Differences are also found in such other structures as the
hypha, zoosporangium, oogonium, egg and its wall and also the position of
the oil drop Thus it is named as Pythiopsis intermedia, because the size
of the zoospores, oogonia and eggs is more or less intermediate between
the two species already described.
Saprolegnia rhatica Maurizio (Plate III)

Hyphse branched, 30 -V thick. Zoosporangia are at times branched
and the branches come out of the empty primary zoosporangia (Fig. 2).
Zoospores 8-9-6^ in diameter. Gemmas formed, complicated (Fig. 4).

Oogonia 72x61-6^ containing 9-12 eggs, generally 12, 19*2^ in
diameter. Oogonial wall not very thick, or pitted, very few pits (usually
2-3) (Figs. 5-7). Antheridia absent.

Growth in culture. On house-fly in tap watergrowth extensive, reach-
ing a length of 1 -5 cm., large number of zoosporangia formed, eggs develop
later; on boiled house-fly in 1% potassium phosphate vegetative growth
extensive, zoosporangia formed but no oogonia; on egg albumen in tap
water vegetative growth vigorous, plenty zoosporangia but on egg albumen
in -1% asparagin, very little growth.

The present specimen of Saprolegnia rhcetica resembles the other two
identical species (Coker, 1923; de Bary), viz., S. torulosa and S. variabilis
in the absence of antheridia and few pits on the oogonia walls. It differs
from the descriptions given by the authors (Maurizio, Minden and de
Bary) who first created these species, namely, S. rhcetica, S. variabilis and
S. torulosa, in minor details of measurements of various organs and number
of eggs. But on the whole the. characters of the specimen are common
to all the three species now considered to be identical, hence the inclusion
of the specimen under the species S. rhcetica.

Collected from Lahore in February, 1939.

Summary

In this fourth paper of the series on Indian Water Moulds, the authors
have recorded and described three water moulds not so far reported from
this country of which, one is a new species. These are Protoachlya paradoxa,
Pythiopsis intermedia nov. sp. and Saprolegnia rhcetica Maurizio.

Before concluding, the authors express their sincere thanks to Dr. B. B.
Mundkur for various suggestions and help with the literature, to Professor
Rapinat for the Latin translation of the diagnosis of the new species and
to Mr. A. Hamid for revising the manuscript.

INDIAN WATER MOULDS V

A New Genus of the Saprolegniaceae: Hamidia Gen. nov.
BY H. CHAUDHURI

(From the Botany Department, Panjab University, Lahore)

Received November 7, 1941

Locality and Isolation

SAMPLES of water with decaying twigs were obtained from two localities,
viz., Tarn Taran (Dist. Amritsar) and Barhamjit (Dist Hoshiarpur) in March

1936, from which the mould was isolated on potato blocks. The growth
on potato was luxuriant,

Morphology of the Fungus

The general morphological characters of the fungus are as under:

The hyphie are delicate, sparsely septate 1 -7-4-4^ in diameter (Fig. 1),
upon which oogomat bodies are borne both in cymose and racemose manner
(Figs. 2-4). Hypha! wall 0'5~0*8/i thick. The long slender hyphae are

generally raccmosely branched, can best be seen in the natural condition,

as the staining and teasing processes upset the arrangement.

The oogcmia when borne singly are attached by delicate and long stalks
(Figs, 5-6), which aire extremely fragile and break even by the slightest
disturbance. These are also borne in cyinose clusters (usually in threes).
The diameter of the oogonia varies from 22 -2-38 -7 /A. The oogonia mostly
contain a single egg (Figs. 7-9), which is quite big and prominent filling the
whole oogonium. The egg has a wall, catches a deep stain as compared
with the hyphse and 18-34*2^ in diameter. The wall of the oogonium is
quite smooth. The centric or subcentric egg comes out

slowly (Figs, 8-9) leaving a hyaline and shrunk capsule behind. The egg
discharge is- slow and almost imperceptible and one has to be vigilant to
watch the gradual emergence of the egg. Germination of egg while still
inside the oogonium has been seen (Fig. 19).

It has noticed- that in bodies similar to oogonia, diameter

18-25 -5 ft, which .may be termed sporangia, 2-7 swarm spores, diameter
6-8-9-5fi f may be formed (Figs, 11-13, 14). These swarm spores are non-
ciliate and germinate immediately on being discharged (monoplanetic)

225

226 H. Chaudhuri

(Figs. 16-17). Occasionally a discharge tube is formed (Figs. 14, 15). The
swarm spores, when they come out, have no properly differentiated wall.
If swarm spores are not discharged, then they germinate inside the mother
wall and S3nd out germ tubes (Figs. 11-13). These swarm spores either
germinate by a single unilateral germ tube or produce tubes bilaterally.
Occasionally it has been seen that only a part of the sporangium
forms 1 or 2 spores and the rest germinates by a long germ tube (Fig. 18).

Growth in Culture

The fungus was isolated from the decaying twigs under water, and
ths growth on fresh potato blocks was luxuriant. This was the only thing
upon which the fungus could be cultured and leucine (-1%) had tonic
effect. Prolonged culture in leucine (-1%) and slightly lower temperature
about 20 C, produced certain abnormal types of gemmae (Figs. 20-21)
though not in abundance. Insect and egg yolk were found to be unsuitable*
The fungus was very sensitive to high temperature and the mycelium lost its

vegetative activity soon.

Discussion

The word " oogonium " has been used here for the rounded bodies
attached to hyphae due to the close resemblances of the structure
of these bodies to oogonium and other characters of the egg. Here, as in
Isoachlya, no antheridia are present. The mere absence of the male
structures in the neighbourhood of the female cells does not debar one from
calling these structures oogonia, so long as these function as such. Thus
these round cells, with a single 4 egg, can easily be regarded as oogonia.

The main characteristics of the fungus, viz., (a) Septate hyphae, (b) Pre-
sence of oogonial bodies, (c) Single egg in an oogonium, (d) Absence of
antheridia, (e) Presence of swarm spores, and (/) Gemmae formation, no
doubt show Saprolegnalian affinities. The mycelium is septate, but the
septa being sparse, one is apt to take it as a non-septate ccenocytic myce-
lium. The simple septatioa is seen in Blastocladiacece but the absence of
the joints and ciliated gametes are radical differences. The fungus suggests
diverse affinities by its markedly striking characters. The delicacy
of the mycelium brings it closer to Monobkpharidales but the absence of
antheridia altogether, is again a primary difference.

The formation of swarm spores, the eggs and the gammas, are charac-
ters of the Saprolegniacea, though, no doubt, the absence of the true
antheridia is a handicap in the proper location of the form. Considering,
however, all its characters, one feels justified in placing it as a new genus
f Saprolegniacea.

Gen. nov.

Indian IValer Moulds *V 227

Diagnosis

Hypiue hyaline, generally raccmoscly branched, delicate, sparsely septate,
bearing oogonia and sporangia both in racemose and cymose manner; best
seen in natural condition. Oogonia borne singly or in clusters (of usually
3). Oogonia when borne singly have long stalks which break at the slightest
disturbance. Oogonium with a single large egg entirely filling it. Egg with
smooth wall; c'lU'fges gradually and imperceptibly. Apandrous; sporangia
resembling ooyoniu also formed, each with 2-7 swarm spores. Discharge
lube may or nuy not be present. Swarm spores, without a properly
differentiated wall and non-eilialc (monoplanclic) germinate soon after
discharge. Undischarged swarm spores germinate inside the sporangium*
putting forth unilaterally or bilaterally produced germ tubes. Gammse also
formed. Growth on fresh potato stalks most luxuriant; egg yolks and
insects unsuitable for growth.

Hamitlia Gen, nov.

Hypiue hyalinar, commumter racemose ramificantes, tenues, sparse
septa ta% oogonia cl sporangia turn racemose turn cymose fcrentcs; quarn
oplimc obscrvari possum in vivo, Oogonia singulatim vel acervatim (prae-
scrtim ternaiim) feruntur. Oogonia singulatim producta longis insident
pcdicciiis, qui faciilimc rumpuntur. Oogonia uno tantum magno ovo com-
plcntur. Ovum, lerso parictc, gradatim et sensim sine sensu emergit.
Fungus est apandrus. Sporangia ctiam, oogoniis similia, producuntur,
quorum unumqitodquc 2-7 zoosporas habeU Tubulus quo sporae liberantur
poles: vel vet abcssc. Zoospone pariete non plene differentiate et

non ciliato (monoplano) germinant cito post liberationem; zoosporae quie
non liberantur, gcrminaiH in sporangio t unilateraliler vel bilateraliter pro-
ductis gcrminationis tubulis. Gcmm 'ctiam producuntur. Crescit lucu-
Icntissimc super vivos trimcos Solani tuberosi; non erescit in medio ex-
ovorum viicllis vel in in&cctis.

h\lica >p* nov, (Plate I)
Chaiaelcrs as above. Hyphar 1-7-4-4/i broad. Oogonia wall smooth^
Oogonia 22 -2-38' 7 ft in diameter; eggs 14-34-2 ^ in diameter. Sporangia

18-28 -9 p in diameter. On decaying twigs under water. Collected by
A. Hamid in March 1936.

Hamidia Indica sp. nov,

Characterse ut supra. Hyphae 1*7-4*4^ latae. Oogonii panetes tarsi;
oogonia 22 -2-38 -7^ diam.; ova 14-34 -2 ft diam.; sporangia 18-28-9/x

228

H. Chaudhuri

PLATE I. Hamidia indica

F,V A g ; S Vegetative h yP ha5 ' x636 - Fig. 2. Arrangement of oogonia. X525.

xf 2 5 ^S^^^^Y^ sporangia. X600. Fig. 4. Arrangement of oogonia.
X525. Figs. 5-6 Oogoma with elongated stalk. X600. Fig. 7. One large egg before

of egg. xS^SKg 10
germinating insii the

Indian Water Moulds V

229

11)

PLATE I, ffamldla Indica (continued)

FIG*. 14-21. Fig, 14. Spores germinating inside a stalked sporangium. X600. Fig. 15.
A swarm spore just before escaping from the sporangium through a discharge tube. x600.
Figs. 1 6' -17. Swarm germinating outside the sporangium. x600. Fig. 18. Sporangium

partly forming and partly germinating into vegetative hypha. x600. Fig. 19. Egg

germinating, x600. Figs. 20-2!. Gemmae, xS25

diam. Occur rit sub aqua super putrescentes ramusculos arborum. Collec-
tus ab A, Hamid, martio 1936,

230 H. Chaudhuri

Type specimens deposited in Panjab University, Botany Department,
Lahore.

Summary

A new genus of Saprolegniacece Hamidia has been isolated, grown
in culture, and described; and a new species Hamidia indica established.
The Latin diagnosis in both cases has been given.

The author expresses his thanks to Dr. B. B. Mundkur for kindly looking
through the manuscript and making valuable suggestions and to Rev. Father
Rapinat and Prof. H. Santapau for the Latin diagnosis.

d at The Bangalore Press, Bangalore City, by O. Srinivasa Kao,
and Published by The Indian Academy of Science*. Bangalore

CONTRIBUTIONS TO THE BIONOMICS, ANATOMY,

REPRODUCTION AND DEVELOPMENT OF THE

INDIAN HOUSE-GECKO, HEMIDACTYLUS

FLAV1VIRIDIS RUPPEL

Part III. The Heart and the Venous System
BY BI-:NI CHARAN MAHENDRA

iDrjiartment oj Analogy, St. John's College, Agra)
Received January 10, 1942

CONTENTS PAGE

1. INTRODUCTION , . , . . . . . _ . . 231 ,

2. Ti niNiour . . . . . . . . _ _ 232

3. THI: Hi ART

(A) Sinus Vcnosus . . . . ,. .. .. 234

(H) Atrium !>extrum . . . ., .. .. 234

(O Atrium Sinistrum . . . . . . . . . 235

(I)) Vcntriculus . . , . . . .. . . 235

(h) Trunci Artcriosi . . .. . . .. .. 238

4. Tin-: ANTMUOK Vi'.N/i' CAV>I-:-

(A) Venn Trachea Us . * . . . . . . . . 240

(B) Vena Jugularis Communis .. .. . . .. 241

5. VI;NA C'AVA POSTMUOR . . . . .. .. . . 242

6* THK Su^RA-Ri-iNAL PORTAL SYSTEM , . . . . . . . 245

1, Till; SYSTEM w TIII>; CAUDAL VEIN . . . . . . . . 246

8, Tin- HEMATIC PORTAL SYSTEM

(A) The System of the Anterior Abdominal Vein . . . . 247

(B) The System of the Epigastric Veins . . . . . . 248

9. Vmm PULMONALES . . . . . . . . . . 249

10. SUMMARY . . . . . . . . .. .. 250

11. BIBLIOGRAPHY . . . . .. .. .. ..251

/. Introduction

ALTHOUGH it is more than fifty years ago that Hoffmann (1890, p. 1010
complained about the paucity of detailed accounts of the venous system
in Lizards a complaint repeated "in 1920 by O'Donoghue we have stil

231

Bl F

232 Beni Charan Mahendra

not much knowledge about this system in a great many families of Sauna.
In particular the deficiency of such information about the family Cek k o-
'

to be'ded, as this family approaches the

this gap

a great deal in its organization. The present account aims to fill
nTur knowledge, as* well as to complete the _ description o the ^
system of Hemidactylus flavmridis, which Bhatxa and Dayal (1933)
with the excellent paper on its arterial system nine years ago.

To mention the more important contributions on the subject Corti
(1853) described the vascular system in "Psammosaurm griseus " ( - Varanus
griseus), while Briicke (1852), Rathke (1857) and Fritsch (1869) gave valuable
accounts of the heart and aortic roots in a number of lizards. Hoi Imann
(1890) and Hochstetter (1893) inter alia and Grosser and Brczma (1^>
particularly dealt with the development of the veins in Lacerta. Orel
(1903) studied the structure and development of the heart in Lacerta and
some other lizards. Beddard (1904-06) made valuable observations on the
veins of numerous lizards, such as Heloderma suspectum, Varanus grlxeus,
V. niloticus, V. exanthematicus, Iguana tuber culata, TiHqua sdncoic/e.i, Ttipi-
nambis, Chameleon, Pygopus lepidopus, Phelsuma madagascariens'ts, Tarcn-
tola annularis, Gerrhosaurus flavig'ularis, Ophisaurus apus, and Amphisb&na
brasiliana. Bruner (1907) described, in connection with the cephalic vessels,
a muscular mechanism for raising the blood pressure in the head. Goodrich
(1916, 1919) pointed out the phylogenetic importance of " the subdivision
of the aortic trunk so as to form two systemic arches crossing at their base
in such a way as to become separated by the interventricular septum".
O'Donoghue "(1918) noted the condition of the septum ventriculorum and
its relation to the openings of the aortic arches in various reptiles, and criti-
cised Goodrich. Bhattacharya (1921) and Thapar (1921) described the
venous system in Varanus bengalensis (now = V. monitor). John (1924)
proved that the flow of blood in Varanus monitor, contrary to the generally
accepted belief, is from the Hepatic Portal to the Renal Portal System
by means of the short cross-connection. Bhatia (1929) described the heart
and the venous system of Uromastix hardwickii in detail. Finally, Benning-
hoff (1938) gave an excellent resume of our knowledge of the heart, and
Gelderen (1933), that of the venous system in the whole Vertebrate series.

2. Technique

The structure of the heart was studied in thick hand-cut sections (both
transverse and horizontal) of formalin-preserved material, in careful dissec-
tions, and in serial sections, 15/x thick, prepared according to the paraffin-
embedding process and stained with borax carmin and haematoxylin,

t of Indian I touzc-Gccko, II. ilaviviridis Ruppellll 233

of the \eins can he made out, even without injection, in a freshly
captured and kitied specimen. Geckos, kept for a long time in captivity,
lose much of their blood and are not so good for demonstration of the
venous system, unless injected, Bealc's Prussian Blue (Lee's Microtowisfs
Vatfc Mccwn, 192K, p. 24S) and l-mery's Aqueous Carmine (ibid., p. 247)
were successfully tried, although the best results were obtained by the
injection of Reexe's l ; i\cd Indian Ink.

>, Ttw Heart

The heart in llcmulavivlm flavhirhlix lies in the mid-ventral line between
the bases of the fore-limbs immediately behind the neck. Such a forward
position is worth noting, as it perhaps represents a primitive condition.
As shown by Rathke {1X57), the farther back does the heart lie in the
plcuropcritoncal cavity, the more highly organised is the reptile. The heart
(Text-Fig, 1) is almost as broad as long, being about 6mm. in width at its
anterior one! in a full-grown individual It is roughly conical in appearance,
the base of the cone being directed anteriorly and the apex posteriorly. The

C t" -V.

..v./.e.

1,4,

G.c

/.p.

r.jt.v.

