222
C. A. WRIGHT
:
[J.L.s.z.
THE APPLICATION OF PAPER CHROMATOGRAPHY TO A
TAXONOMIC STUDY IN THE MOLLUSCAN GENUS LYMNAEA
By C . A. WRIGHT
(Communicated by the Zoological Secretary)
[Read 29 January, 19591
THEincreasing use which is being made of paper chromatography in taxonomic and
genetic studies is a tribute t o thc simplicity and utility of the method.
It is intcnded to apply this techniquc to some of the problems concerning t h e
molluscan intermediate hosts of Schistosomiasis in Africa but in order t o get a greater
understanding of the method and the interpretation of the results obtained a preliminary study has been carried out in the gastropod genus Lymnaen. The main
reasons for this choice are the rehtively ready avdability of material, the clear
p:itterns given by many Lymnnen species and the fact that thc specific identities of a t
least the British members of the genus are quitc easily established by conchological
and anatomical examin:t t'ion.
Thrrc exists already a good deal of literature on the use of chromthtography in
taxonomic work and it is hoped that a review of these papers may prove uscful to
othcr workers.
HISTORICAL
I n 1944 Consden, Gordon & Martin described a partition chromatography method
using paper and there is no doubt that thc enormous popularity of the technique
dates from that time. Howevcr, t ~ searly as 1939 Brown was achicving microseparations by adsorption on blotting paper and even before that time a form of
chromatography on blotting paper was used in thc oil industry t o obtain a rough
idea of the constitucnts in oil mixtures. Once the usefulness of the techiliquc W:LS
fully reslizcd pLper chromatography found almost universal applications in analytical
work and numbers of variations of the basic techniquc were introduced. The
variations were in direction of flow, solvent mixture and grade of filter-paper. Further
methods for obscrving the substances separated were also developed. There are
now several text-books which cover all of these aspects in great detail and there is
no need to discuss them further here. Table I summarizes the techniques which
h:iw heen applied in taxonomic studies.
These studies can be divided roughly into two main sections depcnding on the
approach used. Some investigations have bren based on specific differences in tho
frec amino-wids in the blood of nnimals. This requires some pre-treatment of the
tissues used, while tht, other method depends on the usc of untreated tissue arid the
o b s e r v h m of the unknown substances separated, either by their fluorescence under
ultra-violet light or by their staining reaction with triketo-hydrindene-hydrate
(tiirihydrin).
Thc pioneers in the study of specific variation in free amino-acids were Micks &
Ellis (1981). These authors were investigating the amino-acid content of various
species of mosquitoes in the hope that they would be able to correlate their results
with the susceptibility of the insects to infection with malaria parasites. I n vitro
culture httd already shown that there is i i variation in the amino-acid requircments
of difFerent species of Plasmodium. The samc authors (Miclcs & Ellis, 1952) later
showed that there are qualitative variations in the free amino-acid content of the
developmental stages of mosquitoes and that the total level was lowest in the pupal
APPLICATION OF PAPER CHROMATOQRAPHYTO A TAXONOMIC STUDY
XLIV]
223
TABLEI
1
Author
1
Whatman'
Technique 1
Solvent
l x k T & Ellis, 1951
1
---
--
(1) Water saturated phenol
1
Hadorn R: Mitchell, 1951
1
Ascending
Micks & Ellis, 1952
Free amino-acids.
1
Bozzati-Traverso &
Rechnitzer, 1953
Fluorescent and ninhydrinpositive.
I
Buszati-Traverso, 1953 a
Ascending
_I -
~
~
Micks, 19.54
Kirk, Main & Beyer, 1954
-!
1
Horizontal
Daimevig, 1956
Fox, 1956
l-
March & Lewallen, 1956
!
I
Wright, Harris &
Claugher, 1957
.__
Robertson, 1957
Chen & Baltzer, 195'3
,
(u)
1
Ascending
Horizontal
-p I -
n-r~r(ii)anol/I?~
ammonia (2 : 1)
i -~-
I
3
1
Fluorescent.
16: 4
j
Ninhydrin-positive.
i
I --
,,
,
1
n-propanol/l?; ammonia (2 : 1)
(0) ?~-propat~ol/diethyIamine/water
(85 : 4 : 15)
( c ) ti-bntanol/glacial acetic acid/water
( 4 : 1 : 1)
Fluorescent.
1
1
Ninhydrin-positive.
I
ti-propanol/l~&
ammonia (2 : 1)
1
n-hutanoliglacial acetic acid/water
(100 : 22 : 50)
3
1
Fluorescent.
I
(85 : 4 : 15)
I-Lbzrzl
'
I
Fluorescent and ninhydrinpositive.
(u)
(4 : 1 : 5 )
i
L)esce;ding
T-
I
1
1 ?i-butanol/glacialacetic acid/water
1
Amino-acids.
I
,,
~ T b z c i a acetic
I
acidiwater
(4 : 1 : 5)
f~orizontal I ?z-propanol/l% ammonia (4 : 1)
______
i
1
-____
1
I
1
I winhydrin-positive
~
/glacial acetic acid /nater
( 4 : 1 : 5)
(1) n-bntaiiol/glacial acetic acidiwater
(4 : 1 : 8)
____(2) AOY; ~)iienol
i
1
tr-bntanol/glacial acetic acidiwater
(4 : 1 : 5 )
Phenoliwater (4 : 1)
n - p r o p a n o ~ / ~ammonia
~;
( 2 : 1)
I----__
1 dimensional 1
I
-~
_
-~
(1')
Ascending
,,
1
_
n-butanol/glacial acetlc acidiwater
(100.22 : 50)
i
j
I
Lewallen, 1957
_
2
1(1)>OU/,&ueous' phenol
dimensional
(2) a-butanol/glaeial acetic acid/wat,er
(4: 1 : I )
~
Viswanathan and Krishna
Pillai. 1956
~
n-proyariol~l"/, ammonia ( 2 : 1)
(1) Water saturated phenol
(2) n-)~ntanol/glacial acetic acid/water
(4 : 1 : 1)
i
!
1956
-
Fluorescent and ninhydrinpositive.