(A) (B)

Trxr-Fia. 1

The heart of JfrmUlact \lwfltivlvlritlix. (A) Ventral view; (B) Dorsal view.
.v,r., anterior vena eava ilcfO; C.CJ,)., right common carotid arch ; C.C.S., left common
carotid arch; <*./>.v., Cintimon pulmonary vein; el n fluted region of the sinus vcnosus; G.c.
gubcrnaculum cordi<; /.p,v, left pulmonary vein; L.S.A.t left systemic trunk; L.A., left auricle;
/*./<., pulmonary arch; p.v.c^ posterior vena eava; R.A., right auricle; r./?.v., right pulmonary
vein; RS.A,, right systemic arch ; v./.c. t common jugular vein ; v..y., subelavian vein ; v./.,' trachea!
vein; K, ventricle.
Bla F

234 Beni Charan Matiendra

apex is obtusely rounded and is tied to. the posterior pericardia! wall by a
ligamentous band, the Ligamentum apicis cordis or Gubernacuhtm cordis.
Internally, the heart shows the typical saurian structure. Its cavity
is divisible into a thin-walled sac on the dorsal side (the sinus venoms),
two antero-lateral chambers (the right and left auricles), and a conical thick-
walled sac (the ventricle). The conns arteriosus is absent as a distinct
chamber, having been absorbed in the ventricle.*

(A) Sinus Venosus

The sinus venosus (Text-Fig. 1, B) is a dorso-ventrally flattened
chamber and lies transversely across the part of the auricles directly in
front of the anterior border of the ventricle. It is formed, as in other
lizards, by the confluence of the two anterior vena cavce with the posterior
vena cam, but it shows several peculiarities. Its right half is somewhat larger
than the left, being formed by the union of the right anterior vena cava
with the posterior, the latter sweeping forwards distinctly to the right of the
heart before opening into the sinus venosus. There is no internal ridge
(tuber culum inter venosurri), corresponding to that in the Mammalia, between
the opening of the right anterior vena cava and that of the posterior*
The trachea in Hemidactylus flaviviridis .lies closely adpressed to the medio-
longitudinal part of the sinus venosus, and consequently all this region of the
sinus, when empty, is strongly fluted: a characteristic which has not been
so far recorded in any other reptile. Bhatia (1929) noted a slight median
constriction between the right and left portions of the sinus venosus in
Uromastix hardwickii, but the characteristic groove-like depression in He mi"
dactylus, which evidently serves for the accommodation of the trachea,
cannot be compared with it; there is no constriction of the sinus venosus
in Hemidactylus, comparable to that shown by Bhatia in his diagram of the
heart of Uromastix.

The sinu-auricular aperture is a narrow semi-circular slit in the dorsal
wall of the middle portion of the sinus venosus, lying at right angles to the
anterp-posterior axis of the heart. Its lips, like those in Uromastix hard-
mckii (Bhatia, 1929) are valvular, there being, however, no distinct valves
as described by O'Donoghue (1920) in'Sphenodon punctatus.

(B) Atrium Dextrum (The Right Auricle)

The right auricle is slightly larger than the left. In Sphenodon punc-
(O'Donoghue, 1920), Uromastix hardmckii (Bhatia, 1929),. and a snake

/ n off (1933) has given an account of the manner in which the corns arteriosus

(Bulbus) is believed to have been absorbed in the ventricle.

Development of Indian House-Gecko^ H. flaviviridis Ruppellll 23 5

Ptyas mucosus (Ray, 1934), a small sac-like diverticulum has been described,
arising 'from its antero-dorsal mesial edge. I do not find such a structure
in the heart of Hemidactylus flaviviridis.

The left auricle, although slightly smaller than the right, resembles the
latter in its general appearance. Its postero-ventral portion, however, espe-
cially when full of blood, projects backwards over the left antero-ventral
border of the ventricle to a considerable extent. The common pulmonary
vein opens in the dorsal wall of its posterior portion near the inter-
auricular septum.

(D) Ventriculus

The ventricle is a stout-walled, slightly asymmetric, conical sac with
its apex directed backwards, and is distinctly marked off from the auricles
by a deep coronary sulcus. Its right lateral surface is evenly convex, while
its left one shows a slight concavity.

A study of serial transverse sections show four successive regions in the
ventricle, passing gradually and insensibly into each other. As these regions
have not been so far recognised or described in detail [the only other account
of the saurian heart, based on a reconstruction of serial sections, being
by Ran (1924) who has more particularly studied the origin of the aortic
arches in Tiliqua scincoides}, I give a concise account of these regions here.

In the first place, there is the hindmost, apical portion of the ventricle
(Text- Fig. 2), characterized by the presence of numerous muscular trabecula,

TEXT-FIG. 2

Transverse section passing through the apical part of the ventricle.
lac., lacuna; m.t. 9 muscular trabeculae.

dividing the internal cavity into a series of irregular lacunas. An important
point about this region, not previously observed, is, that the trabecube (at
least the vast majority of them) extend in a dorso-ventral direction, so that the
ventricular spaces are in the form of laterally compressed, vertical chambers.
This would naturally result in the separation of the blood on the right

236 Ben! Charan Maheixdra

of the ventricle from that on the left, while permitting a flow of both kinds
from the dorsal portion of the ventricle, in which the auricles open, into the
ventral portion, from which the aortic arches take their origin (vide infra).

Secondly, there is immediately preceding the apical region the region
of origin of the muscular ridge. This ridge, called " Muskelleiste " by Greil
and other German workers, has been identified by Goodrich (1916, 1919,
1930), O'Donoghue (1918) and Rau (1924) as the septum ventriculorum. As
I do not intend to commit myself to the latter determination, which should
be substantiated by a reference to development,* I prefer to call the
structure " the muscular ridge ".

The muscular ridge in Hemidactylus flaviviridis (Text-Fig. 3) arises as
a prominent structure on the ventral wall of the ventricle distinctly towards

TEXT-FIG. 3
Ventride dissected from the ventral side to show the free edge (near ,/.) o f the muscular ridge

^=r-;s r : ., n

the right and is at its origin, 6bliquely vertical (Text-Fig. 4, A) As it
passes forwards it becomes more and more inclined into a Lkon al direc
ton, and thus separates a small ventro-lateral cavity on the righTside from
a dorso-lateral one on the left (Text-Fig 4 B^ The fnrm *u <<

W rk D '^velopment <** heart seem to be against

Development of Indian House-Gecko, H. flaviviridis Rup P el~III 237

M.R.

(f.C.

(A)

(B)

TEXT-FIG. 4
Transverse sections through the ventricle, showing the muscular ridge.

Section A is caudad to B.
d., dorso-lateral chamber; M.R., muscular ridge; s.r., supplementary ridge.

the dorso-lateral wall of the ventricle towards the right of the muscular
ridge and running in a direction opposite to the latter. As far as I know,
no previous author has recorded this structure. This additional ridge obvi-
ously supplements the muscular ridge.

Thirdly, there is the region of the auricular apertures (Text-Fig. 5),
which both lie dorso-lateral to the muscular ridge and are separated from
each other by an obliquely vertical septum.

M.R.

TEXT-FIG. 5

Transverse section of the ventricle in front of that figured in Text-Fig. 4B.
l.au.ap., left auriculo-ventricular aperture; r.av.ap., right auriculo-ventricular aperture.

238

Beni Charan Mahendra

Fourthly, there is the anterior region (Text-Fig. 6) of the ventricle,
from which the aortic arches take their origin. It is interesting to note that

TEXT-FIG. 6

Transverse section of the ventricle caudad to the -definitive origin of the aortic arches.
ex.M.R., anterior extension of the muscular ridge. Other abbreviations as in previous figures.

the openings of all the aortic arches are situated at the anterior extension
of the free border of the muscular ridge, and thus appear to lie, at different
levels, in the " cavum pulmonale".

(E) Trunci Arteriosi

On account of the absorption of the conus arteriosus, the three aortic
arches take their origin directly from the lumen of the ventricle. Of the
three, the pulmonary arch arises most posteriorly, as a forward prolonga-
tion of the " cavum pulmonale ", and lies at its origin latero-ventrad to
the left of the left systemic arch (Text-Fig. 7). The two systemics can be

v ' TEXT-FIG. 7

Successive transverse sections showing the shifting positions of the aortic trunlcs.
Section A is the most caudad.

Development of Indian House-Gecko, H. flaviviridis RuppelIII 239

traced backwards to a point slightly anterior to the point of origin of the
pulmonary arch, where they intercommunicate with each other. At this
point, the right systemic arch lies dorsally to the other two arches, while
the left one lies below it, slightly dorsal and distinctly to the right of the
pulmonary arch.

Soon after their origin, the aortic arches shift their positions in relation
to each other. The pulmonary trunk gradually assumes a dorsal position.
The left aortic arch shifts from its origin on the right side, at first, to a
distinctly ventral position, and then towards the left side. The right aortic
arch passes over from its original dorsal position to a position on the right
side. The carotid trunk is cut off from the right systemic arch towards its
dorsal aspect slightly towards the left, as the latter is descending to assume
its proper position,

4. The Anterior Vence Cavce

In the present description of the venous system of Hemidactylus flavi-
viridis I have followed, as far as possible, the manner and arrangement
so ably adopted by O'Donoghue (1920) in his exposition of the vascular
system of Sphenodon. The veins are described from the heart towards the
periphery, and a consistent system of numbering them witji alphabets,
sometimes coupled with Roman numerals, is used. As the two anterior
vencE cavse are similar in their disposition, only the left is described
in detail.

The left anterior vena cava (Vena cava anterior sinistra) is an extremely
short stout trunk passing backwards and inwards to open into the left
anterior border of the sinus venosus (Text- Fig. 8). It is formed, immedi-
ately outside the pericardium, by the union of three veins the Vena trache-
alis (A), the Vena jugularis communis (B), and the Vena subdavia (C).
Although there may be slight variations in the mode of union of these three
veins in a vast majority of individuals, the former two trunks definitely
unite together before they are joined by the subclavian. This is clearly
a point of difference from Sphenodon in which all the three trunks are
figured by O'Donoghue as meeting at the same place (O'Donoghue's Text-
Fig. 7). In " Psammosaurus griseus" the anterior vena cava, according to
Corti (1853), is formed by the union of two vessels, Vena jugularis and
V. subdavia, the former of which might perhaps be regarded as representing
the conjoined V. trachealis and V. jugularis communis of Hemidactylus.
A similar condition is shown in a figure of Varanus monitor by Thapar
(1921), although his terminology at places is different from the one used
by O'Donoghue (1920).

240

Beni Charan Mahendra

L.V;

TEXT-FIG. 8

The anterior venae cavas and their tributaries.

Br.V., vena brachialis; Cut.V., cutaneous vein;i.F., vena lingualis; L.v.c.a., left anterior
vena cava ; M. V., ramus from muscles ; Md.V., vena mandibularis ; R.Tr., rami tracheales ; JR..v.c.a* 9
right anterior vena cava; Tr.V.L., left tracheal vein; Tr.V:R. 9 right tracheal vein; V.Az., vena
azygos; V.J.C., venajugulariscommunis; K/./.,venajugularisinterna; F.0., vense oesophageae;
V.Th., vena thyreoidea; V.c.p., vena cava posterior ; V.Co.Cl., venacoraco-clavicularis; V.Co.p.
vena coraco-pectoralis ; V.Subc., vena subclavia.

(A) Vena trachealis

The Vena trachealis (Text-Fig. 8), traced cephalad from the point of
its junction with the Vena jugularis communis, runs forwards dorsad to the
carotid and systemic arches, turning slightly to the middle line so as to
approach the lateral surface of the trachea. It then extends forwards lateral
and parallel to the trachea, receiving minute tributaries from it, as well as
from the thyroid gland (Vena thyreoidea), the oesophagus (Vena cesophagece)
and the adjoining muscles (Rami musculares). After passing dorsal to one
of the posterior arms of the basihyoid, it receives one or two small factors
from the larynx (Vena laryngea), as well as a few from the ventral wall of
the oesophagus (Vence asophagea).

The left tracheal vein, like that in Sphenodon (O'Donoghue, 1920), is
smaller than the right. It begins approximately at the level of the larynx,
while the right one is a stout vessel extending back right from the mandibular
symphysis. Anterior to the place where the left tracheal arises, the right

Development of Indian House-Gecko > H. flaviviridis Ruppel /// 241

tracheal sweeps gradually inwards so as to occupy a mesial position. It
also receives a vein from the tongue (Vena lingualis). *

In Sphenodon punctatus (O'Donoghue, 1920) some of the laryngeal
tributaries of the two tracheal veins anastomose with each other to produce
a venous network, and in Varanus monitor (Thapar, 1921) there are four
anastomoses between the right and the left tracheals. I find no such connec-
tions in Hemidactylus flaviviridis.

As pointed out by O'Donoghue (1920), the tracheal vein is erroneously
termed the external jugular by Parker (1884), a usage which was adopted by
Thapar (1921) in his paper on Varanus monitor. The vein in question, runs
dorsad to the arterial arches and cannot be homologised with the external
jugular in the Amphibia, which latter runs ventrad'to them.

In Lacerta, the left tracheal vein is absent in the adult, although it is
represented in the embryos (Brunei, 1907; Grosser and Brezina, 1895).
During development, however, anastomoses arise between the two tracheals,
so that the right tracheal gradually takes upon itself to drain the left side
also, this leading ultimately to the entire disappearance of the left tracheal
vein. O'Donoghue (1920) claims that the condition in Sphenodon is primi-
tive as it represents the stage in Lacerta when anastomoses have appeared
between the two tracheal veins. The same might perhaps be said for
Varanus monitor. If such claims are tenable, the condition in Hemidactylus
flaviviridis, where there are two tracheal veins with practically no anasto-
moses between them, might be regarded as very primitive.

(B) Vena jugularis communis

The Vena jugularis communis traced cephalad, runs slightly upwards
from the point of its union with the Vena trachealis, extends forwards later-
' ally in the neck and receives small veins from the adjacent muscles. The
part of the common jugular vein extending in the neck, strictly speaking,
corresponds to the Vena jugularis interna of Grosser and Brezina (1895),
Brunei (1907) and O'Donoghue (1920), while the external jugular vein must
be regarded either as altogether absent, or perhaps as represented by one
of the tributaries from muscles, which open into the Vena jugularis communis
in its post-cervical part.

The vena jugularis interna receives, just behind the angle of the jaws,
a large vein, the Vena mandibularis (Vena maxillaris inferior., Grosser and
Brezina) from the lower jaw and its muscles. In front of this point, its main
stem extends on the ventral aspect of the cranium and receives factors from
the brain, palate and other parts of the head. As shown by the develop-
mental studies of Grosser and Brezina on Lacerta and Tropidonotus (1895),

Beni Charan Mahendra

this stem consists of three parts: (a) the posterior part formed from the
anterior cardinal vein, (6) the middle part, derived from the vena capitis
lateralis, and (<r) the anterior part, representing the remnant of the vena
capitis medialis and the orbital sinus.

The subclavian vein is a stout vessel bringing blood from the fore-limb,
shoulder girdle and the ventral and dorsal body wall. Traced distally from
the pointof its union with the jugular and tracheal veins, it runs straight
outwards towards the base of the fore-limb, receiving a Vena coraco-
clavicularis from the dorsal part of the shoulder girdle, a Vena azygos from
the costal and vertebral regions, a Vena coraco-pectoralis from the ventral
region of the shoulder girdle and some factors from the skin. The main
trunk extends into the upper arm as the Vena brachialis. The forma-
tion of the subclavian vein thus resembles that in Sphenodon punctatus
considerably.

5. Vena Cava Posterior

The posterior vena cava (Text-Fig. 9) is a large thin-walled vessel, aris-
ing by the union of the two vena renales revehentes (A) at the level of the
epididymes in the male and at the corresponding region in the female.
It lies rather to the right of the mid-longitudinal line, enters the substance
of the liver at the ventral aspect of the dorso-posterior prolongation of
the right lobe and comes out ventro-mesially a little behind the attenuated
anterior edge of this gland. It receives a number of minute hepatic veins
(B) during its passage inside the liver, and on coming out of that organ,
sweeps round to the right side of the heart to open into the postero-lateral
part of the sinus venosus.

(A) Vena renales revehentes

The ven% renales revehentes (Efferent Renal Veins), which drain the
blood brought to the kidneys, take their origin in" Sauria (as far as hitherto
recorded) in three ways:

(1) Post-renal Origin. Both Thapar (1921) and Bhattacharya (1921)
observed that the vena renales revehentes in Varanus monitor., instead of
taking their origin entirely from the substance of the kidneys, appear to
arise behind them in the form of a post-renal transverse vessel, which
(according to Bhattacharya) even receives a small vein from the dorsal
region of the rectum. A condition somewhat similar to it was described by
Beddard (1904) in Pygopus lepidopus in which "the efferent renals arise
at first as a single trunk very near to the posterior end of the kidneys,

p,v.e

p.v.c

243

v,e.

TEXT-FIO. 9

The posterior veins

<i/i., nmwtomcvtcH revchentes; /.v.r.r, and r.v.r.r,, right and left

renate<( rcvchentc*; ov.i*.. ovariiin vein; /ti j ,r, t posterior vena cava; /.a;?. transverse anastomosis;
.a. t vena ahdommattH anterior; v.a,r., vena anastomotica renalis; v.c., vena caudalis; v.h.a! 9
hepattca advchenn; v,//,c., vena iUacacommuntu; v.arj^ vena cesophagea; v.p. 9 vena pubica;
venit pariclali* latcralit; v./w/,, vena pelvica; v,r.a., vena renalis advehens; v,r,anL t vena
anierlor.

244 Beni Charan Mahendra

and of course between them. This trunk divides into two before reaching
the middle of the kidneys " (see also Beddard's text-fig. 4).

(2) Intra-renal Origin. In Lacerta (Hochstetter, 1893), the venae renales
revehentes take their origin separately in the kidneys, but are connected to
each other by a small anastomosis near the posterior end of that organ.

(3) Sinusoidal Origin. Bhatia (1929) found a large sinus in the middle
region of the conjoined kidneys in Uromastix hardwickii. The sinus receives
several factors from the substance of the kidneys and gives rise anteriorly
to the two vence renales revehentes.

In Hemidactylus flaviviridis, the vence renales revehentes arise in the
second of the foregoing three ways. They commence, by the confluence of
minute efferent renal factors, towards the posterior attenuated ends of the
kidneys and run forwards on the ventral aspect close to the mesial border
of these organs, separated from each other by the dorsal mesentery. The
right vein, as usual in most Sauria and in Sphenodon, is distinctly larger than
the left. Unlike Lacerta, however, in which there is only one inter-renal
anastomosis, and Sphenodon in which there are four such connections be-
tween the venae renales revehentes, the veins in Hemidactylus flaviviridis are
connected to each other by two (rarely one) anastomoses.

Each vena renalis revehens runs forwards in front of the kidney, receiving
some minute tributaries from the mesentery, and comes to lie along the
mesial border of the supra-renal organ of its side. The left vein finishes
here; it discharges into the right vena renalis revehens by a transverse
anastomosis. The right vein, from this point forwards, extends as the
posterior vena cava to open into the liver. The arrangement in Hemidactylus
flaviviridis thus closely resembles that found in Lacerta (Parker's fig. 40,
1884), Uromastix hardmckii (Bhatia, 1929) and Sphenodon (O'Donoghue*
1920), but it differs from that in Varanus monitor, in which (as ascertained
by me by dissection) the two vence renales revehentes, although obliquely
directed from the median line towards the right, are of the same calibre and
contribute equally to the formation of the posterior vena cava.

receive the '

A. I. Venn spermaticv.-The vence spermaticce, which of course are
present only in the male, drain the blood of the testes and
mesorchia. As in Sphenodon (O'Donoghue, 1920), the left
spermatic vein opens into the left vena renalis revehens, while
he right one opens into the posterior vena cava in front of
the latter s junction with the transverse anastomosis from the

of Indian I louse-Gecko, II. flaviviridis RuppelIIl 245

left c!Tercnt renal. This is different from the condition found
cither in Uromastix hardwickii or in Varanus monitor. In
UnwwMi\\ as figured by Bhatia (1929), the right testis is
supplied with four vena? spermaticce* two of which open into
the transverse anastomosis and the rest into the posterior
vena cavn. In Varanm monitor, although Thapar (1921)
and Bhuttaelwrya (1921) do not describe the arrangement,
I find that the right vena spermatiea opens not into the post-
erior vena cava but into the vena renalis revehens of its side.
The disposition of the ovarian veins is similar to that of the
spermatic, but the right ovarian is situated anterior to the left.

A. 11, l*eme supra-nmales revehentw. O'Donoghue (1920) found
that in Splumodon these veins, a series of minute twigs, open
into the vena spermatiea of their side. In Uromastix hard-
wick it, according to Bhatia (1929), each supra-renal body
sends a supra-renal vein to meet the spermatic of its side.
Neither Thapar (1921) nor Bhattacharya (1921) mention
anything in this connection in Varanus monitor. In Hemi-
ttactylus flavivirulis there are several very minute supra-renal
efferent veins corresponding apparently to those found in
SplwmKlon* but they open not into the vena spermatiea as in
that reptile but directly into the vena renalis revehens adjacent
to them.

A. Ill* Vcntt ovitfacales. In the female, a number of minute oviducal
veins can be traced running across the broad ligament to open
into the vena renalis revehens. The disposition of these veins
thus represents an extremely simplified condition, different
from that described in Iguana tuhcrculata (Beddard, 1904),
Chtmwlctm vulgar is (Beddard, 1904), Pygopus lepidopus
(Beddard, 1904), and Helodemut suspectum (Beddard, 1906).

(I. The Supra-renal Portal System

To quote from O'Donoghuc (1920), "the presence of this system has
already been noted in the Varanidiz by Corti, Hochstetter and Beddard,
in which genus it is very well developed. It is subject to a great deal of
variation in the Lacertilia, as Beddard has shown, and although usually
present and often well marked, as in the Varanida* Iguana tuberculata,
Qphisattrus apus and Amplrisbctna bra^iliana, it may be considerably reduced,
e.g., Tiliqim scincoidcs, or even absent altogether, save as an abnormal

246 Beni Charan Mahendra

variation, as in Chameleon vulgaris. In Sphenodon it has apparently retained
more primitive relations than in the other forms described, and this throws
useful light on the question of its constitution." Its main factor is a vein
termed differently by various authors. Hochstetter (1893) called it Vena
deferentialis, while Beddard (1905) termed it the posterior cardinal vein.
O'Donoghue (1920) suggests that it should be called. Vena supra-renalis
advehens. *

In Hemidactylus flaviviridis, I find that the supra-renal portal system
is absent.

7. The System of the Caudal Vein

The vena caudalis (Text-Fig. 9), as usual in the Sauria, runs micl-
longitudinally ventrad to the caudal artery in the tail and .receives tributa-
ries from the caudal muscles in each segment. Anteriorly, it comes to Ho
on the mid-ventral aspect of the posterior tapering, conjoined ends of the
two kidneys and bifurcates, approximately at the level of the anus, to form
the paired vence renales advehentes. The point of bifurcation of the vena
caudalis in Sphenodon punctatus] as described and figured by CTDonoghue
(1920), lies definitely behind the posterior ends of the kidneys; while in
Uromastix hardwickii, the vena caudalis, according to Bhatia (1929), " enters
the kidney where it becomes split up into Y-shaped diverging limbs running
forward and becoming partially buried in its substance." The condition in
Hemidactylus corresponds to that found in Uromastix rather than in Sphe-
nodon. Close to the point of its bifurcation, the vena caudalis receives a pair
of inguinal veins, bringing in blood from the inguinal region.

The two vence renales advehentes, unlike those in Sphenodon, arc equi-
sized. They extend forwards on the ventral aspect of the kidneys, each
receiving a vena cloacalis and a vena rectalis during its course, and disappear-
ing in the substance of the anterior lobe of the kidney a little behind its
anterior border.

From each vena renalis advehens, almost at its mid-point, a branch runs
outwards to the cleft between the anterior and posterior lobes of the
kidney, passes upwards in this cleft and joins the iliac vein. Such an
anastomosis occurs also in Uromastix hardwickii (Bhatia, 1929), Sphenodon
punctatus (O'Donoghue, 1920) and Lacerta (Hochstetter, 1893, Plate 16,
fig. 12), being more superficial in the last genus, but this sort of disposition
differs conspicuously from that found in Varanus monitor (Bhattacharya,
1921), Pygopus lepidopus (Beddard, ,1904), and Chamceleon vulgaris (Hoch-
stetter, 1893; Beddard, 1904).

t of Indian House-Gecko, H. fkviviridis RuppelIII 247

X. The Hepatic Portal System

Although the anterior abdominal and epigastric veins form part of
a single system afferent to the liver, I follow (TDonoghue in treating them
separately for the sake of clarity and convenience.

(A) The System of the Anterior Abdominal Vein

The Vena abdominal^ anterior runs mid-longitudinally along the ventral
body wall and is formed by the union of the paired vemc pelviac in front
of the pelvis. It receives a small tributary from the duodenum a little in
front of the place of its formation, extends forwards to receive the Vena
hepatica advehens ( Vena porta), and enters the left lobe of the liver.

A. Vena pelrica.Thv pelvic vein arises as a branch of the Vena iliaca
comnnmis at the outer aspect of the kidney, and extends, from its point of
origin, downwards and mcsialwards to join its fellow of the other side to
form the vena ahtfaniinalis anterior. During its course, it receives the follow-
ing tributaries:-

A. I. Vena parictalis laterals (Lateral abdominal vein of Beddard).
It arises as a delicate vessel approximately at the level of the
middle of the liver, passes backwards in the angle between
the dorsal and ventral body wall and receives tributaries
from the intercostal muscles. It opens into the pelvic vein
at the place where the latter is curving downwards towards
the mid-ventral line.

A. 11. Vena puhica*- This is a small vein coming from the ventral
aspect of the pelvis near the pubic symphysis.

A. III. Vena vested. The vesicular vein conies from the ventral wall

of the urinary bladder.

B. Vena iliaca communis.This vein, called by Hochstettcr Vena
ischiadica, extends from inside the hind-limb towards the outer border of
the kidney and divides into three branches:

B. I. Vena pelviea, which has been described above.

B. II. Vena renalis anterior. In Sphenodon punctatus (O'Donoghue,
1920), this vein, "'composed of several twigs from the first
kidney lobe," opens into the vena anastomotica renalis. In
Hcmidactylus ftaviviridis, however, I find that it is an inde-
pendent branch of the vena iliaca communis, and is altogether
separate from the vena anastomotica renalis.

B. II I. Vena anastomotica renalis. This is a vein running from the
common iliac into the cleft between the anterior and post-
erior kidney lobes and joins the vena renalis advehens.

248

Beni Charan Mahendra

(B) The System of the Epigastric Veins

In Hemidactylus flaviviridis, this system is composed of a large trunk,
Vena hepatica advehens (A), joining the anterior abdominal vein, and a
small vein Vena cesophagea (B), from the oesophagus and cardiac end of the
stomach. Besides these, there is a small parietal vein, collecting blood from
the ventral aspect of the vertebral column and opening into the dorsal side
of the right lobe of the liver a little in front of the entry of the posterior
vena cava. This last has not been recorded previously.

A. Vena hepatica advehens. The formation of this vein in Sauria,
as shown by the work of many authors, is subject to a great deal of
variation. In Hemidactylus flavivindis (Text-Fig. 10), it is formed, a short

v.oes.

v.pan.

v.r.

TEXT-FIG. 10
The hepatic portal system.

Development of Indian House-Gecko, H. flaviviridis R'uppelIII 249

distance behind its junction with the anterior abdominal vein, by the
union of two factors: the vena gastrica anterior (A. I) and the Vena intestine-
gastero-linealis (A. II).

A. I. Vena gastrica anterior. Thh is a large vein arising near the
anterior end of the stomach and running longitudinally back-
wards on its wall, receiving a number of minute tributaries.
Unlike the one in Sphenodon punctatus (Beddard, 1905;
O'Donoghue, 1920), it has nothing to do with the anterior
abdominal vein, not to speak of its being regarded as a
continuation of the latter in any sense.

A. II. Vena intestino-gastero-linealis. This is a large trunk formed

by the union of two veins: (i) the Vena gastero-UneaUs, which
receives tributaries from the posterior dorsal half of the
stomach wall, the spleen and the pancreas, and (ii) the Vena
intestinalis posterior* which collects blood from the duodenum,
Heum, rectum and cloaca.

B. Vena awphaRcu. The ctsopageal vein arises by the union of an
anterior with a posterior tributary on the wall of the oesophagus and cardiac
region of the stomach. It runs in the gastro-hepatic omentum to open
into the tapering anterior end of the liver towards the left of the emerging
posterior vena cava. Although not described in Sphenodon punctatus, it
occurs in Unmiastix hanlwickii (Bhatia, 1929) and seems to correspond with
the gastro-liepattc vein in Varanus monitor (Thapar, 1921), Varanus griseus
(Hochstetter, 1X93), Varanus vxanthematicus and Varanus niloticus (Beddard,
1906). Beddard (1906) laid stress on its limitation to a single vein in the
genus Varanus as a noteworthy difference from many other Lacertilia. The
same condition, however, was reported by him in the two geckos, Phelsuma
nwiagtiMaruwis and Tarentola annularm (Beddard, 1904), and it will prob-
ably be found a general feature of the family Gekkonidte.

9. Verne Pulmonaks

Each pulmonary vein runs on the ventro-lateral border of the lung,

receiving numerous minute factors from it. It leaves the lung a little way
back from the latter's anterior end, extends mesialwards, and meets its
fellow to form the common pulmonary vein behind the heart. The latter

(Text-Fig K B) runs over the left side of the heart parallel and mesial to
the left pulmonary artery, crosses the left part of the sinus venosus and opens

into the left auricle.

250 Beni Charan Mahendra

10. Summary

The author gives a detailed account of the heart and venous system of
Hemidactylus flaviviridis. The more important features discovered are as
follows :

(1) The heart is situated considerably forwards in the pleuro-peritoneal
cavity. The sinus venosus is strongly fluted so as to accommodate the trachea
which lies closely adpressed to it. There is no mesial constriction in the
sinus venosus as described in Uromastix hardwickiL The right auricle is
devoid of the sac-like diverticulum. The lumen of the ventricle is divisible
into four regions passing insensibly into each other : (a) the apical portion,
subdivided by numerous trabeculae, (b) the region in which the muscular
ridge originates, (c) the region of the auricular apertures, and (d) the region
of the aortic trunks. There is, in addition to the muscular ridge, a supple-
mentary ridge opposite to it in direction. The openings of the aortic arches
are situated at the free border of the muscular ridge.

(2) The disposition of the aortic arches has been described.

(3) The vena trachealis and F. jugularis communis meet each other,
and the combined trunk, thus formed, joins the vena subdavia to form the
anterior vena cava.

(4) The left tracheal vein is smaller than the right, and there are hardly
any anastomoses between them a condition presumably more primitive
than in other lizards.

(5) The external jugular vein is absent, so that the internal jugular drains
the whole cephalic region.

(6) The vena renales revehentes arise intra-renally and are connected
to each other by two anastomoses. The right one is larger than the left
and extends forwards, after receiving the blood of the left side by
a transverse anastomosis, as the posterior vena cava.

(7) The left spermatic vein opens into the left vena renalis revehens, while
the right one opens into the posterior vena cava. '

(8) There are a number of minute oviducal veins opening into the vena
renales revehentes.

(9) The suprarenal portal system is absent.

(10) The point of bifurcation of the caudal vein to form the 'vena renales
advehentes, lies anterior to that in Sphenodon. The two vena renales adve- '
hentes are equi-sized.

(11) A branch of the vena renalis advehens runs in the cleft between the
anterior and posterior lobes of the kidney to join the iliac vein.

Development of Indian House-Gecko, H. flaviviridis RuppelIII 251

^ (12) The vena renalis anterior is a branch of the vena iliaca communis,
independent of the vena anastomotica renalis.

(13) The vena gastrica anterior is a separate vein and has nothing to do
with the anterior abdominal vein.

(14) The vena gastero-linealis and F. intestinalis posterior unite to form
a common trunk.

(15) There is a single cesophageal vein.

(16) The right and left pulmonary veins unite to form the common
pulmonary vein behind the heart,

I. Beddard, F. E.

5 .MM* -, . . ,-

7. BenninghofF, A.

8, Bhatia, M. L.

10. and Dayal, J,

1L Bhattacharya, D. R.

12. Brllckc, E.

13. Bruner, H. L.

14. Corti, A.

BIBLIOGRAPHY

4 * Contributions to the Anatomy of the Lacertilia. I. On
the Venous System in Certain Lizards/' Proc. Zool' Soc
London, 1904, 1, 436-70.

44 Contributions to the Anatomy of the Lacertilia. II. On
some Points in the Structure of Tupinambis " ibid *1904
1, 465-70.

44 Contributions to the Anatomy of the Lacertilia. III. On

Some Points in the Vascular System of Chameleon and

other Lizards," Ibid., 1904, 2, 6-22.

" Some Additions to the Knowledge of the Anatomy, princi-
pally of the Vascular System, of Hatteria, Crocodilus, and

certain Lacertilia," ibid., 1905, 2, 461-89.
44 Some Notes upon the Anatomy of the Yellow-throated

Lizard, Gerrhosaunis flavigularis," ibid., 1905, 2, 256-67.
" On the Vascular System of Heloderma, with Notes on that

of the Monitors and Crocodiles," ibid., 1906, 2, 601-25.
" Das Hera," Bolk, Goppert, KalHus und LuboscWs Handbi

tL vergl Anat. d. Wirbeltiere, 1933, 6.
" On the arterial system of the lizard Uromastix hardwicki.

Gray, 1 * Jour. Morph. and Physio/., 1929, 48, 281-316.
" The venous system of a Lizard Uromastix.Jiardwickii (Gray),"

Zool. Anz., 1929, 85, Heft 1/2, 15-27.
44 On the arterial system of the lizard Hemidactylus flaviviridis

Riippel (the wall lizard)," Anat. Anz., 1933, 76, Nr. 23-24

417-37.

" Notes on the Venous System of Varanus bengaknsis," Jour.

and Proc. Asiat. Soc. Beng. (N.S.), 1921, 17, No. 3, 257-61.
44 Beitrage zur vergl. Anatomic und Physiologic des Gefass-

systemes der Amphibien," Denkschriften der Wiener

Akademie, 1852.

*' On the cephalic veins and sinuses of Reptiles, with a de-
scription of a mechanism for raising the venous blood-
pressure in the head," Amer. Journ. Anat., 1907, 7.

De Systemate Vasorum Psammosauri grisei, 1853,

252

15.
16.

Fritsch, G.
Gelderen, Ch. van

17. Goodrich, E. S.

18.

19.

20. Greil,A.

21. Grosser, O., and Brezina, E.

22. Hochstetter, F.

23. Hoffmann, C. K.

24.