I
( a ) n-propa~iol/l%~
ammonia (2 : 1)
( b ) n-butanol/glacial acetic acidiwater
(4 : 1 : A)
({I)
i_____
___
Micks, 1956
%
aytr',
1
n-propanol/l";, ammonia ( 2 : 1)
( a ) n-propanol/lY: ammon!a (2 : 1)
(6) n-but.anol/glacial acetic acidiwater
(4 : 1 : 5 )
Buzxati-Traverso, 1953 b
-
Amino-acids.
b) n-butanol/glacial acetic acidiwater
I
I
_
Fluorescent and ninhydrinpositive.
( I ) Water saturated phenol
(2) 2 , 4 lutidine/water (3 : 1)
1
Ball & Clark, 1953
I
-
l 1
____
1
2
dimensional
Free amino-acids.
n-propanol/l% ammonia ( 2 : 1)
I
-__
Substances
separated
(1) 70% n-propanol
--__--____
(2) Saturated aqueoiia phenol
1
~
Ninhydrin-positive.
Fluorescent,
324
C. A. WRIGHT:
[J.L.S.Z.
stagcs. Ball & Clark (1963) found qualitative differences in the amino-acid patterns
of sevcr~lspecies of Culex using extracts of starved adult insects but Micks (1954),
working on the Culex pipiens complex found that all of the species which he examined
had the same qualittitive pattern but that there were Bpecific quantitative differences.
An interesting point in this paper is the marked quantitative difference shown Iletween
hibernating C. pipiens from Europe compared with recently emerged, unfed specimens
from America. Micks (1966) found qualitativc differences in the free amino-acid
composition of representatives of different orders of insects but only quantitative
variations within gencra. Micks & Gibson (1957) described the use of a photovolt
transmission densitometer for measuring maximum density curves of one dimensional
chromatograms of ninhydrin-positive material a8ndthus give a numerical value to the
specific quantitative differences observed.
In the second category of experiments, those based on the separation of unknown
materials, the work of Hadorn & Mitchell (1951) on Drosophila melanogaster is the
earliest. These authors studicd the ninhydrin-positive and fluorescent patterns
given by different growth stages and mutants of Drosophila. They found marked
age a.nd sex differences, also differences between mutants. They identified several
of the ninhydrin-positive substances and found that some of the fluorescent materials
were related genetically and chemically t'o the red-eye-pigment. Buzzati-Traverso
(1953 a) also working with Drosophilu found little corrcspondcnce between the
ninhydrin-positive and fluorescent patterns and based most of his olwervations on
the 1;Ltter. He found, as did Hadorn & Mitchell, that the resultant pci.tterns were
unaffected by the gut contents of cither larvae or adults ; his results showed age
differences in female specimens but not in males. This author also introduced the
idoa of the basic chromatographic pattern which is the maximum number of distinguishable spots for a given material and solvent. Variations in this basic pattern
;m: in the size and intensity of the spots, their colour or position does not change ;
the intensity of a spot may be so low that it is no longer distinguishable and therefore
apparently absent. Huzzati-Traverso was able to observe differences due to single
genes, also to distinguish re
ive heterozygous forms from dominant homozygotes.
In some reciprocal crosses a strong maternal infliience wii,s observed. I n other papers
thc same author developed further this approach to the identification of recessive
gene heterozygotes (Buzzati-Traverso, 1053 h) and, in collaboration with Rcchnitzer
(Bueznti-Travcrso & Rechnitzer, 1953) uscd paper chromatography for the first
time on untreated squashes of musclc tissue of fishes. Both the fluoresccnt and
niuh ydrin-positive patterns were iised in this work and the authors suggested that,
although the nature of the substances being scparated was unknown, they hclieved
t h a t a'mino-acids did not play an important part. They chimed that geographical
raccs of some species of marine fishes could be distinguished by this technique.
Kirk, Main & Beyer (1054)app1ie.d the principal of using fresh squashcs of muscle
tissues to a study of Australian land-sritds ; they wcrc the first workers t o use :I
horizontal disk technique of chromatography in taxonomic work. They found the
ultra-violct fluorescent patterns obtained from eech of seven species of snail were
characteristically different and that for two of the species they were uniLble t.o find
significant variations due to age or geographical locality. For one of the species
they found no variations in specimens ra,iscd in the laboratory on different diets.
Dannevig (1955) used squashes of fresh muscle of several species of marine fishes and
fitudied the ninhydrin-positive patterns. She found specific differcnces between
various gadoid, clupeoid and flounder species and suggested that it was possible t o
detect a difference bet>wecnthe in-shore and deep-water races of cod. Hybrids
bctwccn two species of flat-fish gave :L combination of the parent patterns and
showed considenhle maternal influence. Pox (1956)applied two-dimensiond chromatography t o fresh squashes of Drosophih melunogaster (two-dimensional techniques
ha.d already been used by Ball & Clark (loc. cit.) and Micks and his co-workers in
their amino-acid investigations) and was able to demonstrate the existence of a
XLIV]
APPLICATION OF PAPER CHROMATOGRAPHY TO A TAXONOMIC STUDY
225
‘ sex peptide ’ in males which was absent in females. He was able to ‘ map ’ 23
spots, most of which were identified. Fox also showed that the separation of substances from squashes of female insects was poor in the first dimension but that the
application of the second solvent achieved a separation demonstrating the presence
of the same substances (with the exception of the ‘ sex peptide ’) in both sexes. March
& Lewallen (1956) examined nine different strains of house-fly and found that the
variation between the ninhydrin-positive patterns obtained was as great as that
between the patterns of three species of flies belonging to different genera. These
authors were unable to correlate their findings with insecticide resistance in some
of the strains. Lewallen (1957) found sex and population differences in the ninhydrin-positive chromatogranis of two species of anopheline mosquitoes in California.
Varty (1956) applied paper chromatography to some of the taxonomic problems of
the Adelges insects. He found that the patterns obtained from eggs differed from
those given by larval or adult stages but that adults gave constant specific patterns
regardless of the food tree. He suggested that the red fluorescent substances he
observed were tannin compounds and the blues were anthocyanins. This is open
to some doubt since neither of these groups of compounds is known to occur in animal
tissues. If they were derived from the food then it is surprising that they show
constant results when the host plant is varied unless the gut metabolism of certain
species always produces the same breakdown products. Viswanathan & Krishna
Pillai (1956) used tissue crushes from Indian fishes and applied both horizontal and
ascending techniques. Their results from two species of Surdinella were equivocal
but they claim to have demonstrated clear differences between two species of
Leiognathus, also two species of Cnranx. They maintain that these results are given
even ‘ when the fish are not quite fresh ’. Robertson (1957) working with various
insects showed that the findings in one group did not necessarily agree with those
from another. For instance he was able t o detect sex differences in the pupae of the
mosquito Aedes aegypti but not in the adults of the hymenopteran Illrccrocentrus
uncylivorus. I n third-instar larvae of the hymenopteran Pristiphora erichsonii an
effect due to diet was obrerved, but this was not seen in any other species examined.
Population differences were not found in a lepidopteran or a dipteran. Although
differences a t the specific levcl were observed in most of the groups studied these
differences were small and could not be extended to taxonomic levels above genera.
I n attempting to differentiate hetween species of the coleopteran genus Laemophloetis
Robertson applied tests for reducing substances to his chromatograms and suggedted
that the possibility of amino-sugars playing an important part should be considered.
The most recent work seen in this field is that of Chen and Baltzer (1958) who have
carried out two-dimensional chromatography of deproteinized extracts of echinoderm
larvae and who claim t o have found specific differences in the ninhyclrin-positive
materials. These differences were not detected in hydrolysates of the protein
residues from which the extracts were made. They claim that the specific differences
persist in a11 larval stages despite alterations in intensity. The species used belong
to three different families and it is not, therefore, possible t o decide whether these
differences are truly specific or not. The results which the authors illustrate are
open to question in the light of Berg’s (1950) statement that unfertilized echinoderm
eggs give different p3tterns from the developing larvae and Kavanau’s (1953)
demonstration of diflerential changes in amino-acid content during cleavage and
through t o the early pluteus stages.
An analysis of some of thc results recorded above can help t o resolve several of
the conflicting statements which have been made and will also help t o evaluate the
techniques used and the results they give.
Micks (1956) pointed out the necessity for caution in the interpretation of the
results of amino-acid investigations since Pratt & Auclair (1948) have shown that
sensitivity to the ninhydrin reaction is not uniform. Thus glutamic acid and glycine
can be detected in amounts as small as 0.1 pg. while the minimum quantity of histidine
226
C. A . WRIGHT:
[J.L.s.z.
monochloride which can be demonstrated with ninhydrin is 25 p g . This factor almost
certainly accounts for the discrepancy between Micks' (1954) findings and those of
Ball & Clark (1953). It also means that investigations of the specific free aminowid content of animals must be carried out under very carefully controlled quantitative conditions. The physiological condition of the animals under examination
must also be closely comparable. This is well illustrated by the great difference
between the patterns obtained by Mieks (1954) from hibernating Culex pipiens
from Italy and those given by recently emerged American specimens of the same
species. It is likely that in the hibernating state the lowered metabolic rate will
result in a considerable reduction in the concentration of free amino-acids but this
reduction will not necessarily be uniform. This supposition is supported hy the
observations of Hadorn & Mitchell (1951) and Micks & Ellis (1952) that total free
amino-acid concentrations are a t their lowest in the pupal stages of insects. The
findings of Pox (1956) concerning the different separations obtained betwecn thc
sime substances in one dimensional chromatograms of males end females of the same
specics throws doubt on the value of applying too accurate methods of measurement
to the intensity of spots as described by Micks & Gibson (1957). It is more difficult
t o assess thc rcsults obtained from the fresh-tissue squush technique. The observations of Hadorn & Mitchell (1951) and Ruzzati-Traverso (1953 a) are probably the
most detailed and they agrcr on most points. Ruzzati-Traverso & Rechnitzer
(1953) and Dannevig (1955) claimed that population diffcrences in marine fishes
could he detected while Kirk, Main & Beyer (1954) were unable to find such differvnccs in widely separated populations of two land snails. A similar discrepancy in
findings occurs between the report of population differences in Californian mosquitoes
by Lewullen (1867) while Robertson (1957) was unable to demonstrate geographicel
variations in two species of Canadian insects. Undue importance should not be
given to the findings of Kirk and his co-workers in this respect since the two species
of gastropod concerned were Helix aspersa and l'heba pisana, neither native t o
Australia, and therefore relatively recent introductions in which one would not expect
to find marked population differences. It is not possible a t present to comment
on t h e fact that Viswanathan & Rrishna Pillai (19%) used tissue squashes from fish
dead for some time apart from noting that in the present work on snails it has been
found that the ninhydrin-positive patterns from dead material are different from those
given by living specimens. The problem of the effect of gut contents is obviously
important, particularly in work on small animals. Hadorn & Mitchell (1951) and
Ruzzati-Traverso (1983 a) both found that intestinal contents did not influence the
patterns given by Drosophila but these workers were using a standard culture medium.
The possible influence of gut contents on the results rrcordrd by Varty (1966) has
bccn mentioned, also the observation of Robertson (1957) that in one species a
food effect was observed. Most workers who have studied mosquitoes have used
unfed laboratory-reared spccimcns. With freshwater snails it has been found that
contamination of a tissue crush by gut contents or part of the digestive gland usually
gives additional bands on the fluorescent chromatogram.
MATERIALSAND METHODS
All the resultts recorded in this work have becn obtained from living snails. The
origins of the specimens are listed below. It has not yet proved possible to achieve
good results with preserved material and the results from dead specimens are unreliable.