25.

27.

29.
30.

Ihle, Nierstraz, van Kampen
and Versluys
John, C.

Beni Charan Mahendra

"Zur vergleichenden Anatomie der Amphibienherzen,"

Reichert und Dubois Reymond's Archiv., 1869.
" Venensystem, mit einem Anhang liber den Dotter-und

Placentarkreislauf," Bolk, Goppert, Kallius und LuboscVs

Handb. d. vergl. Anat. d. Wirbeltiere, 1933, 6.
" On the Classification of the Reptilia," Proc. Roy. Soc., B,

1916, 89, 261-76.
"Note on the Reptilian Heart," Jour. Anat., 1919, 53, pt. 4,

298-304.
Studies on the Structure and Development of Vertebrates, Mac-

millan & Co., London, 1930.
" Beitrage zur vergleichenden Anatornie und Entwicklungs-

geschichte des Herzens und des Truncus arteriosus der

Wirbelthiere," Morph. Jahrb., 1903, 31.
" "Uber die Entwicklung der Venen des Kopfes und Halses

bei Reptilien," ibid., 1895, 23.
" Beitrage zur Entwicklungsgeschichte des Venensystems der

Amnioten. II. Reptilien (Lacerta, Tropidonotm)" ibid.,

1893, 19.
" Reptilien. II. Eidechsen und Wasserechsen," Bronn's KL

u. Ordn. des Thier-reichs, 1890.
Vergleichende Anatomie der Wirbeltiere, Berlin, 1927.

26. O'Donoghue, C. H.

28. Owen, R.

Parker, T. J.
Rathke, H.

31. Rau, A. Subba

32.

33.
34,

Skramiik, Emil v.
Thapar, G. S.
Wiedersheim, R.

" On the alleged flow of blood from the renal portal to the

hepatic portal system in Varanus bengalensis" Proc. Ind.

Sci. Congr., 11 th session, 1924.
" The Heart of the Leathery Turtle, Dermochelys (Sphargis)

coriacea, with a Note on the Septum ventriculorum in the

Reptilia," Jour. Anat., 1918, 52, 467-80.
"The Blood Vascular System of the Tuatara, Sphenodon

punctatus" Phil. Trans. Roy. Soc., London, 1920, 210, B,

175-252.

On the Anatomy of Vertebrates. Vol. 1 . Fishes and Reptiles,
London, 1866.

A Course of Instruction in Zootomy, London, 1884.

" Untersuchungen Uber die Aorta wurzeln und die von ihnen

ausgehenden Arterien der Saurier," Denkschr. Kais. A/cad.

Wiss., 1857, 13.
" Observations on the anatomy of the heart of Tiliqua scin-

coides zndEunectes murinus" Jour. Anat., 1924, 59, Pt. 1,

60-71.

" Uber die fiihrende Stelle im Reptilienherzen," Zeitschr. /.

vergl. Physiol, 1932, 16, Heft 3, 510-14.
." On the Venous System of the Lizard Varanus bengalensis

(Daud.)," Proc. ZooL Soc., London, 1921, 487.
Comparative Anatomy of Vertebrates, translated by . W. N.
^ Parker, London, 1907,

CYTOLOGICAL STUDIES IN INDIAN
PARASITIC PLANTS

II. The Cytology of Loranthus
BY L. S. S. KUMAR AND A. ABRAHAM

(Botany Department, College of Agriculture, Poona)
Received February 20, 1942

IN a previous paper in this series (Kumar and Abraham, 1941 a\ we
described the cytology of Striga. The present paper briefly deals with the
cytology of Loranthus longiflorus, Desr.

Loranthus species are hemi-parasites and the present material was
collected locally from a mango tree. Due to the presence of large quantity
of mucilage and tannin, proper fixation of the buds was difficult. The
anthers in the immature condition are fused together by their margins to
form a tube. Excellent smear preparations were obtained by dissecting out
the anther tube and laying it with the inner side in contact with the slide
and smearing with a scalpel, applying a little extra pressure, and imme-
diately fixing in Nawaschin's fluid or in La Cour 2 BE. Staining was
done by the usual iodine-gentian violet method.

Mitosis

The somatic chromosomes were studied from sections of the young
ovule. There are eighteen chromosomes, two of which are satellited while
the others have nearly median constriction (Fig, 1).

Meiosis

Meiosis was studied entirely from smear preparations. In smears
stained with acetocarmine the spiral structure of chromosomes was evident
from leptotene stage onwards. But as the chromosomes are not large enough
it was not possible to critically follow all the details of structure.

At diplotene nine bivalents are clearly seen. The nucleolus persists up
to this stage, though only faintly stained, and one of the bivalents is
usually attached to it.

The chiasma frequency at diplotene is very low and most of the bi-
valents are held together by a single chiasma. In acetocarmine preparations
it was seen that in such cases the major spirals of the chromosomes are free

253

B2fl

254 L. S. S. Kumar and A. Abraham

from each other, suggesting that the mechanism of spiralisation is of the
" balanced type " (Abraham, 1939) or " anorthospiral " (Kuwada and
Nakamura, 1940).

Though the free arms of the bivalents are seen diverging from each other
at the diplotene stage (Fig. 2), by diakinesis they come together again and
at metaphase (Fig. 3) they are seen close together. It is not possible to say
whether this is a fixation effect or not; but it is significant that it was
observed in a number of cases both in permanent smears as well as in tem-
porary acetocarmine preparations.

The second division is normal and nine chromosomes were counted
at both metaphase plates in the same mother cell.

Nucleolus

There are two nucleolar chromosomes seen in the somatic complement
and at diplotene one bivalent is seen attached to the nucleolus. An inter-
esting feature seen from leptotene to pachytene is the presence of what
appears like nucleolar buds. As the chromosome threads are very fine and
very distinctly stained, it was possible to study the relationship of these
bodies to the chromosomes. In Fig. 4, a nucleolar bud is seen in contact
with the nucleolus and two leptotene chromosomes attached to it. In
Fig. 5 the nucleolar bud is connected to the nucleolus by two very fine

TEXT-FIGS. 1-8

Fig. 1 . Somatic chromosomes from cell of ovule (2 w=18). Note two satellited chromosomes.
Fig. 2. Diplotene, showing nine bivalents, seven of which possess only one chiasma each.
Fig. 3. Metaphase showing nine bivalents. Figs. 4-8. Nucleolus and knob-like structure on
nucleolar chromosomes (vide text). All figures are camera lucida drawings made from permanent
preparations. Magnification of Fig. 1 i$ x 3,000, while all the other figures are x 2,000.

Cytotogical Studies in Indian Parasitic Plants //

255

threads, which may be the continuation of the chromonemata. In deeply
stained preparations the small nucleolar bud is always *seen in contact with
the nucleolus, probably due to the masking of the gap due to retention of
stain, as in Fig. 8. In Figs. 6 and 7, there are two nucleolar buds each
attached to a chromosome. These clearly indicate that what has been
called a c nucleolar bud ' for the sake of convenience of description may not
actually be the result of budding of the nucleolus, but may be only an
accumulation of nucleolar matter at the constriction between the satellite
and the main body of the nucleolar chromosome. Whether this is really
so or only a knob of the type seen in Euchlcena chromosomes (Longley,
1937) could not be determined. In Sesamum orientate, we found a second-
ary nucleolus originating from the primary nucleolus and persisting through-
out both divisions of meiosis and ultimately getting enclosed in one or other
of the four daughter cells (Kumar and Abraham, 1941 b). But the knob-
like structures seen in the present case are most clearly visible from leptotene
to pachytene and later they disappear, even before the main nucleolus
disappears. The origin of nucleoli and their possible role in nuclear and cell
division have been discussed in detail elsewhere (Kumar and Abraham,
1941 c).

Summary

The cytology of Loranthus longiflorus is briefly described. There are
eighteen chromosomes in somatic cells and two of these are satellited.
Nine bivalents were seen at the first meiotic division. The presence of a
knob-like swelling on the nucleolar chromosomes near their attachment
to the nucleolus -is seen from leptotene to zygotene.

Abraham, A.

Kumar, L. S. S., and Abraham,
A.

Kuwada, Y., and Nakamura, T.
Longley, A. G.

LITERATURE CITED

" Chromosome Structure and the Mechanics of Mitosis and

Meiosis. I. Mitosis in Lilium" Annals of Hot., 1939,

N.S. 3, 11, 545-68.
44 Cytological studies in Indian .Parasitic Plants. I. The

Cytology ofStriga" Proc. Ind. Acad. Sci., Sec. B, 1941 a,

14, 509-16.
44 A cytological study of sterility in Sesamum orientale L."

Ind. J. of Genetics and Plant^Breed., 1941 b, 1, 44-60.
"The organization and behaviour of nucleoli in Sesamum

orientale^ Cytologia, Dec. 1941 c, 12, No. 1.
44 Behaviour of chromonemata in mitosis IX," ibid., 1940,

10, 492-515.
44 Morphological characters of Teosinte chromosomes,"

Journal of Agri. Research, 1937, 54, No. 11, 835-67.

ON THE* BIOLOGY OF RED SPIDER MITE
(TETRANYCHUS TELARIUS LINN.)
IN BALUCHISTAN

BY NAZEER AHMED JANJUA, M.Sc. (HONS.)

(Entomologist, Department of Agriculture, Baluchistan, Qmttd)

Received November 15, 1941
(Communicated by Dr. Hamid Khan Bhatti)

Introduction

THE Red Spider Mite (Tetranychus telarius Linn. Family Tetranychidae,
Acarina), which is widely spread all over the world, is fairly common in the
hilly tracts of Baluchistan where it is one of the major pests of some of
the deciduous fruit trees, vegetables, cultivated plants, etc. In view of its
importance, a detailed study of the biology of the mite has been made during
the last three years and the results are recorded in this paper. The writer
feels indebted to Sri. M. C. Cherian, Government Entomologist, Coimbatore,
and Dr. Khan A. Rahman, Government Entomologist, Punjab, for their
valuable criticism; to Mr. A. M. Mustafa, Agricultural Officer in Baluchi-
stan, for his able guidance; to the Imperial Institute of Entomology,
London, for the identification of the species and to , M. Sabir Janjua,
Entomological Fieldman, Quetta, for his help in the field and laboratory.

Distribution

The Red Spider Mite is cosmopolitan; it is widely distributed between
the tropical zones and North Germany. It is also found all over North
and South America, Hawaii Islands, South Africa, Australia and Palestine.
It is fairly common in Punjab and South India. In Baluchistan, it is widely
distributed in the hilly tracts, its attack being particularly severe in the
districts of Quetta-Pishin and Loralai. This is the first record of the pest
from this province.

Host Plants

The Red Spider Mite is markedly polyphagous, its food plants num-
bering over 300 different species in various parts of the world (Zacher,
1928). These include weeds, shade and forest trees, ornamental plants,
garden and field crops. Its attack is particularly severe on plants grown
in green-houses in Europe and America. McGregor (1917) has recorded
183 host plants of the mite in America where it is a serious pest of cotton,
deciduous fruit trees, citrus varieties and a majority of vegetables. In
256

Biology of Red Spider Mite (T. telarius Linn.} in Baluchistan 257

Europe its host plants are weeds, shrubs, fruit trees, ornamental and green-
house plants. Sixty-three host plants of the pest have been registered in
Palestine which include fruit trees, vegetables, fodder crops, ornamental
plants, wind-break and forest trees (Klein, 1936). Its attack is, however,
more severe on Citrus trees. In South India, Cherian (1938) has recorded
the mite on a variety of plants, the more important of which are ganja
(Canabis sativa), castor (Ricinus communis), tomato, Cambodia cotton (Gos-
sypium hirsutum), rose and jasmine (Jasminum sambac). In Punjab, it is a
serious pest of a large majority of plants such as lady's finger (Hibiscus
esculentus\ " tinda " (Citrulls vulgaris var. fistulosus), pulses such as
" mothe " (Phaseolus aconitifolius), " mung " (P. mungo),' " mash " .
(P. radiatus), " desi-sem " (Canavalia ensiformis), " ghiatori " (Luffa
cegyptica) and sweet-potato (Ipomcea batatas) (Rahman and Sapra, 1940).

The following 32 host plants have so far been observed by the writer
in Baluchistan. Those that are severely attacked are : almond (Prunus amyg-
dalus), apple (Pyrus malus), apricot (Prunus armeniacd)., carnation (Dianthus
caryophyUus), celery (Apium graveoleus), cherry (Prunus cerasus), chrysan-
themum, geranium, grape-vine (Vitis vinifera), nectarine (Prunus persica var.
Nucipersicd), peach (Prunus persica), pear (Pyrus communis), plum (Prunus
communis), poplar (Populus sp.), potato (Solanum tuberosum), rose (Rosa
muschatd), strawberry (Fragaria spp.), tomato (Solanum lycopersicum),
walnut (Juglans regia) and willow (Salix spp.), while those that are less
attacked are : ash (Fraxinus excelsid), cabbage (Brassica oleracea), cucumber
(Cucumis sativus), dahlias (Dahlia spp.), egg-plant (Solanum melongend), lettuce
(Lectuca sativa), melon (Cucumis melo), pea (Pisum sativum), turnip (Brassica
Rapa), verbena (Verbena spp.), violet (Viola spp.) and water-melon (Citrulus
vulgaris). The above-mentioned plants, however, represent only a part of
the total number of host plants in this province and future research is sure
to reveal further host plants and swell the list greatly.

Injury

The mites feed upon the undersides of the leaves which they first
cover with silken threads and then suck the sap by piercing the epidermis.
A small white spot develops around each feeding puncture which at first
shows only on the underside of the leaf; later on the area becomes trans-
lucent as the spots coalesce. When these punctures become numerous, the
foliage loses its healthy green colour, turns pale and appears sickly. The
leaves so affected do not function normally and often turn rust-red before
they crumple and dry up. In severe infestations, the mites spin webs of such
thickness over the foliage that the leaves practically cease to function. The

258 Nazeer Ahmed Janjua

buds may also be attacked, and even if they are not, they may still fail
to produce normal flowers if the leaves are badly infested. Infestations,
when very severe, not only result in the plants being badly webbed but
also cause them to die.

Developmental Stages and Seasonal History

The female after hatching from the egg passes through three quiescent
and three active stages, while the male goes only through two quiescent
and two active stages. The stages in the life-history are as follows :

Female Male

Egg Egg

Larva Larva
Quiescent Stage 1 . Quiescent Stage 1

Protonymph Protonymph

Quiescent Stage 2 Quiescent Stage 2

Deutonymph Adult
Quiescent Stage 3
Adult

The Egg (Fig. 1). The egg is round, spherical in shape and when
newly laid is semi-transparent, globular and whitish. It gradually turns to
a light straw colour and shortly before hatching becomes opaque and
pink. On an average the egg measures 0-108 mm. in diameter.

The eggs are deposited singly on the underside of leaves amongst the
fine strands of webbing spun by the female. They are hardly visible to
the naked eye. The largest number of eggs deposited by a single female
was 72, and the largest number of eggs laid during a single day was 15.
Eight, nine and eleven eggs a day are frequently laid during April and May.
A single female on an average lays 37 eggs ten females laying 370 eggs.
During winter season the incubation period of the eggs was 6 days From
May to October the incubation period averaged 3-6 days. One brood
deposited in July hatched in 2 days. The average length of the incubation
period for the entire season was 4-9 days.

The Larva (Fig. 2).-When the larva is about to hatch, the shell of the
egg splits and the larva which is round and of the same s4 as egg,

n f* 6 - ? y 7 the e y e ' s P ot - n an average the larva measures
0- 15 mm. in length and 0- 12 mm. in width. measures

1 ' tClanus

Baluchistan 259

The Red Spicier (Tttranchychus tctarius L.

Larva. 2CB

Protonymph, <20t).

Adulf ma It, / 200.

Adulr fewilc, x 284,

Fro. 7. Palp of female. ) Highly

; ^-^/-t leg of male. LUified.
Ho, 9. Claw of ie^s of female. J

260 Nazeer Ahmed Janjua

The average time required for the larva to attain maturity was 2-6 days
for the whole year. The larv* developed in 3-4 days from January to March,
in 2-3 days from April to May and in 1-2 days from June to September.

When the larva attains its growth, it enters a resting stage (Quiescent
Stage 1) The mouth parts are thrust into the tissue of the plant, the front
two pairs of legs are extended parallel to each other and projected forwards
while the hind pair of legs extend along the sides of the body. During the
months of December, January and February this resting stage will last for
about one day but during the warmer months this resting period lasts only
a few hours.

Protonymph (Fig. 3). The larva undergoes a moult, when the larval
skin, underneath which a new skin has been developed, breaks and discloses
the protonymph. In addition to being larger, this differs from the larva in
that it has four pairs of legs instead of three pairs. The dark spots at
the sides of the body are slightly larger and the bristles on the body are
longer. The abdomen becomes yellowish with brown spots on the sides,
cephalothorax and legs are of the same colour but the tips of the legs are
reddish. On an average the protonymph measures 0-22 mm. in length and
0-15 mm. in width. *

The nyumphs of this stage are somewhat more active than the larvae
but their habits are similar. They begin feeding soon after the moulting
is completed. The average time required for the completion of this instar
was 2 1 days. As with the larvae, this instar lasted much longer during the
colder months. It required 2-3 days from January to March; l-l-J- days
from April to May; and about one day from June to September. They
pass through a resting or premoulting period (Quiescent Stage 2) which
lasts for an average of one day during the winter months and for only six
hours during the more active period.

Some protonymphs become much elongated and develop to male while
the females remain more or less rounded in shape. The sexes are distin-
guishable from each other immediately on hatching from the egg by the
structure of the palps and the claws of the first leg (Figs. 6, 7, 8 and 9).

Deutonymph. It has been observed that the males do not pass through
this stage but develop directly from the protonymphs. The females, how-
ever, pass through this additional stage which differs from the first in that
the body becomes larger and longer. The abdomen becomes yellowish
brown with black spots on the sides; cephalothorax and legs are of light
yellow colour while the tips of the legs remain reddish. The average
length of females of this stage is 31 mm. and width - 19 mm, The average

Biology of Red Spider Mite (T. telarius Linn.} in Baluchistan 261

length of time required for this nymphal stage was 2-6 days; during cooler
months about three days and in summer months about one day. This
instar also passes through a short resting period. (Quiescent Stage 3) before
the last nymphal skin is shed.

Adults (Figs. 4 and 5). The adult male is easily distinguishable from
the female by its attenuated shape. The female is broader and longer than
the male. The average length of 20 females is 0-42 mm. and breadth 0-25
mm., while the average length of an equal number of males is 0-25 mm.
and 0-13 mm. wide. The females vary considerably in colour, ranging from
a light green colour to black. Occasionally light yellow and red individuals
arc observed, but most of the individuals are green in colour with darker
spots at each side of the body. Cephalothorax and legs are yellowish
while the tips of the legs are reddish. The variations in colour in the male
are very slight. Most of the individuals are light green in colour with small
black spots at the side of the body and a prominent red eye-spot. After
feeding, black areas appear rather far forward on each side of the dorsal
surface of both sexes and a string of dark pellets makes their appearance
posteriorly in the duct of the excretory organ. In the female these dark
globular products of digestion become diffused over the whole abdomen
and give the mite a black appearance shortly prior to death.

In several cases the males were observed assisting the females to shed
the last nymphat skin and in these cases copulation took place immediately
following the female's complete freedom from the nymphal skin. After
mating, the females begin feeding almost immediately and pass through
a short prc-oviposition period which was on an average 1 *5 days; in summer
ov {position began after 24 hours, in spring and autumn the period was
2 days while in winter it went up to even 3 days. Generally speaking, males
and females possess the same longevity, which varies with the various seasons
of the year. In winter, the adults lived upto 25 days, in spring up to 20 days,
in summer up to 10 days and in autumn up to 18 days. The average egg-
laying period extended through a period of 15*1 days. The longest ovi-
position period for a single female was 25 days.

In winter the mites are gregarious, often gathering in vast numbers
In one place, where they spin a common web in which they constantly move
about. They are found chiefly in cracks in the soil and shelter under rocks,
crevices in the bark of trees and shrubs and among trash and weeds. All
the are met during the winter season and on very cold days when

the temperature goes far below the freezing point, they become inactive
but even then sunlight will induce their multiplication and they resume their

262

Nazeer Ahmed Janjua

ctivities. It has been estimated that there are approximately 21 generations
of the mite in a year in the Quetta Valley (5,500 ft.). Generations develop-
ing during the winter months required a much longer time (22-28 days)
than those developing during the summer months (9-12 days).

. The life-history may be summarized as follows :

Summarized Life-history Data of Red Spider Mite (Female)

Stage in life-history

Maximum
(days)

Minimum
(days)

Average
(days)

Incubation period of eggs

4.9

Larval period
Quiescent Stage 1

4
1

1
4 hrs.

2-6
12 hrs.

Protonymph Stage
Quiescent Stage 2

3
1

1
6 hrs.

1-9

14 hrs.

Deutonymph Stage

2-1

Quiescent Stage 3

3 hrs.

10 hrs.

Life of adult

18-7

Pre-oviposition period
Number of eggs deposit

sd per fer

nale

J
21

1-5

In some females parthenogenesis was observed and the unfertilized
females were found laying eggs.

Natural Enemies. At Quetta, Adalia decempunctata Linn. (Family
Coccinellidae) and Chrysopa sp. (Family Chrysopidae) are the natural enemies
of the Red Spider Mite, but these are not found in large numbers as to
check the pest.

Cherian, M. C.

Klein, H. Z.

McGregor, E. A.

Rahman Khan, A., and Sapra,

A. N.
Zacher, F.

REFERENCES

Agric. & Live-stock in India, 1938, 8, 537-40.
Bull. Agric. Res. Sta. Rehovoth, 1936, 21, 37-63.
U.S.A. Dept. of Agric. Farmer's Bull, 1917, 831.
Proc. Ind. Acad. Sci., 1940, II, 177-96.

NachrichtenbL f. d. Deutsch. Pfl. Schutzdienst. Nr. t 1928, II.

y, by G. Srinivasa Rao. Superintendent-
by The Jndian Academy of Sciences, Bangalore,

/Vz P. Majumdar

Proc. Ind. Acad. Sd., B, vol. XV, PL VI

Girija P. Majiimdar

Proc. Ind. A cad. Set., J3, vol. XV, PL

STAGES IN THE SPERMATOGENESIS OF

SIPHONOPS ANNULATUS MIKAN. AND DERMOPHIS

GREGORII BLGR. (AMPHIBIA : APODA)

BY B. R. SESHACHAR

(Department of Zoology, Central College, Bangalore)

Received March 12, 1942
(Communicated by Prof. A. Subba Rau, D.SC., F.R.M.S.)

FOR some years past the author's interest has been aroused by the peculiar-
ities in the gametogenesis of the members of the Apoda and has found ex-
pression in a number of papers on the various aspects of its study. India is
the home only of four genera of Apoda of which two are very rare. The
more common genera (Ichthyophis and Uraotyphlus) which have been studied
by the author have given indications of the importance of the study which has
tempted him to continue his investigations and extend them to the other genera
of Apoda. The present paper is the result of this effort and marks an attempt
to consolidate our available knowledge of the spermatogenesis of this inter-
esting group. In this the author has not been as successful as he would have
wished to have been. The material which forms the subject-matter of
this paper is not Indian and evidently had not teen treated for the study of
the chromosomes. The chromosomes, their number and behaviour, have
formed a very important part of my earlier studies on the spermatogenesis
of Ichthyophis and Uraotyphlus and it is therefore with considerable regret
that I state that I am unable to write on this aspect of the question. But
feeling that it might be a very long time before fresh material may come my
way, I have thought it fit to describe the general process of spermatogenesis
in Siphonops annulatus and Dermophis gregorii and compare it with that in
Ichthyophis glutinosus and Urceotyphlus narayani.

A single male specimen of each of these two species came into my
possession. For the specimen of Siphonops annulatus I am grateful to
Prof. A. Subba Rau who obtained it in turn from Prof. J. P. Hill's
collection in the University College, London. The specimen of Dermophis
gregorii is the property of Dr. L. S. Ramaswami who kindly allowed me
to examine the form and remove the testis. I am grateful to both Prof.
A. Subba Rau and Dr. L. S. Ramaswami for placing at my disposal this
valuable material.

263

264 B. R. Seshachar

The specimen of Siphonops annulatus was fixed in corrosive sublimate
acetic and later transferred into alcohol. The fixation of Dermophis gregorii
is not known. Both of them had been lying in alcohol for a long time
before they came into my possession but the preservation of the former is
very much the better of the two. It has given me all the information I wanted
on the spermatogenesis of this species, except the chromosomes. The
preservation of D. gregorii is not very satisfactory but some of the stages
stand out clearly and have lent themselves for a correct and clear interpre-
tation.

I wish to thank Prof. A. Subba Rau for his kindness and encouragement
throughout the course of this study and for his many helpful suggestions.

Structure of the testis.All the Apoda agree in the disposition of the
testis. It occurs as a number of beaded structures strung together along
the collecting duct as has akeady been observed in Ichthyophis and Urceo-
typhlus and as noticed again in Dermophis and Siphonops. The number
of testis lobes, however, is subject to variation, not only in the different
species but also in the different individuals of the same species. I found
only three lobes on each side in the specimen of D. gregorii that I dissected.
I cannot say if this is the normal number found in the species. At any rate
it represents the lowest number of testis lobes found by me in any Apodan
example. The size of the individual testis lobes is also subject to variation
and the remarks I made about the significance of this variation in Ichthyophis
and Urceotyphlus apply to the present genera also.

The microscopic structure of the testis of the two genera under examin-
ation shows a close similarity with that of Ichthyophis and Urceotyphlus.
The external appearance of each testis lobe shows a number of rounded
elevations which have been described by earlier workers (Spengel, 1876;
Tonutti, 1931) as resembling a bunch of grapes. Each of these elevations
represents a locule and a number of such locules make up the testis. In
regard to the size of the locules the genera under examination differ a little,
those of Dermophis being slightly the larger of the two.

In the matter of the microscopic structure of the testis, the Apoda exhibit
two distinctive characters. First, the disposition and size of the locules
both show a very distinct and clear departure from either the urodelan plan
on the one hand or the anuran plan on the other. The locules are very
large in the Apoda as compared with the Urodela or the Anura and are
separated by clear thin septa (Fig. 1). Each locule is filled with a fatty matrix
in which are embedded cell groups in different stages of spermatogenesis.
This matrix is very characteristic of the Apoda and has been found by me

Spermatogenesis of S. annulatus Mikan. & D. gregorii Blgy. 265

-in all the four genera examined and has also been found by Tonutti (1931)
in Hypogeophis. The presence of this matrix imparts an appearance to
the testis which is different from the crowded nature of the cells in the
testis of either the Anura or the Urodela. In the latter groups, the
comparatively small sized locules and the large number of cells (in different
stages of spermatogenesis) give a packed appearance to the entire organ,

which is not seen in the testis of the Apoda at any time of the year or in any
period of spermatogenesis. Never at any time have I seen the locules of
the testis packed fully with cells as in the other two groups of Amphibia.
Even at the height of its activity much of the space in the locule is occupied

266 - B. R- Seshachar

by the fatty matrix and it is only in this matrix that the germ cells
lie embedded. Indeed, it would appear that this matrix is essential lor
the development of the cells themselves.

The second point of interest is the uniformity of this testis structure
throughout the group Apoda. So far the structure of the testis of five genera
is known; four studied by me and one (Hypogeophis) by Tonutti (1931) and
in all these the peculiar and unique microscopic appearance of the testis
described above, is seen. In this respect, therefore, the Apoda is a close
knit homogeneous group.

To summarize: the unique features of the testis of the Apoda consist
in (a) the segmented nature of the organ which is resolved into a number
of lobes strung along the collecting duct and which extends over a greater
part of the length of the animal on either side of the alimentary canal ; (h)
the large size of the locules of the testis; these lobules are not tubular as in
the Urodela or the Anura but are more or less spherical and vary in number
according to the size of the testis lobe; (c) the peculiar arrangement, in
groups, of the sex cells in the locules. Each locule exhibits a number of
cell groups each of which is in a certain stage of spermatogenesis and such
groups occur scattered and embedded in the fatty matrix that fills the locule.
Generally the cell groups in earlier stages of spermatogenesis occur near
the periphery of the locule while those in later stages and those undergoing
spermateleosis occur nearer the centre of the locule. This arrangement
is unique in the Amphibia, in the other two groups of which neither the fatty
matrix nor the scattered arrangement of cell groups is found, and where
cells in different stages of spermatogenesis are packed together in the avail-
able space found in the narrow tubules.

The question may be asked if it is possible to account for this variation
m the external and internal structure of the testis of the Apoda. A partial
answer may probably be found for the former. The lobed nature of the testis
may be a result of the elongation of the body of the animals of this group
Even this is only a partial answer; for, while elongation of the body brings
about, on the analogy of other animals, an elongation of the organs and
also an asymmetrical development of the organs of the two sides, it does
not ordinarily produce a segmentation of the organ. It is probable that
he reason for the lobmg of the testis in the Apoda is a more fundamental

th^rZ^A Geand T ^ 10 ked f r in the ancest ^ Condition of
the group. An answer to this cannot be provided at this stage.

m The origin and significance of the second structural feature of the testis
even more obscure. The very large size of testis *

Spermato genesis ofS. annulatus Mikan. & D. gregorii Blgr. 267

with those of the other groups of Amphibia is as inexplicable as the develop-
ment of the peculiar matrix filling the locules,

The relation between the collecting duct and the locules has been
described by me in Ichthyophis and Urceotyphlus where I have shown that
the longitudinal collecting duct runs through the testis following the contour
of the locules and giving off short side branches to the latter. The locules
of Dermophis gregorii resemble those of Ichthyophis and Urceotyphlus in
size while those of Siphonops annulatus appear to be much smaller
comparatively. Spengel (1876) in his observations on the structure of the

testis of Apoda indicates the relation between the testis locules and the collect-
ing duct such that the latter runs in the centre of the testis with the locules
arranged radially around it. In Ichthyophis and Uraotyphlus this regular
relationship between the two could not be distinguished and it was observed
that the collecting duct followed irregularly the interstices of the locules
giving off smaller ducts to the locules. I find the same kind of arrangement
in Dermophis also. In Siphonops on the. other hand, a very slight trace of this
central position of the duct and the peripheral position of the locules is seen
(Fig. 17). The main collecting duct appears to be central, with the locule

268

B. R. Seshachar

arranged peripherally. But it must be mentioned that this arrangement i
not constant in Siphonops as seen in Fig. 2.

Spermatogonia. My remarks regarding the origin of primary spermato-
gonia in Ichthyophis hold good for the two genera under examination.
I have pointed out how in Ichthyophis the cells lining the ducts of the testis
undergo transformation and so become germ cells. Large numbers of these
cells were constantly found at the mouth of the duct in the locule in Ichthyo-
phis and Urceotyphlus. I believe, in the two genera under examination also
the duct mouth forms a constant source of primary spermatogonia in the
adult (Fig. 3). Some of these cells at the rcouth of the duct become large

with conspicuous spherical and polymorphic nuclei while others invest
them and become the follicle cells. From the close similarity which the
sections of the testis of Siphonops and Dermophis present to the sections of
the testis of Ichthyophis and Umotyphlus, I have reasons to believe that the
origin and behaviour of the primary spqrmatogonia is very similar.
Arising in this position the primary spermatogonia migrate along the wall

Spermato genesis ofS. annulatus Mikan. & D. gregorii Blgr. 269

of the locule taking up positions along it (Fig. 19) where they start to divide
and grow and pass through meiosis.

The size of the primary spermatogonium is subject to great variation,
according to its age and activity. In Siphonops it varies from 25 to 40 microns
while in Dermophis the variation is between 16 and 30 microns.

The problem of polymorphism of the nuclei of primary spermatogonia
has been dealt with by me in sufficient detail in Ichthyophis (1936) and in
Urceotyphlus (1939) and I need not dwell on it here at any great length.

My conclusions regarding polymorphism are amply borne out by my obser-
vations on Siphonops and Dermophis. The spherical form of the nucleus
denotes an earlier condition and the polymorphism (Figs. 6 and 20), which
is never very pronounced and which is similar to that encountered in other
Apoda indicates a condition of particularly heightened metabolic activity.
The nucleus however, always reverts to its spherical form just before
division (Figs. 7, 8 and 22). The .number of nucleoli is subject to great
variation and from a condition where there are one or two nucleoli (Fig. 5)

270 B. R. Seshachar

to one where there are several (Figs. 4 and 21), all gradations occur.
As in Ichthyophis and Uraotyphlus the staining reactions of the nucleus of
the primary spermatogonium also vary according to its spherical or poly-
morphic condition, being deeper in the former and fainter in the latter.

Of the cytoplasmic bodies I am able to speak only of the centrosome ;
for this, along with the contained centrioles, is the only object that is at all
preserved in the material. Even the centrioles are not always preserved well
enough to be seen clearly. This is especially so in regard to Dermophis where
the fixation is not as good as in Siphonops. In this latter material, however,
the centrioles are quite clear and occupy the centre of the archoplasmic
area which bears the same relationships with the nucleus and cell in general
as in Ichthyophis and Umotyphlus (Fig. 21). These relationships leave me
in no doubt as to the topography of the different cytoplasmic inclusions of
the primary spermatogonium, which, had they been well fixed and preserved,
would have revealed the same arrangement as in Ichthyophis and Urceo-
typhlus.

A word about the amitotic divisions of the nucleus of the primary
spermatogonium. In my paper on Ichthyophis (1936) I discussed this
matter fully and subscribed to the view of Wilson (1928) that amitosis here,
as elsewhere, means nothing more than a fragmentation of the nucleus with
an attendant increase in the nuclear surface and that in no case could a divi-
sion significance be attached to it. In Ichthyophis I did find, though extremely
rarely, a few isolated instances of binucleate spermatogonia. In Siphonops
during my examination of the very limited material at my disposal I found
a single primary spermatogonium with two nuclei (Fig. 23). I was
unable to determine the nature and condition of the cytosome but I feel that
it is unnecessary to deviate from the conclusions drawn in case of Ichthyo-
phis, that amitosis here, as in Ichthyophis, has no division significance and
means nothing more than a temporary change involving an increase of
nuclear surface.