A modification of the method of Kirk, Main & Beyer (1954) has been used
throughout (Wright, Harris & Claughcr, 1957). The original method of Kirk and
his co-workers did not give consistent results nor did it give a sufficiently good
separation of the fluorescent bands in those species with a complex pattern. The
XLIV]
APPLICATION OF PAPER CHROMATOGRAPHY TO A TAXONOMIC STUDY
227
majority of the technical objections to horizontal chromatography using filter-paper
,disks are overcome by the use of the apparatus described by Kawerau (1956). The
most valid objection that can be made is that the Rf values in each sector of the same
disk are not identical. This is due to the rolling of the paper during manufacture,
the flow of solvent being faster in one direction resulting in a slightly elliptical front ;
it was pointed out (Wright et al., 1957) that the actual distance moved by the solvent
front on the disk affects the Rf values of the bands and thus in those sectors of the
paper in which the front has moved the greatest distance the Rf values of the bands
will be a little higher than in the others. This may be of significance in analytical
work but it is of no importance here and this disadvantage is far outweighed by the
clearness of the separations which can be obtained by using this technique. I n
'either ascending or descending methods the spots tend to be diffuse and poorly
defined even after a long run while very neat, clear-cut bands can be obtained on a
disk with a run of only a few hours.
A number of grades of paper have been tested and so far, none better than
Whatman No. 3 has been found ; the next best is perhaps No. 3 MM but the fluorescent
bands tend t o have more diffuse edges when this grade is used. Only one solvent
has been employed throughout this work, the butanol/glacial acetic acidlwater
(100 : 22 : 50) mixture used by Kirk et al. (loc. cit.). At room temperatures there is
no separation of this solvent into phases provided that the water is the last ingredient
added. The majority of t h e chromatograms have been run a t laboratory temperatures (15-22' C.) although some work has been done in a constant temperature room
at 22" C. For critical work there is no doubt that a steady temperature is desirable.
The time for which a chromatogram is allowed t o run is variable according to the
type of pattern given by the species being examined. For most Lymnaea species
a run of about four hours at 20" C has been used but where the pattern has only a
few bands, some of which are only faintly fluorescent, a run of about one hour's
duration may be found useful. For species such as Helix aspersn it is necessary to
use the maximum run possible, about six hours on 26.5 cm. diameter disks, in order
t o separate clearly all of the bands present in the pattern.
For examination of the fluorescent patterns an Osram 125-watt mercury vapour
lamp with a Wood's glass outer bulb has been used. Chromatograms have been
tested for fluorescence a t wavelengths throughout the ultra-violet spectrum. The
strongest fluorescence is obtained in the 3650 d band and this is the principal wavelength emitted by the Wood's glass bulb used. Weaker fluorescence also occurs
in the 3100 d and 2700 d bands.
Originally the method of Kirk et al. was followed in that the posterior tip of the
snail's foot was used for the crush preparation ; subsequent work has shown that the
substances giving the fluorescent pattern are not derived from the tissues of the snail
directly but from the mucus secretion of the body surface. The slime-trail mucus of
the pedal gland in Helix aspersa gives no fluorescent substances but the pattern
obtained from the general body surface secretion shows all of the bands which are
present in chromatograms of tissue crushes and a clearer separation is obtained in
the absence of tissue. The material for chromatographic examination is obtained
simply by removing mucus from the body surface of the snail with a fine paint-brush
or wooden splinter and applying it to the paper near the apex of a sector of the disk.
Gentle pressure with the smoothed end of a piece of glass rod ensures penetration
of the mucus into the papers which are then allowed to dry overnight a t room temperature. If drying is too rapid the mucus may form a hard lump in the paper
which interferes with the flow of solvent and results in a distorted pattern.
Because some species of Lymnaen give scarcely any fluorescent pattern, chromatograms have been treated with a variety of reagents in an attempt to reveal nonfluorescent compounds, also in the hope that some progress might be made in the
identification of the fluorescent materials. The ninhydrin-positive substances
separated from tissue crush preparations have not been found to show clear specific
228
C. A. WRIGHT
:
[J.L.s.z.
differences with one possible exception mentioned below. The reagents which
have been used on chromatograms of mucus are ninhydrin, ammoniacal silvcr nitrate,
iodine vapour and Partridge's (1948) modification of the Elson-Morgm reaction.
Sources of Material
L. stugnalis
1. Basingstoke canal, Hampshire.
2 . St Rlbans, Hertfordshire.
3 . Flatford Mill, Essex.
4.Near Glasgow, Scotland.
5 . Malham Tarn, Yorkshire.
ti. Holland (exact locality unknown).
L. trunuxhla
1. Laboratory bred stock, Veterinary Research Laboratory, Weybridge,
Surrey.
2 . Eakring, Nottinghamshire.
3. Tjndfield, Sussex.
L. glabra
1. Near Darlington, Porkshire.
2. Anglcscy, Wales.
3. Lindfield, Sussex.
L. palustris
1. Laboratory bred stock, Veterinary Research Laboratory, Weybridge,
Surrey. (Original source- -Byfleet, Surrey.)
2 . Bromborough Pool, Wirral peninsula, Cheshire.
3. Eiisthiim, Wirrnl peninsula, Cheshire.
4. Maidenhead, Berkshirc.
5 . Haywttrdn Heath, Siiasex.
6. Grantham Canal, Stathern, Leicestershire.
L. pereyrn
1. Laboratory bred stock, Veterinary Research Laboratory, Weybridgc,
Surrey.
2 . Yiltdown, Susscx.
3. Lindfield, Sussex.
4.Shropshire Union Canal, Backford, Cheshire.
A. Epping Forest, Essex.
ti. Nottinghani Clanal, Wollaton, NottJingharnshire.
7 . Malham Tarn, Yorkshire.
L. auriculariu
1. Shropshire Union Canal, Backford, Cheshire.
2 . Nottingham Canal, Wollaton, Nottinghamshire.
L. auricularia rufescens ( 2 )
Laboratory bred at Veterinary Research Laboratory, Weybridge, Surrey,
from stock obtained from Wah Bridge, 30 miles from Rawalpindi on t h e
Peshawar road, North West Frontier Province, Pakistan.
XLIV]
APPLICATION
OF PAPER CHROMATOGRAPHY TO A TAXONOMIC
STUDY
2298
L. auricularia rubignosa (?)
Laboratory bred at Veterinary Research Laboratory, Weybridge, Surrey,
from stock from two sources in the North West Frontier Province of
Pakistan :
1. Turangzai village (about 34" 10' N., 71" 50' E.).
2 . Amagnar, a creek off the Kabul River north of the Peshawar-Rawalpindi
road.
L. natalensis
1. Laboratory bred stock, Veterinary Research Laboratory, Weybridge,
Surrey. (Origins uncertain.)