The onset of division brings about a conversion of the polymorphic
nucleus into a regular spherical condition and so far as I can see, the changes
that occur in the nucleus and cytoplasm are similar to what I have reported
already in Ichthyophis and Urceotyphlus. The nucleus shows blocks of chroma-
tin connected by filamentar processes and these are the forerunners of the
definitive chromosomes. A stage of the kind is shown in Fig. 7. The dif-
ference between the nucleus in a state of rest and a stage just prior to division
can be seen on comparing Figs. 21 and 22. In the former, the nucleus appears
granular, the granules filling the cavity pf the nucleus more or less evenly,
while in the latter, the nucleus appears like a vesicle the chromatin having

Spermato genesis of S. annulatus Mikan, & D.
5

gregorii Blgr, 271
6

277 B. R. Seshachar

into a few coarse masses leaving conspicuous clear spaces
Th"Ser=nce was a.so observed in the two genera

studied earlier.

The primary spermatogonium divides by mitosis and the products of
division remain together just beneath the locule scutum. The number of
d visions a primary spermatogonium passes through before meiosis starts
Ts Teen a matter of interest to me and I have determined that m Ichthyo-
Ms the number of divisions is eight, while in Urceotyphlus it is more irregular
the primary spermatogonium passing, sometimes, through six divisions and
sometimes through seven.

In the present two genera also I tried to count the number of divisions
the primary spermatogonium passed through before meiosis set in and so
far as the material at my disposal enabled me, and based on the countings
of the metaphase plates of the first meiotic divison, I found that in Sipho-
nops the number of divisions was seven and in Dermophis it was slightly
irregular, being either six or seven. From an examination of the conditions
obtaining in the four genera of Apoda I have so far studied, I am in
a position to conclude that the number of divisions a primary spermato-
gonium in the Apoda passes through before meiosis, though subject to some
variation^ is six, seven or eight. So far it is only in Ichthyophis I have
noticed evidences of eight divisions.

Spermatocytes. After the completion of the divisions by mitosis, the
cells, which are now the primary spermatocytes, enter on the meiotic phase.
At first, as in Ichthyophis and Urceotyphlus, these cells are arranged along
the periphery of the locule in two rows (Fig. 24) and form a compact mass.
But as meiosis proceeds, the cells leave their peripheral position and migrate
inwards into the locule, the individual cells of the same group occurring
together but the cell groups themselves being separated from one another

and lying in the matrix filling the locule (Figs. 1 and 2).

I have observed both in Ichthyophis and Urceotyphlus a stage of rest
intercalated between the last division of the spermatogonia and the prophase
of meiosis of the spermatocytes. In this condition the nucleus shows a
large number of blocks of chromatin. But there is no attendant diminu-
tion of basophily of the nucleus as has been observed in a number of Amphi-
bia, notably in Rana (Witchi, 1924) and in Bufo (Saez and others, 1936).
In Siphonops, where this stage could be observed with great clearness, it
was even more pronounced than either in Ichthyophis or in Urceotyphlus
(Figs. 9 and 25).

Spermato genesis of S. annulatus Mikan. & D. gregorii Blgr. 273

The first stages of meiosis appear to be similar to those described in
the two Indian genera. The leptotene bouquet is built up as in Ichthyophis
and Urceotyphlus. The polar orientation of the leptotene threads is evident
even from the start and as in the above two genera, the threads begin to be

formed at the pole, the rest of the nucleus displaying unthreaded granules.
The pachytene stage is clear and conspicuous with its thicker threads (Figs.
10 and 26) and forms, as in Ichthyophis, by far the most stable stage of meio-
sis and is one of longest duration. Gradually the polar orientation of the

74 B. R. Seshachar

threads of the pachytene nucleus is lost and soon the threads lie anyhow
inside the nuclear cavity spanning it (Figs. 11 and 27). Meanwhile splits
and spaces are appearing inside each thick bivalent chromosome and the
duality of each of these is clear and evident (Figs. 12 and 28).

Associated with the diplotene stage has been described in the Apoda
a conspicuous stage of diffusion of chromatin where the individuality of
the earlier diplotene chromosomes is temporarily lost in an indistinguish-
able network characteristic of all the Apoda. The ' diffuse ' stage has been
described in a variety of plants and animals and while it appears as a
more normal phenomenon in the development of the oocyte in animals,
its occurrence in spermatogenesis is relatively rare but more interesting.
Chickering (1928) has given a detailed account of this stage in the
spermatogenesis of Belostomatidae (Hemiptera) and his observations coincide
with mine in the Apoda (1937, 1939). Chickering traces the development of
diffusion in Lethocerus step by step in which the first step is marked by a
separation of the two univalents at intervals, their transverse movement and
a fine branching. MyFig.15 of Ichthyophis (1937) and his 59 of Lethocerus
are strikingly similar. And Chickering concludes " a coarse reticulum is
formed by a continuation of this process", a statement which is very simi-
laf to my description of the process in Ichthyophis. The extension of my
observations of this stage to Urceotyphlus and now to Siphonops and
Dermophis substantiate my conclusions arrived at in case of Ichthyo-
phis and I have reasons to believe that the 'diffuse' stage is a universal charac-
ter of the Apoda, following the diplotene stage and arising in the same manner
as that described by me in Ichthyophis. A nucleus in the 'diffuse' stage is
shown in Fig. 13.

The 'diffuse' stage is of fairly long duration at the end of which the
chromosome bivalents emerge gradually from the network characteristic
of the diffuse condition. The final stages of this condensation show the bi-
valents still long and thin and bearing a large number of transverse filamentar
processes (Figs. 14 and 29). The chiasmata can be made out clearly now
and it is seen that in some of the larger bivalents the chiasmata are quite
large in number. This character of the large number of chiasmata in the
early stages of diakinesis was noticed in Ichthyophis and also in Urceotyphlus
where 6 to 7 chiasmata were observed. In these two animals it was noticed
that the number of chiasmata were gradually reduced till the largest bivalent
m either form did not have more than four chiasmata in the final condition
Unfortunately I am not, on account of the unsuitability of fixation of the
material, able to describe the history of the chiasmata or trace the fate of
the chromosomes into metaphase.

Spermatogenesis of S. annulatus Mikan. & D. gregorii Blgr. 275

The division stages succeed each other rapidly, and, intercalated be-
tween the first and second meiotic divisions there is a definite stage of rest
(Figs. 15 and 30) of fairly long duration as observed by me in Ichthyophis
and Urceotyphlus. The spermatids are formed after the second division
(Fig. 16).

The following measurements give the diameter of the nucleus in the
different stages of spermatogenesis in the two genera under examination.

Stage '

Siphonops annulatus

Dermophls gregorii

microns

Primary spermatocytes at rest

9 to 10

5 to 6

Pachytene stage

11-5 to 12

10 to 11

* Diffuse ' stage

12-5 to 13-5

11 to 12

Diakinesis

15 to 16-5

13 to 13-5

Secondary spermatocyte

10 to 10-5

6 to 8

Spermatid

6 to 7

4 to 5

Summary

The testis structure of the two genera described here shows that it
conforms to the plan outlined for Ichthyophis glutinosus and Urceotyphlus
narayani except that in Dermophis gregorii very few testis lobes were
seen. The testis locules are smaller in Siphonops annulatus when compared
with those of the other three genera. The locules are filled with a matrix
which in Ichthyophis and Urceotyphlus were determined as containing fat.
In this matrix are embedded the germ cells in groups in different
stages of spermatogenesis. The primary spermatogonia are found at the
mouth of the duct in the locule and are believed to have arisen, as in Ichthyo-
phis, from the cells lining the duct epithelium. Their nuclei may be
spherical or polymorphic, the latter condition indicating a high degree of
metabolic activity. Just before division, however, the nucleus resumes its
spherical or oval contour. After a number of divisions, varying between six
and eight, the cells, now primary spermatocytes, embark on the meiotic
phase after a brief period of rest. The leptotene and pachytene stages follow,
after which, the nucleus is marked by a 'diffuse' condition in which the
chromosome bivalents lose their identity temporarily and the whole nucleus
presents the appearance of a resting stage. When the bivalents emerge
from this network, their chiasmata are clear and in the larger bivalents they
are quite large in number though they are probably reduced later as in
Ichthyophis and Urceotyphlus. After a brief interkinesis the second division
occurs giving rise to the spermatids.

276

B, R. Seshachar

Chickering, A. M.

Saez, F. A., Rojas, P., and
de Robertis, E.

Seshachar, B. R.

Spengel, J. W.
Tonutti, E.
Wilson, E. B.
Witschi, E.

BIBLIOGRAPHY

. . " Spermatogenesis in Belostomatidae. II. The chromosomes

and cytoplasmic inclusions in the male germ cells of Belo-

stoma flumineum Say, Lethocerus americanus Leidy and

Benacus griseus Say," Journ. Morph., 1927, 44, 541.
" Untersuchungen uber die Geschlectszellen der Amphibien

(Anuren). Der Meiotische prozess bei Bufo arenarum"

Z. Zellforsch, 1936, 24, 727.
. . " The Spermatogenesis of Ichthyophis glutinosus Linn. I.

The Spermatogonia and their divison," Z. Zellforsch, 1936,

24, 662.
. . " Germ cell origin in the adult Caecilian Ichthyophis glutinosus

Linn.," Ibid., 1937, 26, 293.
. . " The Spermatogenesis of Ichthyophis glutinosus Linn. II.

The Meiotic divisions," Ibid., 1937, 27, 133.
. . " The Spermatogenesis of Uroeotyphlus narayani Seshachar,"

La cellule, 1939,48*63.
. . Das Urinogenital system der Amphibien," Arb. Zool. Zootom.

Inst. Wurzburg, 1876, 3.

.. "Beitrag zur kenntnis der Gymnophionen. XV. Das Urino-
genital system," Gegenbaur's Morph.Jb., 1931, 68.

. . The cell in Development and Heredity, Macmillan, New York,
1928.

. . " Die Entwicklung der keimzellen der Rana temporaria L.,"
Z.Zellforsch, 1,523.

EXPLANATION OF FIGURES

1 . Longitudinal section of a testis lobe of Dermophis gregorii. The large locules are separated

by thin septa and contain cell elements in various stages of Spermatogenesis. Groups
of interstitial cells are also seen, x 50.

2. Transverse section of a testis lobe of Siphonops annulatus illustrating the general plan of

structure, x 70.

3. A part of the longitudinal section of a testis lobe of Dermophis gregorii showing a group

of primary Spermatogonia at the mouth of the duct, x 266.

4. Siphonops annulatus. A primary spermatogonium with a spherical nucleus and several

nucleoli. Two nucleoli extruded into the cytoplasm are also seen, x 3100.

5. Dermophis gregorii. A primary spermatogonium with a slightly polymorphic nucleus

x 3100.

6. Siphonops annulatus. A primary spermatogonium with a polymorphic nucleus, x 2266.

7. Siphonops annulatus . A primary spermatogonium preparing for division. X 2266.

8. Siphonops annulatus. A primary spermatogonium in prophase. x 2266.

9. S. annulatus* A primary spermatocyte with nucleus in a stage of rest, x 3100.

10. S. annulatus. A pachytene nucleus, x 3100.

11. S. annulatus. Beginning of the diplotene stage. The loss of polar orientation of the

bivalents is seen, x 3100.

12 S. annulatus ; Early diplotene. Splits have appeared at intervals along the bivalents.
X 3100.

11. K. Seshachar

Proc. IntL Aca<L Set., />', vol. XI', /'/. fX

ORIGIN OF INTRALOCULAR OOCYTES IN MALE

APODA

BY B. R. SESHACHAR

(Department of Zoology, Central College, Bangalore)

Received March 12, 1942
(Communicated by Prof. A. Subba Rau, D.SC., F.R.M.S.)

THREE years ago I reported the occurrence of three oocytes in the testis of
Urceotyphlus narayani (Seshachar, 1939). The ova which were all intra-
locular were fairly well advanced in development and were characterised
by their large size, the germinal vesicle condition of their nuclei and the large
number of nucleoli. That was the first time oocytes were found in the testis
of any Apodan example and it was concluded that they were formed as
transformations of primary spermatogonia. This conclusion is now amply
borne out by the discovery in Ichthyophis glutinosus of an ovicell in the testis
which bears all the characters of a transforming spermatogonium and I am
now able to corroborate Witschi (1934) that intratubular ova in the testis
are derived from a transformation of primitive gonia.

During a recent examination of fresh material of Ichthyophis glutinosus
I noticed in a locule of the testis a cell which bore all the marks of an
oocyte, but which had not advanced in development so much as the oocytes
described by me in Urceotyphlus. This material happened to have been
fixed in Kolatschev's fluid and the sections had been mounted unstained.
The ovicell was large and extended over four sections each of seven
microns thickness. Its appearance in two of them has been figured here.
In one, the nucleus is seen ; in the other, what is obviously the archoplasmic
region is figured.

The oocyte is larger than a normal primary spermatogonium though
much smaller than the ova described in the testis of Urceotyphlus narayani.
It has a definite shape which is maintained by the development of a con-
spicuous envelope, two features which at once led me, along with the large
size, to distinguish the cell from the primary spermatogonium. Moreover,
while the primary spermatogonium is always found either at the mouth of
the duct or at the periphery of the locule, the oocyte projected into the
cavity of the locule, a position which is never ordinarily occupied by the pri-
mary spermatqgonium. The nucleus is slightly polymorphic and has a single
nucleolus. The size of the nucleus is similar to that of the primary spermato-
gonium but the cytoplasm definitely showed evidences of increase in volume.
The Golgi elements which are seen in. the section figured in B, are in the
form of numerous discrete crescentic bodies of various sizes arranged in a
ring around a more or less clear space, the archoplasmic area. In the

278

Origin of Iniralocular Oocytes in Male Apoda

279

10 M-

G. Golgi body;

M. Mitochondria;

N. Nucleus;

primary spermatogonium, the Golgi apparatus is in the form of a compact
body investing the sphere (Seshachar, 1936). This difference between the
normal primary spermatoffonium and the oocyte just described is very
clear and striking. The intensity of osmication of the structures in the two
cells also differs. In the oocyte the Golgi bodies were only slightly black-
ened while in a primary spermatogonium close by, the Golgi apparatus
was deeply black. The mitochondria however, remained scattered in the
cytoplasm.

From the above description of the oocyte it will be clear that it repre-
sents one of the stages through which a primary spermatogonium passes
in its transformation into an oocyte. The small polymorphic nucleus and
the single nucleolus (there are usually many in the fully developed oocyte)
are characters of the primary spermatogonium while the large size and
definite shape of the cell and its thick envelope remind us of the oocyte. It
is evident that the first changes that take place during ' oviform degeneration'
affect the cytoplasm. The increase in its quantity as well as the development
of a definite envelope are only two of these changes. The Golgi apparatus
which occurs as a compact body in the primary spermatogonium has become
broken up here into discrete elements. The mitochondria appear to be the
only structures which are unaffected; they occur scattered in the cytoplasm
as they do in many cases in the primary spermatogonium.

Seshachar, B. R.

Witschi, E.

REFERENCES
. . Z. Zellforsch, 1936, 24, 662.
. . Proc. Ind. Acad. $cL, 1939, 10, 213.
, . Biol Rev., 1934, 9, 460.

ANALYSIS OF RASPURI AND BADAMI VARIETIES
OF MANGO (MANGIFERA INDICA) GROWN IN

MYSORE*

BY C. SRIKANTIA AND N. L. KANTIENGAR

(Department of Physiology and Biochemistry, University Medical College, Mysore)
Received March 12, 1942

Two varieties of the mango fruit, Raspuri and Badami, are largely grown
and eaten in Mysore in the earlier part of the mango season which lasts
for about three months from March to June. The badami variety
is preferred to the raspuri and it is the popular belief that the former
does not bring about any digestive trouble. In order to know the
difference in composition that might exist between the two varieties we
undertook a chemical analysis of the edible portion of the fruit. While
doing so, instead of taking only the fruits sold in the market for ana-
lysis we thought it would be interesting to investigate the variations of several
factors like pH, mineral contents, sugars, etc., during its development.
Ranganathan and co-workers (1937) have determined moisture, mineral
matter and carbohydrates in the green and ripe mangoes grown in Coimbatore
and Salem. Shri Ranjan and Jha (1940) have estimated the mono- and
di-saccharides in the edible portion. The former authors have calculated
the value for carbohydrates by difference and the latter have used Pavy's
solution for the determination of sugars. It is unnecessary to discuss
here the relative merits of the methods used for sugar determinations.
However, we have used Lane and Eynon's copper titration method for
reducing sugars (1923) and Hanes for finding out the ratio of fructose to
glucose (1929).

Experimental

Collection of samples. Four healthy trees, two badami and two ras-
puri, were selected for experiment in the Mysore Municipal Farm. Samples
were collected between 7-30 and 8 A.M. every week for three months till
the fruits were plucked for storage. The fruits were weighed immediately
and the edible part cut, making use of stainless steel knife. The skin and
the stone were discarded. An average sample was made use of for the
analysis. As for the ripe fruit, samples were bought in the local market.

* Read before the Indian Science Congress, January 1941.

280

Analysis of Raspuri and Badami Varieties of Mangd

Moisture. 15-20 gm. were dried at 50-51 C. to a constant weight
in an electric oven thermostatically controlled.