2. Salisbury, Southern Rhodesia.
3. Khartoum, Sudan.
L. lessoni
1. Helena River, Western Australia.
2 . Perup River, Western Australia.
ACKNOWLEDGMENTS
I wish to express my thanks to the following people for collecting and sending living
material for examination : Dr E. T. Abdel-Malek, Khartoum ; Dr A. Berry, Glasgow ; Mr P . F. Holmes, Malham Tarn ; Dr s. B. Kendall and M.r J. W. Parfitt,
Veterinary Laboratories, Wcybridge ; Mxs N. F. McMillan, Liverpool ; Dr M.
Mirolli, Verbania Pallanza, Italy ; Mr G. W. Pitchford, Nottinghamshire and Dr
W. S. S. van der Feen van Bentham-Jutting, Amsterdam, Holland. I am also very
grateful to Mr D. Claugher for his technical assistance throughout the work.
RESULTS
Four main groups of results were obtained and the species belonging t o these groups
are treated together for convenience in presenting the results ; this does not necessarily infer that there is any relationship between the species so grouped.
Between Rf 0.35-0-45 on mucus chromatograms of all the Lymnaea species studied
a double band of strongly reducing substances reacted with ammoniacal silver nitrate
to give a brown colour later changing to black and with iodine vapour t o give a
light yellowish-brown colour which fades easily. These bands often overlap into a
fluorescent band but do not coincide exactly with any of them. Because of their
ubiquitous nature thew reducing substances are not mentioned in each of the following
sets of results.
The Rf values given are the means for the front and back edges of the fluorescent
bands from a number of chromatograms. The standard variations from the mean
values are not included because too much reliance on Rf values may be misleading.
They are intended to serve merely as an indication of the relative positions of the
bands.
L. stagnalis, L. truncatula and L. glabra. (Table 11)
All three of these species a t first gave no detectable fluorescent patterns at all.
The only difference found between them was that when tissue crush chromatograms
were sprayed with ninhydrin the original spot for the first two species gave a greyish
colour while that for L. glabra gave a marked bluish-purple. Later, using mucus
only and a run of one hour a t 22" C. two faintly fluorescent bands were detected for
each of the three species. It was also noted that the fluorescent colours of the mucus
spots before running were bluish for L.stagnalis, blue for L. truncatula and pale yellow
for L. glabra.
230
0. A. WRIGHT
:
[J.L.s.z.
TABLEI1
___ __
Pluorescont
colour
.--.-
L. stagnalis
0.42-0.49
L. trzlncatula
-
__
0 . 41-0 49
0.55-0.67
Elson-Morgan
.-
Faint purple
Faint, blu e
Faint purple
..
____._
L. ~ I C L ~ C U
i
0.39-0.47
0.53- 0 . 6 3
Ninhydrin
1
i
..
Bright yellow.
-~---_-----1
0.59-0.66
_-
I
~-
Faint purple
..
..
I
..
I
Faint
greenish-yellow.
Faint
greenish-yellow.
1
~
I
Thcre can be little doubt that the substances here separated are the same in all
three species but the strong yellow reaction to the Elson-Morgan test given by the
outer band in L. stagnalis indicates that this substitnce is present in relatively greater
concentration in this species. The fact that there does not appear t o be any increase
in the intcnsity of fluorescence of this band in L. stagnalis suggests that the ElsonMorgan positive substance and the fluoresccnt material may not be identical but
merely have the same Rf value under the chromatographic conditions used.
L. peregra, L. auricularia, L. a. rufescens (?), L . u. rubiqinosa (?) and L. natalensis
(Table 111)
The basic pattern given by all of these species is similar and consists of four
fluorescent bands. To simplify the discussion of the results the bands have been
given Greek letters, from thc lower Rf upward, a,b, y , 6.
Only the Rf values of the fluorescent band are given. Wherc ninliydrin- or ElsonMorgan-positive substances were found to be not coincident with the fluorescent
pattern they are simply shown in the tablc below as lying between the fluorescent
bands. The p band in till of these species has a greenish-blue colour if viewed by
transmitted daylight and gives a light brown reaction with ammoniacal silver nitrate.
This band can be identified in chromatograms of embryo snails removed from the
egg-capsules.
The patterns of the first two of these species are almost identical but thcre is a
clcar fe&ure distinguishing them. In L.au&ularia the y and 6 bands are of approximately equal intensity and, although in many cases both may be absent, one is
never present without the other. I n L. peregra, on the other hand, the 6 band is
always brighter than the y and it gives a faint reaction with the Elson-Morgan reagent
in about 50 per cent of the specimens examined. I n ubout 30 per cent of the specimens the y hand is too faint for its Rf value to bc read with certainty. The ct band
may be absent in both species and is seldom observed in tissue crush prepzirations
due to the tailing flare below the p band which is usually present if this tcchiiique is
employed.
The material originating from Pakistan has been examined for me by Dr Bengt
Hubendick and, in privace communication, he has informed me that while the&
is no doubt that all of the material belongs to the L. auricularia super-species it is
not possible to name the specimens with accuracy. The subspecific names used
here are mcrcly those which seem most appropriate but they are followed by a
question-mark to indicate the doubt that exists concerning them. The material of
L . auriculurin rufescens ( 2 ) has given a pzttern similar to that of the British 11. awri,cubria but the y and 6 bands :we yellow. If small quantities of mucus from this
XLIV]
APPLICATION
TO A TAXONOMIC
OF PAPER CHROMATOGRAPHY
231
STUDY
TABLEI11
1
i
I
~
Fluorescent
colour
Rf
I
L. peregra
a
,!i ,
0.30-0.3.5
0.41-0.46
y
0.52-0.57
6
0.63-0.67
Blue
____ __-__-_
______
I L.aurzculariu
1
Faint blue
Hrightblue
,
Faintblue
~
~
1
,9
0.31-0.37
0.41-0.45
;j
0.50-0.32
~
Faint blue
Bright blue
..
..
..
..
..
Pink
Light pink
..
Bliie
I
1-
I
..
~
Greenish -yell0w.
Light pink.
-
-
..
..
I
~
,
Elson-Morgan.
_____-
i
a
Ninhydrin
---
..
Light pink
..
..
L.a. rufescena
'
I
(?)
a
i
6! I
0.30-0.36
0.40M.44
0.50-0.55
~
Faint blue
Bright blue
Yellow
..
..
Pink
Greenish-yellow.
~
..
..
..
..
..
0'31-0.36
0.40-0.43
0.56-0.59
I
~
Faint blue
Bright blue
Light blue
L. nutulertsis
a
,9
Greenish-yellow.
I
I
1'
11
I
..
..
I
0.64-0' 66
I
..
..
>,
Faint blue
Bright blue
Blue
---
..
I1
..
Light pink
..
Pink
..
I
..
..
..
..
Greenish-yellow.
species are run for about six hours the fluorescent colour of these bands is faint blue
suggesting that the colour difference may be a matter of concentration of the fluorescent substances. On the other hand, the 6 band in this form does not give a positive
Elson-Morgan reaction as it does in L. peregra and this would be expected if it were
the same substance present in greater quantity. L. a. rubiginosa ( 2 ) gives a pattern
not unlike that for L . peregra in that the 6 band is sometimes a little stronger than
the y but the whole pattern in this form is weaker, even the p band lacking the
brilliance which is usual in this group.
L. natalensis has basically the same pattern as the rest but the 6 band has not
been observed and the y band is very bright, sometimes almost as intense as the p.
It has been observed that t h e Rf values of the p band are a little higher in chromatograms of mucus than they are in tissue crush preparations. It is possible that
some of the substances present in the crush have a retarding effect on the movement
of this band, This raises the question of whether a substance will always have the
same Rf value under given solvent and paper conditions regardless of the other
compounds present in the original mixture. The evidence of Fox (loc. cit.) suggests
that it will not, in that the same substances in male and female Drosophila are not
separated in the same way in the two sexes.
232
[J.L.s.z.
C. A. WRIGHT:
L.palustris
This species has proved the most interesting of those studied in that not only
is its chromatographic pattern in no way related to that of any of the other species
examined but it is the only one in which clear population differences and a possible
seasonal variation have been detected.
The first specimens examined were laboratory-bred material derived ten years
before from a wild population near Byfleet in Surrey. Tissue-crush chromatograms
were prepared in September and the following fluorescent pattern was observed :
Rf
Yluorescerit colour
I
0.46-0.53
0.50-0.59
Light blue.
I
Pink.
Lilac.
0 50-0.65
0.760.84
The pink fluorescent band is scarcely detectable in many of the specimens and is
only faint in the rest. The yellow fluorescenceis not the same as that in L.a u r ~ c u l a r ~ ~
rufescenfi ( 1 ) but is more of an ochre colour. Exposure to ammonia vapour makes the
lilac band visible in daylight with a yellow colour and changes its fluorescent colour
t o yellow. This treatment intensifies the fluorescence of the pink band but converts
its colour t o a greenish-yellow. The lilac band also gives a yellow colour with the
strongly alkaline solution I of the Elson-Morgan reagent and with ammoniacal silver
nitrate but it becomes colourless once more on treatment with acid.
No further material was seen until March when about 24 specimens from the
Wirral peninsula in Cheshire were examined, Mucus chromatograms prepared from
this material gave the following results :
T~'1uorescent
colour
0.34-0' 43
0 * 52-0.55
0.59--0.61
Paillt blue
Pink
Lilac
I
,
~
Nirihydrin
Pale pink
..
--
~
I
Elson-Morgan
-I
Faint pink.
..
1
I
Yollow (with
Mulution I
Tissue crush preparations failed t o reveal the yellow band seen in the earlier
muterial. The faint blue was barely visible and its Rf was a good deal lower. Thc
pink fluorescent band was much stronger and clearly separated from the lilac. The
Rf of the pink was a little lower than before but that of the lilac remained unchanged.
Similar results were obtained from a different population from the Wirral and another
from Leicestershire in May. Also in May small samples from Maidenhcad, Berkshire
and Hibywards Heath, Sussex wcre cxamincd and both of these lots showed scarcely
any trace of the light blue fluorescence while the pink showed only as a faint fringe
on the inside of the lilac band which was in the usual position. Again no trace of
the yellow band was found either in mucus preparations or tissue crushes. A further
supply of specimens from the same laboratory bred stock as the original material
thcn examined and a pattern similar t o that given by the last two batches was
obtained. The pink band was a little more in evidence but still only as a fringing
flare 011the inner edge of the lilac and not as a clearly discrete band as in the materid
from Cheshire. These results were given by both tissue-crush and mucus prcpara-
XLIV]
APPLICATION OF PAPER CHROMATOGRAPHY TO A TAXONOMIC STUDY
233
tions. No change had been made in the routine maintenance technique of the
laboratory bred material since September and the patterns did not vary between
various size groups in the sample. Anatomical examination has failed to reveal
any obvious morphological differences between the populations studied. The
variation in the Rf values of the light blue band may be further evidence of the
effects of the presence of mixtures of substances on the movement of one particular
component.
L. lessoni
An attempt to send living specimens of this species from Western Australia,
where it has only recently been found, was unsuccessful. I am indebted to Dr
J. A. L. Watson of the Zoology Department, The University of Western Australia,
Perth for making tissue crushes from living material and sending them to me for
examination. The pattern obtained from these tissue crushes was unlike that of
any of the previous species, it consisted of a fluorescent flare extending t o the solvent
front and behind it a weak absorption band. Behind the absorption were three blue
bands of different intensities but so close together that the front of one is the rear
edge of the next. The table shows the relative positions of the bands in this pattern.
I
'
Ii
Fluorescent colour
___---_
____
Rf
0.21-0.35
0.35-0.43
0.43-0.51
0.51-0.61
0.61-1
I
Blue.
I,
Faint blue.
Absorption.
Blue flare.
~.
No tests were carried out on these chromatograms since the results would not be
comparable with those from mucus preparations in the other species studied.
DISCUSSION
Hubendick (1951) has shown that, with the exception of Lanx, there is no real
justification for any of the large number of generic names which have been used
in the Lymnaeidae. Hubendick recognizes certain super-species, notably the L. auriculuria group, but points out that on the whole affinities within Lymnaea are difficult
t o determine.