Total mineral matter. 10-15 gm. were heated slowly in a porcelain
crucible till carbonisation took place and then ashed and weighed to con-
stant weight. A qualitative analysis of the ash showed the presence of
carbonate, phosphate, chloride, iron, manganese, sodium and potassium.

pH of the expressed juice. 100 200 gm. were mechanically pressed
in a fruit juice extractor and pH determined electrometrically using a quin-
hydron electrode. The values were checked by the colorimetric method.

Titratable acidity of the extract with alcohol. The extract prepared as
given below under sugar estimation was titrated against 1 N NaOH using
phenolphthalein as the indicator.

Sugars. 100 gm. were extracted with 200 c.c. of neutral 95% alcohol
at laboratory temperature in a percolator for 12 hours; the liquid was
separated from the solid and kept aside. The residue was subjected to hot
extraction with 125 c.c. of 80% alcohol in a Soxhlet apparatus for 16 hours
continuously. The two extracts were mixed, the alcohol from the mixture
was distilled off under reduced pressure (8-10 cm.) at 35 to 38 C. The
residue was made up to 200 c.c. with water. The solution was treated as
given by Archbold and Widdowson (1931), and the reducing sugars estimat-
ed by Lane and Eynon's method (1923) using their sugar table. As the de-
leaded solution was free from colouring matter, it was not boiled with
charcoal. The ratio of fructose to glucose was determined by using alkaline
ferricyanide (Hanes, 1929; Widdowson, 1931). Sucrose was estimated by
hydrolysing with 10% citric acid, neutralising with NaOH and estimating the
total sugars by Lane and Eynon's copper titration method. Starch was not
detected.

The following table gives the results of the analysis.

B2a

282

C. Srikantia and N. L. Kantiengar

for 100 gm.

&.S

^iz

ctf

rt
P

f b

6
Z

"2
'o

vj* ca O

Badami (Municipal Farm)

30-3

28-5

2-20

87-7

474

0-31

2
3
4
5

6-4
13-4
20-4
27-4

14
14
14
14

42-0
69-0
89-7
165-0

3-51
4-95
5-26
5-64

88-8
87-1
88-4
89-6

0-2810
2986
2208

-2137

602
572
566
638

0-88
90

0-44
44

13
15
11
02

Rain

4-5

186-0

4-02

89-7

-2012

566

11-5

207-5

5-24

85-5

2025

654

8
9

18-5
25-5

14
14

250-0
244-3

5-34
3-20

83-5
81-0

1794

2787

602

532

74
66

48
60

22
46

Rain

1-6

241-0

5-48

81-3

-.2693

372

8-6

267-6

5-84

79-8

2874

468

1-13

15*6

226-5

5-60

75-1

-3349

264

0-83

1-53

Market Sample

11-6

286-0

6-7

81-1

3354

2-64

2-22

4-98

19-6

240-0

5-2

78-8

4322

2-14

1-86

3-72

26-6

286-0

6-3

81-4

4582

2-35

1-60

5-17

241-0

6-3

80-7

4366

2-23

1-77

4-01

Average for market
sample

6-1

80-5

4156

. 2-34.

1-86

4-47

Raspuri (Municipal Farm)

30-3

18-2

3-99

87-6

412

.. r

0-19

6-4

50-0

3-27

88-5

2500

528

13-4

85-0

5-16

88-5

2824

592

0-88

0-40

20-4

113-0

5-43

88-1

2324

606

1-00

46 ;

27-4

187-2

5-57

89-0

2298

634

0-72

Rain

4-5

176-0

4-71

89-4

2507

585

11-5

226-5

4-81

87-0

2668

588

18-5

257-0

5-10

85-6

2302

522

1 -34

25-5

282-5

3-08

84-0

2661

512

Rain

1-6

276-5

5-18

82-4

-2458

432

-68

8-6

292-6

5-29

80-1

2907

388

15-6

265-0

5-90

78-7

2846

324

1-22

1-00

1-28

Market Sample

11-6

233-3

7-51

81-7

3071

2-60

2-00

6-76

19-6

364-2

5-87

77-6

3690

2-48

1-47

4-53

26-6

345-0

6-03

81-3

3384

1-65

1-53

5*60

261-0

6-21

84-0

2-31

1-98

4-74

Average for market

sample

6-4

81-1

3382

2-26

1-74

5-41

MB. The blanks in the above table indicate quantities not estimated.

283

284 C. Srikantia and N. L. Kantiengar

Summary

Analysis of the edible portion of the badami and raspuri varieties of the
mango fruit grown in Mysore has been made during the different stage of
its development, with a view to finding out the change in moisture content,
ash, pH, titratable acidity, the reducing sugars and sucrose.

The values obtained are tabulated to show the differences between the
two varieties at different stages. It is particularly of interest to note that
the badami variety contains a greater percentage of minerals and less of
the sugars than the raspuri.

Acknowledgement

We wish to record here our thanks to Mr. S. Seshagiri Rao, Health
Officer, Mysore City, for placing at our disposal the fruits from the trees
grown in the Municipal Farm.

REFERENCES

Archbold and Widdowson . . Biochem. /., 1931, 25, 863.

Hanes * . . ibid., 1929, 23, 99.

Lane and Eynon . . /. Soc. Chem. Ind., 1923, 42, 32 T.

Ranganathan, Sundararajan and 2nd. Jour. Med. Res., 1937, 24, 700.
Swaminathan

Shri Ranjan and Jha . . Proc. Ind. Acad. ScL, 1940, 11 , 266.

ENZYMIC PROTEOLYSIS*

Part V. The Liberation of Cystine
BY M. DAMODARAN AND T. K. KRISHNASWAMY

(From the University of Biochemical Laboratory, Chepauk, Madras)
Received January 29, 1942

INVESTIGATIONS on the time course of the liberation of cystine and other
ammo-acids from proteins during enzymic digestion were carried out by
Abderhalden and Reinbold (1905). In these experiments pancreatin was
allowed to act upon edestin, portions of the digest were removed at definite
intervals and dialysed. The non-dialysable residue gave negative reactions
for tyrosine after 24 hours, for tryptophane after 4 days and for cystine
(" Schwefelblei probe ") after 8 days, indicating that these three amino-acids
were present entirely in the free condition at the end of the respective periods
noted. Recently the rate of liberation of cystine has been studied more
quantitatively with the aid of two widely used methods, the Folin and Marenzi
method, based upon the reduction of Folin's uric acid reagent by thiol
compounds, and the Sullivan reaction using ^-naphthaquinone sulphonate.
Employing these colorimetric procedures Jones and Gersdorff (1933) found
that during peptic digestion of casein the Sullivan reaction remained
negative throughout while the Folin and Marenzi reagent gave high colour
values which rose to a peak in the early stages of digestion and then fell to
a constant value. In parallel experiments in which hydrolysis was effected
by means of 20% HC1 the Folin and Marenzi method showed similar
abnormal values which were, in the early stages of hydrolysis, much higher
than the total cystine content of casein. Jones and Gersdorff came to the
conclusion that pepsin does not split free cystine from casein and that the
high Folin and Marenzi^ colour values obtained with peptic digests are
produced by some compound or compounds other than cystine. In their
opinion two factors, a stable and an unstable type of chromogenic compound,
were involved; the possibility was also suggested that substances such as
furfural, pyruvic acid, levulinic acid etc., might be responsible for colour
formation. With egg albumin using the same reagent for the estimation

* Papers I IV in this series appeared in the Biochemical Journal, 1877, 32; 1919, 32 ; 2105,
32 ; 122, 35.

285

286

M. Damodaran and T. K. Krishnaswamy

of cystine Calvery et al (1936) obtained high chromogenic values but in
contrast to Jones and Gersdorff appear to be of opinion that these values
represent in fact cystine set free by pepsin.

In tryptic digestion there is evidence to show that cystine is rapidly -
liberated. Pollard and Chibnall (1934) using Prunty's (1933) modification
of the Sullivan reaction found that about 72 and 52% respectively of the
cystine in rye grass and cocksfoot proteins were present in the free condition
after 72 hours' digestion with trypsin. These experiments also gave indi-
cation of the decomposition of cystine during prolonged digestion. In
experiments on the tryptic digestion of casein carried out by Jones and
Gersdorif (1939) 80% of the cystine was liberated in 24 hours, but a large
part of this was destroyed on account of the alkalinity of the medium
(pH 8-9). At pH 6*6-6-8 about 40% of the cystine as determined by the
Sullivan method was in the free condition in 24 hours and almost complete
liberation took place in 120 hours. In these experiments also the Folin
and Marenzi method gave " high and erratic values ".

In the experiments now described a study has been made of the liber-
ation of cystine from eight proteins during the^action of pepsin dnd trypsin
in succession. As in the experiments of Jones and Gersdorff (1933) none
of the eight proteins studied gave a positive Sullivan reaction during peptic
digestion, but with the uric acid reagent all gave high colour values. The
value in the case of casein (Fig. I) reached a maximum at 161% of the total
cystine of casein in the first hour of digestion and then fell gradually to a
constant value of about 104%. Egg albumin (Fig. II) showed a similar peak

200;

160
120-

40-

Pepsin on Casein

Cystine O Folin and Martnzl
__. ^ Casein

Time (hours)

FIG. I

Peptic' Dig^ Cystine^. Folin and Marenzi

Anacaret

Gliadin

Egg Albumin

Time (/tours)

120

FIG. II

value of 86-5% at the end of 12 hours. The remaining six proteins (Figs.
II and III) showed no such maxima, the Folin and Marenzi colour values
increasing gradually in the course of digestion and finally remaining at a
constant high level. In the case of anacardein this constant value was higher

Enzymic Pro feo lysis V

287

again than the cystine content of the protein. In every case \\ was found that
colour production with the Folin and Marenzi reagent could be completely
inhibited by H.CHO and HgCl 2 ; as inhibition by these reagents has been
shown by Shinohara (1936) and Lugg (1932) to be specific for thiol groups

150!
120
90

TVypsirt '"' (tliadin i 0- Fohn and Maren:

Cysfiru? {-&-" Sullivan

* (/tours)

FlG, Ill

24 W 72 % 120 144 1GS 192 21G 240

FIG. IV

it can be concluded that the chromogenic substances formed during peptic
digestion are cystine complexes, most probably large polypeptides, capable
of producing a higher intensity of colour than cystine itself. In the case of
casein and egg albumin which show pronounced maxima in the hydrolysis
curves it has to be assumed that complexes formed in the earlier stages
of digestion are later split up into substances with less colorigenic power.
In tryptic digestion there were similar discrepancies between the values
obtained by the two methods. Folin and Marenzi method gave with all
the proteins, with the exception of edestin, values higher than the total
cystine content, gliadin (Fig. IV), watermelon globulin (Fig. V) and ana-
cardein (Fig. VI) showing maxima in the early phases of digestion similar

' tn anci
in* -Ar Sullivan

Mtroprusside

FIG. V

psin on Anacardem

Fo/t'n ancf
Cy^'/m^ ( ](2t~ Sullivan

Nitroorussidt
AminoN ~

r~24 48 72 06 120 144 168 192 216 240

288 M. Damodaran and T. K. Krishnaswamy

to those shown by casein and egg albumin in peptic digestion. As colour
production in tliese digests also could be completely inhibited by H.CHO
and HgClo it is to be inferred that the chromogenic substances are cystine
peptides.

Sullivan values also showed maxima in two cases, viz., casein and fibrin.
However, while the shape of the curves with Folin and Marenzi values is to
be explained on the assumption of the formation and subsequent break-
down of highly chromogenic polypeptides the rise and fall of the Sullivan
values observed with these two proteins is to be ascribed to destruction of
free cystine. In view of the evidence for the destruction of cystine produced*
by Jones and Gersdorff (1939) at the end of the digestion period aliquots of
the digests were hydrolysed completely with acid and cystine determinations
carried out by the Sullivan method. With most proteins it was found that
there was considerable destruction, the value obtained in the digests being
much lower than on an equivalent amount of the intact protein. The
extent of destruction varied from protein to protein being highest with casein,
amounting to almost half; egg albumin and watermelon globulin showed no
destruction while the other proteins occupied intermediate positions. In
addition to the Folin and Marenzi and Sullivan methods, the nitroprusside
reaction (Krishnaswamy, 1942) was also made use of in studying the progress
or tryptic digestion. The values for disulphide groups thus obtained were
usually intermediate between the values obtained by the other two methods
but it does not appear that any special meaning can be attached to
these data.

From a comparison of the rates of pep tide splitting (increase in amino
nitrogen) and cystine liberation (as determ : ned by the Sullivan method)
it will be obvious that in some digests cystine splitting takes place much
more rapidly than peptide hydrolysis so that there is justification for
the view that this amino-acid occupies an " exposed position " in these
proteins. The data regarding the extent of peptide hydrolysis at the stage
when cystine liberation had reached the maximum are summarised in Table I.
Making allowance for the amount of cystine destroyed during digestion it
will be seen that practically all the cystine in anacardein, watermelon
globulin and edestin are set free in the early stages of digestion (48-72 hours)
when peptide hydrolysis has proceeded to only about 50%. From gliadin
95% of the cystine is set free in 96 hours when the extent of protein hydro-
lysis is 58%. With the animal proteins, fibrin, egg albumin and serum
globulin, however, there is no such marked discrepancy between the rates
of cystine and amino nitrogen liberation, cystine present in the free condi-
tion ranging from 74-80% when peptide hydrolysis is approximately 70%.

Ensymic Pro tea lysis V 289

The significance of these observations with reference to the position of
cystine in the protein molecule requires further elucidation.

TABLE I
Liberation of Cystine and Amino-'N

Hdestin
Gliadin
Anacurdein
Watermelon

Casein

Fibrin

JHgg albumin t

Serum globulin

tein

Time
hr.

Peptide

Hydrolysis
o/
/o

Free cystine %
(corrected for
destruction)

59-3

97-2

58-7

95-4

56-9

100-9

glot

ulin

52-2

99-1

49-5

90-2

68-6

78-8

120

65-2

74.4

lin

168

71-3

80-7

Experimental

Materials. C&stin, edestin, gliadin and fibrin were prepared accord-
ing to standard methods. The serum globulin used was the euglobulin
described in Plimmer (1933). Anacardein was prepared according to
Damodaran and Sivaswamy (1936), and the watermelon globulin by the
method of Krishnan and Krishnaswamy (1939). B. D. H. preparations of
egg albumin, pepsin and trypsin were used.

Enzymtc Digestion, The following procedure based upon that employed
in previous work from this laboratory (cf. Damodaran and Ananthanaraya-
nan, 1938) was uniformly employed with all proteins studied. , To 20 g.
protein dissolved in about 950 ml. of N/20 HC1 was added, after
adjustment of pH to 1 -8, 1 g. pepsin in 50ml. of N/20 HC1 and the solution
made up to 1 L At the end of 7 days 450 ml. of the digest was brought
to pH 8-3 with NaOH, 0-5g. trypsin dissolved in a small amount of water
added and the solution made up to 500 ml.

For control Ig. pepsin was dissolved in 950 ml. of N/20 HC1, the pH
adjusted to 1*8 and the solution made up to 1 1. After 7 days 450 ml. were
brought to pH 8-3 and made up to 500 ml. after the addition of 0-5 g.
trypsin dissolved in water. The values for amino-N and cystine in the digests
were in all cases corrected for the values in the control experiments.

Methods of Analysis. Amino-N was determined by formol titration
according to Sorensen (Abderhalden, 1923). 5 ml. aliquots of digests were
withdrawn at intervals, neutralized to pH 6-8 and the resulting solution

290

M. Damodaran and T. K. Krishnaswamy

after treatment with 10 ml. of neutralised formalin titrated with standard
N/10 NaOH. ,

Cystine was determined by the Folin and Marenzi (1929) method as
modified by Tompsett (1931), the Sullivan method (Sullivan and Hess,
1937) and by means of nitroprusside (Krishnaswamy, 1942). Aliquots
measuring 2 to 5 ml. were removed according to the concentration of cystine
present. Further details were exactly as described in the original papers.
In casein digests with low cystine content determinations had to be made
with added standard. In the accompanying tables, A, B and C represent
Folin and Marenzi, Sullivan and Nitroprusside values respectively.

For calculation of the extent of peptide hydrolysis amino nitrogen
determined according to Sorensen was compared to the amino nitrogen on
complete hydrolysis, this value being calculated from N-distribution values
given in Plimmer (1917). For calculation of % cystine splitting total cystine
in each protein was determined experimentally after acid hydrolysis.
For ascertaining the extent of the destruction of cystine during digestion the
digests were completely hydrolysed with acid and cystine determinations
made in these hydrolysates.

Total Cystine in the Proteins. 0-5. to 3 g. of protein (to contain about
10 mg. cystine) was hydrolysed with 20 parts of 20% HC1 for 8 hours, the
hydrolysate repeatedly distilled in vacuo to expel the acid, decolorised with
5 g. kaolin and made up to 50 ml. 5ml. aliquots were used for the deter-
mination of cystine by the three colorimetric methods already mentioned.
From the values given in Table II it will be seen that with completely hydro-
lysed proteins, with the exception of casein, the three methods give fairly
concordant values.