The present study has contributed little to the existing knowledge of the relationships within Lymnaea but it has enabled an assessment t o be made of the usefulness
of the chromatographic technique when applied to this type of problem. The
apparent similarity between the patterns of L. stagnalis, L. truncatulu and L. glabra
is undoubtedly misleading. These three species are in no other way closely related,
in fact, on the basis of the structure of the prostate, they fall into different groups.
L. glabra has a simple tubular prostate, considered by Hubendick to be the primitive
condition, L. truncatula has a single prostatic fold and L. stagnalis belongs to the
most advanced group with a multifolded prostate.
The relationship between the chromatographic patterns of L. peregra, L. auricularia and its subspecies and L. natalensis is supported by anatomical evidence.
Hubendick (loc. cit.) has stated that the close relationships of L. peregra cannot be
definitely decided but that the species may probably be derived from L. auriculuria.
He goes on to say that it is difficult to explain how this speciation occurred since the
range of L. auricularia covers almost completely that of L. peregra. Since the
only really satisfactory means of distinguishing the two species is by the length of
the spermathecal duct and intermediate forms have been seen there is little doubt
234
0. A. WRIGHT:
[J.L.Y.z.
of the close relationship between the two. The separation of the two species niay
possibly have occurred during one of the glacial periods with L. peregr, confined t o
the westward of an ice barrier and L. auricuhr'ia t o the east. This hypothesis
explains the greater eastern distribution of L. auricularia and the relatively greater
itbundance of L. peregra in western Europe and is borne out by the finding of fossil
L. peregra in Britain in deposits dating from the a,nte-penultimate inter-glacial
while L. auricularia has been found only in post-glacial deposits (Zeuner, 1945).
A great deal of further work will be needed t o analync the situation within the
L. au'ricularin superspecies in Asia. The two examplcs described above are too
isolated to merit further discussion a t present. Hubendick has included L. natalensis
in the auricularia superspecies and this course sccms quite justified. Perhaps
chromatographie investigation will help to decide whether L. natalensis is really
an homogeneous species or a complex of' closely related forms.
Of L. palur&s Hubendicli says, 'L. palustris seems t o lie a relatively distinct
species without near relatives in the EuriLsim area. I n North America, however,
some forms similar to L. palustris have evolved.' Chromatography definitely supports the lack of relationships ofthis species to others in the British fauna. It may
be that the population differences revealed in England provide the key t o the speeitttion which has occurred in the Ncarctic region. There is no doubt that the type of
phtttern shown by L . palustris lends itself to easy observation of population differences.
It may be that intensive work will reveal similar differences in other species but i t
must be remembered that the variations between populations noted in palustris is
as great as the species diffcrcnces in the peregra/auricularia group. Of the variation
betweeri the pat8t,ernsobtained from laboratory bred matcrid of the same stock in
spring and autumn little can he said at present. No similar difference has been
observed in the CUSR of L. peregra or L.stagnalis. The most reasonable explanations
seem to lie either in metabolic changes in the snails in the autumn in preparation for
hihernation or in the sexual activity of the animals in the spring. Only further
carefully controlled work can elucidate this point.
The results obtained from L. lessoni are too isolated for profitable discussion at,
the moment. They give hope that chromatography may assist in sorting out the
relatively poorly known species complexes in Australasia.
Perhaps the most significant result of this work is the demonstration of specirsspecific substances in the body-surface mucus of snails. There is reason to suppose
that among snails, which are non-visual animals, species recognition may be of i~
chemotactic nature. If this is so then chromatographic analysis of body-surface
mucus mag be revealing, at leiist in part, the mechanism by which inter-specific
cross-fertilization is prcvcntcd. The chromatographic piitterns mcby, therefore be
taxonomic characters of more fundamental importance than the morphological ones
at present in use. Further, chromatography may help to elucidate the ehemotactie
means by which the miracidial stages of trematode parasites select the appropriate
molluscim intermediate host. This work has possihly provided some evidence tto
this effect. When it was discovered that! both the African and Pakistani intermediate
hosts of Eascioln gigan,tica ( L . nntnlen.qis and L.auricularia rufescens ( 1 ) ) showed a
bright blue band in their chromatographic patterns between Rf 0.40-0.45 and t h a t
this band was present also in L. peregrn of British origin a request was made t o
1)r S. R. Keiidall of the Ministry of Agriculture Central Veterinary LiL\)or:Ltory tJo
t:xpose L. pereym t o miracidia of Y. gigantica of Pakiatimi origin.. I n a private
communication Kendall & P a r f t t inform. me that this has been done and a few
shed between 132 and 146 days after infection. The infection only
bccamc established in a few snails and, after shedding cercariae the infection was
apparently thrown off, subsequent disscction of the snails revealing no redim. I n
:L control experiment miracidia from the same batch produced a heavy infection in
their usual host, L. nuricularia rufescens ( ' 1 ) and cercariae were shed after 110 days.
This is particularly intcrcsting in the light of Kendall's (1950) work on the. suscepti-
XLIV]
APPICATION OF PAPEX. CHROMATOGRAPHY TO A TAXONOMIC STUDY
235
bility of British Lymnaea species to Fascioh hepatica. He found that, in addition to
L. truncatula, i t was possible to infect L. stagnalis, L. glabra and L. palustris with
subsequent shedding of cercariae from a t least a few individuals of each species.
Although miracidia were seen to attack L. peregra and early development of sporocysts occurred in several individuals the redia stage was reached in one specimen
only and cercariae were never shed. No infections were observed in any of the
L. auriculuria exposed. As yet no L. auricularia of British origin have been exposed
t o infection with F . gigantica. If the infection develops in this species, and there
is every reason to believe that it will, a clear group relationship will be demonstrated
in that the Lymnaea species with the bright blue band at Rf 0 . 4 0 4 4 5 in their chromatograms are refractory to infection with F . hepatica but susceptible in varying
degree to F . gigantica. This evidence does not, of course, provide direct support
for the idea of chemotactic host selection by miracidia but it does indicate a possibly
fruitful line for further investigation.
The few chemical tests which have been applied t o chromatograms have not
helped much in the further identification of the fluorescent substances. The universal presence of the double band of reducing substances between Rf 0-35-0.45
revealed by ammoniacal silver nitrate treatment recalls a t once the double band
found with the same reagent a t a lower Rf in Laemophloeus spp. by Robertson (1957).
The lower Rf may be accounted for by differences in technique. Robertson suggests
that since a ninhydrin reaction is obtained a t the same place on his chromatograms
the substances may be amino-sugars. I n tissue squash preparations of snails a ninhydrin reaction is obtained a t about the same Rf as the silver nitrate-positive material
but this is not the case in most mucus preparations and suggests that Robertson's
observations may be due to using whole insects in which a ninhydrin-positive substance moves closely with the reducing material. I n L. palustris however, the light
blue fluorescent band partly coincided with the silver nitrate positive substances and
in the same region a pale pink ninhydrin reaction was obtained as well as a light
pink with the Elson-Morgan reagent. This combination of results does definitely
seem to indicate the presence of an amino-sugar. The ninhydrin reactions obtained
from mucus chromatograms have not shown any signs of specificity or much constancy in their appearance. There is reason to suspect that most of the ninhydrinpositive substances are derived from contamination of the mucus by palial fluid
rather than from the mucus itself. The pale greenish Elson-Morgan reaction a t
Rf' 0.60 has been detected in all the species examined except L. pulustris and it is
probably this substance that is present in relatively greater quantity in L. stagnalis
t o give the bright yellow colour recorded. The positive Elson-Morgan reaction of
the 6 band in L. peregra is interesting in that it is the only one of the brightly fluorescent substances to give a definite result with this reagent. Until chemical identification of the fluorescent substances has been achieved little progress can be made
in attempts to demonstrate the presence of possibly related non-fluorescent compounds in species such as L . stagnalis, L. truncatula and L . glabru.
Few papers dealing with the biochemistry of mucoid substances in snails have
been seen. Masamurie & Yosizawa (1950) have shown that in Helix the foot mucus
contains glucosamine and galactose while glucosamine, mannose and galacturonic
acid have been isolated from snail mucus mucin. Baldwin & Bell (1938) and Bell &
Baldwin (1941) have worked with galactogen derived from the albumin glands and
egg-capsules of Helix pomatia and demonstrated the presence of l-galactose as one
of its components. During the present work chromatograms of the gelatinous
covering of lymnaeid egg capsules were run, but no fluorescent compounds were
detected. An observation of some possible significance is that the mucus from the
snail species which give a brightly fluorescent pattern is at least faintly coloured when
placed on paper. The yellow of palustris mucus has been shown to disappear when
treated with acid and to reappear with alkaline reagents and it is this substance which
gives the lilac fluorescent band (in acid pH) characteristic of the species. The bright
236
C. A. WRIGHT:
[J.L.s.z.
blue (j?)fluorescent band of the peregra/au,ricularia group has a bluish-green colour
in daylight but this does not appear t o be affected by pH.
No attempt has yet been made t o apply two-dimensional chromatography to
thc free amino-acids in snail blood. The indication of specific differences between
the ninhydrin-positive materials of L.d i g n u l i s and L. glabra suggest that the technique may be useful. One important fact must, however, be borne in mind if this
type of chromatography is to be used. The sensitivity of the ninhydriri reaction to
vwying concentrations of amino-acids hns been discussed above and the fact that
small amounts of somc substances may be overlookcd has been stressed. Micks &
(%son (1957) have partly attributed thc success of the technique in entomological
work to the relatively large quantities of amino-acids present in insect blood but
Duch5teau et ul. (1952) have shown that the concontration of free amino-acids in a
freshwater lamellibranch mollusc (Anodontn cygneae) is lcss than onc-twelfth of that
in the marine lamellibranchs Mytilus edulis and Ostrea edulis. A similw diffcrcnce
was shown by the same authors betwccn a marine sipunculid (Sipunculus nudus)
and a freshwater annelid (Hirudo medicinalis). If this low concentration of frcc
amino-acids in freshwater forms applies t o gastropods as well it is possible that some
modification of existing two-dimensional techniques of chromatography will bc
required to detcct specific differences.
The main conclusion that can be drawn from this study is that the method of
paper chromatography can be useful in some cases for species discrimination, in
others for population studies, possibly also for work on host-parasite relationships
between snails and larval trematodes. That the results can be misleading is amply
illustrated and the possibility of seasonal variations in patterns must be given consideration in future applications of the technique.
SUMMARY
1. The literature on the use of paper chromatography in taxonomic work is
reviewed bricfly.
2. The technique of paper chromatography on disks is described and the method
has been applied to a number of species of Lymnaea.
3. The chromatographic patterns of the species studied are described and discussed.
4. The implications of the discovery of species-specific substances in the body
surface mucus of snails are discussed, both from the taxonomic and parasitological
points of view.
5. The possibilities of applying two-dimensional chromatography to the free
amino-aeide of snail blood are discussed.
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BALL,G . H. & CLARK, E. W. 1963. Species differences in amino acids of C'ulex mosquitoes.
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BXLL,D. J. & BALDWIN,
E. 1!341. The chernistry of galactogen from Helixpotnatin. 1-galactose
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APPLICATION O F PAPER CHROMATOGRAPHY TO A TAXONOMIC STUDY
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ADDENIIUM
Since the preparation of this pqmr a serios of three communications under the general title
" Biochemical Bases for the Tasoriomy ot' Snails " has been publishocl in the Bull. Soc. A i n i s S c i .
I'ozrian, 14, 1!158. 'l'hc p q x r s arc : 1. ;\Iicht:jda, J., Clirornatographic ar~alysisof some fresh.
water snails. 2. Miohejd;t, J. mid UrbArislii, J. An a t h m p t a t a chromatographic amalysis of
some species of snails. 3. Mic.lirjdn, J . and Turbariska, E. Uiffercnces in Chromatographic
patterns of various organs and tisaucs. Spaco does not pcrrnit a discussion of this important
work, however, somo of the results described by these authors differ widely from some of those
described in the present work. Re-examination of my material does riot lead me to makc any
modifications t o my own reported results at the momont.
JOURN. LINN. S0C.-ZOOLOGY,
VOL. XLIV.
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