TABLE II

Cystine Content of Proteins

Cystine %

Protein

Casein

0-42

0-28

0-29

Edestin

1-44

1-41

1-48

Gliadin

2-52

2-39

2-61

Anacardeian

1-45

1-38

Watermelon globulin

1-19

Egg albumin

2-69

2-15

2-69

Serum globulin

2-41

2-05

2-53 ,

Fibrin

2-02

1-84

2-28

Enzymic. Proteo lysis V

291

j *

fi*fV' ***"

,VB/flMtf

" .' ' H T ' W V/"% ' ii"r < i"ii w ltrt jijjf

120 144 168 192 21O40

FIG. VIII

,s>a aio UMO

FKI. IK

24 48 72 9G 120 144 168 192 216 240

FiC; X

ISO

"ft, Trypsin on Hbrln

Amino-N HE}-

Folin and Marenzl

Sullivan

Mtroprusside

344 168 192 216 240

FlQ. XI

D&truction of Cystine during Tryptic Digestion. ^10 ml. of the digest
In each case was treated with 17 ml. of cone. HC1 and refluxed for 8 hours.
The acid was removed by repeated distillation in vacua, the solution de-
colorised with kaolin and made up to 25 ml 5 ml. aliquots were taken for

292

M. Damodaran and T. K. Krishnaswamy

Sullivan determination, standard cystine being added in the case of aliquots
with low cystine content. The results are given in Table III.

TABLE III
Destruction of Cystine

Protein

Cystine %
in
Protein

Cystine %
in Protein
after diges-
tion

% Destruction

Edestin

1-41

0-97

31 <3

Gliadin

2-39

1-96

18-2

Anacardein

1-38

.1-02

26-7

Watermelon globulin

1-19

Casein

0-28

0-15

45-8

Fibrin

1-84

1*73

5-9

Egg albumin

2-15

Serum globulin

2-05

1-89

7-9

PROTOCOLS

TABLE IV
Pepsin and Trypsin on casein

Hydrolysis %

Time

hr.

Pepsin

Trypsin

Peptide

Cystine
A

Peptide

Cystine

0-5

135-7

0-75

142-9

1-0

5 : 9

161-1

1-5

127-5

6-5

114-3

47 -Y

7-2

'" 105-4

35-6

60-7

7-9

104-3

8-5

103-9

38 : 6

60-4

9 : 8

49-5

90-2

11-9

57-4

114

13-9

60-9

123-2

58-2

15-6

63-8

133-9

43-6

120

16-9

66-8

146-1

32-9

144

17-3

69-0

30-9

168

17*3

103*- 9

70-9

192

72-7

216

74-4

240

75-6

264

76-0

146* 1

3o'-9

288

76-0

146-1

30-9

Rnzymic Proteolysis V

293

TABLE V
Pepsin and Trypsin on Edestin

Hydrolysis %

Time

Pepsin

Trypsin

hr.

Cystine

Pcptidc ! c ^ mc

Peptidc

1 :

2-1

39-2

78-0

5-6

44-5

81-6

7-2

45-8

34 "-0

86-5

K-4

42 2

6 :

37-3

42 : 4

ii>7

51-4

46-8

I .V 9

56-3

59-0

14'H

59-3

66-2

15-5

61-4

J20

16-0

62-9

144

16-2

64:1

16H

J6-2

42-0

65-1

1*)2

66-1

216

66-8

240

67-5

264

67-5

90-0

66-2

TABLE VI
Pepsin and Trypsin on GHadin

Hydrolysis %

psin

Trypsin

Cystine
A

Peptide

Cystine

35-6

105-9

42-9

107-9

51-9

33*- 9

110-9

33*-5

56-9

. .

60-3

* .

39-0

114 : 2

120-0

4i*

50 : 2

73 : 2

48 : 9

112-9

60-7

78-3

82*4

55-1

105-0

67-3

84-1

90-4

58-7

77-4

88-3

58-7

90-4

62-7

90'-4

65-4

67-3

68-0

68-7

68^7

105'-

77-4

90-4

294

M. Damodaran and T. K. Krlshnaswamy

TABLE VII
Pepsin and Trypsin on Anacardein

Hydrolysis %

Time

Pepsin

Trypsin

hr.

Peptide

Cystine

Peptide

Cystine

0-5

63-5

4-5

70-6

112-7

6-4

73-7

130

7-4

77-3

35-6

42-6

8*3

79-4

9-0

85-8

, t

10-9

4i : 2

133 : 3

47 : 6

57 : 2

12-9

105*

50-6

137-6

65-4

75-9

13-7

112-9

54-5

144-7

74-4

83-0

14-5

56-9

150

82-5

84-4

1.5-0

60-2

117-2

85-1

120

15-5

62-0

107-1

144

9 ^

63-1

97-9

168

15-5

112-9

64-1

192

65-4

216

66-3

)

240

67:0

264

)

288

67-0

107-1

82-5

85-1

TABLE VIII
Pepsin and Trypsin on Watermelon Globulin

Hydrolysis %

Time
hr.

Pepsin

Trypsin

Peptide

Cystine
A

Peptide

Cystine

0-5

43-0

0-75

45-0

1-9

46-1

ll6'-9

1-5

50-2

3 : 3

51-3

117-6

5-2

52-9

35 ; 2

122-7

36 : 6

7-2

8-7

39-*5

126*- 7

45-4

69 : 5

11*5

47-6

131-1

81-2

84-0

12-6

52-2

121-9

99-1

85-7

13-5

59-4

108-4

87-4

14-2

63-5

93*0

120

15-0

65-8

144

15-4

67-3

168

15-4

52-9

68-3

'<>*.

192

69-0

216

69-5

240

69-8

264

288

69-8

108-4

99*1

93-0

Enzymic Proteotysi

s -

295

TABLE IX

Pepsin and Trypsin on Egg Albumin

Hydrolysis %

Time

Pepsin

Trypsin

hr.

Peptide

Cystine
A

Peptide

Cystine

B | C

0-5

41-4

i : 3

42-6

1-5

54-0

2 : 7

61-4

93 :

38 : 5'

3 : 8

70-0

39 ' 5

94-2

4-8

75-6

5-7

77-2

7-2

78-4

43 : 7

107-2

28 : 7

46 : 5

8-1

84-7

10-2

86-5

11*5

75-6

51-6

115-3

45*-7

65*-2

13<5

74-6

55-7

119-1

52-3

70-0

15-2

73-5

59-0

61-6

74-4

16-7

62-0

70-2

80-0

120

18-7

65-2

74-4

, 144

19-4

68-1

168

19-4

73'- 5

71-2

192

73-3

216

75-3

240

76-3

264
288

76-8
76-8

119-1

74* 4

TABLE X
Pepsin and Trypsin on Serum globulin

Hydrolysis %

Pepsin

Trypsin -

hr.

Cystine

Peptide

Cystine

Peptide

0-75
1

22-1
40-2

103 * 9

1-25

,2
3

3 : 4
6-8 .

45-9
50-3
69-2

34 : 4

120 '-5
145-3

35 : 9

62 : 4

8-2

72-2

* *

37-5

6
24
48
72
96
120
144
168

li'-i

13-2
14-9
15-4
15-8
16-0
16-0

72-2

47-8
54-1
59-1
64-3
68-2
70-2
71-3

77 -^

52-7
54-6
62-2
64-1
66-5
68-4 .
72-7

74-1
81-5
91-2
91-2
88-3
79 -0

192

71.5

216
240

74-6

75-3

145-3

72-7

79'

288

296

M. Damodaran and T. K. Krishnaswamy

TABLE XI
Pepsin and Trypsin on Fibrin

Hydrolysis %

Time

Pepsin

Trypsin

hr.

Peptide

Cystine
A

Peptide

Cystine

0-5

34-2

3-5

39-2

83 : 2

7-5

41-9

94-6

64 : 1

8-2

44-1

40 : 6

100-2

9-3

105-4

10-4

110-3

46 : 1

39 :

64 : 7

13 : 5

53-4

53-8

65-3

15-5

60-5

,i ,

62-0

65-9

17-3

65-5

63-1

60-3

18-5

68-6

72-8

120

19-2

70-2

65-8

144

19-9

71-7

60-3

168

20-3

44-1

73-3

)

192

74-5

)

216

75-3

240

75-8

288

75-8

110-3

60-3

66'-3

Summary

A study has been made of the rate of liberation of cystine and ammo
nitrogen from eight proteins during successive digestion by pepsin and
trypsin.

Assuming the specificity of the Sullivan reaction it can be concluded
that no free cystine is present in peptic digests.

The same reaction shows that cystine is rapidly set free during the
action of trypsin. From the vegetable proteins (edestin, gliadin, anacardein
and watermelon globulin) cystine is liberated much more rapidly than other
amino-acids, practically the whole of the cystine in the protein being present
in the free condition at a very early stage of proteolysis. Such complete
liberation does not take place with the animal proteins (serum globulin,
egg albumin and fibrin) ; with these, rate of cystine splitting approximates
more closely to peptide hydrolysis. In the course of tryptic digestion at
alkaline pH destruction of cystine takes place, the extent of . the destruction
Varying from protein to protein.

The Folin and Marenzi reagent gives high colour values with both
peptic and tryptic digests. These values have no relation to the free cystine

Enzymic Proteolysis V 297

present, being frequently in great excess of the total cystine content of
the protein. This colour production is due to cystine complexes and not
to non-nitrogenous substances as has been previously suggested.

BIBLIOGRAPHY

AbdcrhaMcn . . Handbuch der Biologischen Arbeitsmethoden,

Eiweissabbauprodukte (Berlin), 1923, 245.
and Rcinbold . . HoppeSeyl. Z., 1905, 46, 166.

Calvcry, Block and Schock . . Jour. Blol. Chem., 1936, 113, 21.

Damodaran and Ananta- Biochem. /., 1938, 32, 1877.

Narayanan

- and Sivaswamy . . Ibid., 1936, 30, 604.

I'olw and Maren/i . . Jour. Blol. Chem., 1929, 83, 103.

Jones and ticrsdorir . . Ibid., 1933, 101, 657.

.. Ibid., 1939,129,207.

Kmhnan and Krishnaswamy . . Biochem. /., 1939, 33, 1284.

Kmhtmwamy * Proc. Ind. Acad. Sci., 1942, 15, 135.

Lygg .. Biochem. J., 1932,26,2160.

Flimmef . . The Chemical Constitution of Proteins, Part I

(London), 1917, 132.

, . - - .. Organic and Biochemistry (London), 1933, 490.

Pollard and Chtbnall . . Biochem. /., 1934, 28, 326.

Prunty . . Ibid., 1933, 27, 387.

Hhmohara * . Jour. Blol Chem., 1936, 112, 697.

Siillivan and lies* . . Ibid., 1937, 117, 423.

Tomptwtt Biochem. /., 1931, 25, 2014.

358-42 Printed at The Bangalore Press, Bangalore City, by G. Srinivasa Rao, Superintendent,
and Published by The Indian Aeademy of Sciences, Bangalore.

INDEX TO VOL. XV (B)

AUTHORS' INDEX

Abraham, A. . . See Kumar and Abraham.

Ayer, A. Ananthanarayana The external morphology of the brain of semnopithecus

entellm (a comparative study), 43.
Ayyangar, G. N, Ranga- The grain sorghums of the durra group, 133.

swami, Ayyar, M. A.
Sankara, Narayanan, '
T. R. t and Nambiar, A.
Kunhi Koran
Ayyar, M. A, Sankara

Barter)!, M. L.

Bhalcrao, G, IX

Chttudhuri, H,
Chaudhuri t H,, and
Bancrji, M. L.

Chiplonker, G. W.

Damodaran, M., and

Krishnaswamy, T. K.

Hamid, Abdul
lyengar, N. K.

Janjua, Nazeer Ahmed

Kantiengar, N. L.
Khan, Abdul Wahid
Krishnaswamy, T. K.
Kumar, L, S, S., and
Abraham, A.

Lai, M. B.

See Ayyangar and others.

See Chaudhuri and Banerji.

The genus cephalogonimusm India and Burma, 178.

Indian water moulds V, 225.
Indian water moulds IV, 216.

Age and affinities of the bagh fauna, 148.
Enzymic proteolysis, Part V, 285.

Indian water moulds III, 206.

Trypsin-kinase in blood, 106.
Anti-tryptic components of blood, 112.
Prothrombin and plasma trypsin, 123.

On the biology of red spider mite (tetranychus telarius
Linn.) in Baluchistan, 256.

See Srikantiah and Kantiengar.

See Rahman and Khan.

See Damodaran and Krishnaswamy.

Cytological studies in Indian parasitic plants, II, 253.

The egg-capsule of the millipede, thyroglutus malayus
Attems (syn. thyropygus malayus Carl.), 58.

299

300

Mahendra, B. C.

Maheshwari, P.

Maheshwari, P., and Singh,

Balwant
Majumdar, Girija P.

Nambiar, A. Kunhi Koran
Narayanan, T. R.

Paul, M. D.

Raghavan, T. S., and

Srinivasan, A. R.
Raghavan, T. S., and

Srinivasan, V. K.
Rahman, Khan A., and

Khan, Abdul Wahid

Raraakrishnan, T. S.

Raman, C. V.
Seshachar, B. R.

Singh, Balwant
Srikantiah, C., and
Kantiengar, N. L.
Srinivasan, A. R.
Srinivasan, V. K.
Swamy, B. G. L.

Thirumalachar, M.

Contributions to the bionomics, anatomy, reproduc-
tion and development of the Indian house-gecko,
hemidactylus flaviviridis Riippel, Part III, 231.

The embryo-sac of euphorbia heterophylla L. A re-
investigation, 158.

On the internal bundles in the stem of rumex patientia
L., 153.

The origin of siphonostele in three species of selagi-
nella spr., 172.

See Ayyangar and others.
See Ayyangar and others.

Studies on the growth and breeding of certain seden-
tary organisms in the Madras harbour, 1.

Cytological studies in datura, I, 61.

A contribution to the life-history of vahlia viscosa,
Roxb., and vahlia oldenlandioides Roxb. 83.

Bionomics and control of Aeolesthes holosericea F.
(Cerambycidse : Coleoptera), 181.

A study of the life-history and control of batocera
horsfieldi Hope (Lamiidse : coleoptera) a borer
pest of walnut tree in the Punjab, 202.

A leaf spot disease of zingiber officinale by phyllo-
sticta zingiberi n.sp., 167.

New concepts of the solid state, 75.

Stages in the spermatogenesis of siphonops annulatus
Mikan. and dermophis gregorii Blgr. (amphibia :
apoda), 263.

Origin of intralocular oocytes in male apoda, 278.

See Maheshwari and Singh.

Analysis of raspuri and badami varieties of mango

(mangifera indica) grown in Mysore, 280.
See Raghavan and Srinivasan.
See Raghavan and Srinivasan.

Female gametophyte and embryogeny in cymbidium
bicolor Lindl., 194.

Phragmotelium mysoremii,\ new rust on Indian
raspberry, 186.

TITLE INDEX

Aeolesthes holosericea F. (Cerambycidae : Coleoptera), bionomics and control
(Rahman and Khan), 181.

Bagh fauna, age and affinities (Chiplonker), 148.

Batocera horsfieldi Hope (Lamiidae: Coleoptera), a study of the life-history and

control (Rahman and Khan), 202.
Blood, anti-tryptic components (lyengar), 112.
Blood, trypsin-kinase (lyengar), 106.

Cephalogonimus, the genus, in India and Burma (Bhalerao), 178.

Cymbidium bicolor Lindl., female gametophyte and embryogeny (Swamy), 194.

Datura, cytogenetical studies, I (Raghavan and Srinivasan), 61.
Durra group, the grain sorghums (Ayyangar and others), 133.

Enzymic proteolysis, V (Damodaran and Krishnaswamy), 285.

Euphorbia heterophylla L., the embryo-sac a reinvestigation (Maheshwari), 158.

Hemidactylus flaviviridis Riippel, Indian house-gecko, contributions to the binomics,
anatomy, reproduction and development, III (Mahendra), 231.

Mango (rnangifera indicd) grown in Mysore, raspuri and badami varieties, analysis
(Srikantia and Kantiengar), 280.

Oocytes, intralocular, origin, in male apoda (Seshachar), 278.
Organisms, certain sedentary, in the Madras harbour, growth and breeding,
studies (Paul), L

Parasitic plants, Indian, cytological studies, II (Kumar and Abraham), 253.
Phragmotelium mysorensis, a new rust on Indian raspberry (Thirumalachar), 186.
Phyllosticta zingiberi n.sp., a leaf spot disease of zingiber officinale caused by

(Ramakrishnan), 167.
Prothrombin and plasma trypsin (lyengar), 123.

Rumex patientia L., stem of, on the internal bundles (Maheshwari and Singh), 153.

Selaginella spr., three species of, the origin of siphonostele in (Majumdar), 172.
Semnopithecus entellus, brain, the external morphology (a comparative study)
(Ayer), 43.

301

302

Siphonops annulatus Mikan. and dermophis gregorii Blgr. (amphibia : apoda),

stages in the spermatogenesis (Seshachar), 263.
Solid state, new concepts (Raman), 75.

(Tetranychus telarius Linn.), red spider mite, in Baluchistan, on the biology

(Janjua), 256.
Thyroglutus malayus Attems (syn. thyropygus malayus Carl.) millipede, the

egg-capsule (Lai), 58.

Vahlia viscosa Roxb. and vahlia oldenlandioides Roxb., a contribution to the life-
history (Raghavan and Srinivasan), 83.

Water moulds, Indian, III (Abdul Hamid), 206 ; IV (Chaudhuri and Banerjee),
216 ; V (Chaudhuri), 225.

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