J. Zool., Lond.

(1968) 156, 1-8

Feeding, digestion and food storage in two species of temnocephalid

flatworms (Turbellaria : Rhabdocoela)

J. B. JENNINGS

Department ofZoology, University of Leeds

(Accepted 13 February 1968)

(With 1 plate in the text)

Histological and histochemical methods have been used to study feeding, digestion and food
storage in two species of temnocephalid flatworms.
Temnocephala brenesi, from Costa Rica, and T. novae-zealandiae, from New Zealand,
are ectocommensal on the gills of freshwater decapod crustaceans and feed on a variety of
small aquatic organisms. The prey is captured by means of prehensile tentacles, and is
either ingested intact or held to the mouth whilst the pharynx ruptures the integument to
draw in the body contents.
In both species the alimentary system is of the typical rhabdocoel form, with a doliiform
pharynx and saccate intestine. The gastrodermis is differentiated into glandular and phago-
cytic components, with some of the gland cells subgastrodermal and embedded in the paren-
chyma surrounding the intestine.
Digestion occurs by a combination of extra- and intracellular processes; endo- and
exopeptidases, acid and alkaline phosphatases and lipases are secreted in sequence and can
be demonstrated histochemically.
Deposits of fat occur in the vitellaria and eggs, and glycogen is laid down in the testes
and the musculature of the pharynx and sucker.
The ectocommensal life has not caused any significant change in the general pattern of
nutrition characteristic of free-living flatworms and no tendency towards a parasitic existence
has been found.

Contents
Page
Introduction .. 1
Material and methods 2
Observations .. 3
The food and feeding mechanisms 3
Structure of the gut 4
Digestion 4
Enzymes in the vesicula resorbiens 6
The food reserves 6
Discussion 7
References 8

Introduction
Studies on the nutrition of rhabdocoel Turbellaria (summarized by Jennings, 1968)
have shown that free-living species are carnivorous and feed on protozoa, rotifers, nema-
todes and small oligochaetes and crustaceans. The food is usually swallowed alive and
1 I
2 J. B. JENNINGS

intact, but in some cases organisms too large for this are gripped by the flatworm whilst
the pharynx sucks out body fluids and soft tissues. Digestion may be extracellular, intra-
cellular, or both, and fat forms the principal food reserve.
Little is known, however, of the nutrition of those rhabdocoels which live either on or
within other invertebrates and it is not clear how far differences in the mode of life have
influenced the basic nutritional pattern. Only one species, Syndesmis franciscana (Lehman)
from the gut and coelom of echinoids, has been investigated from this point of view and
in this instance the principal departure from the typical pattern lies in the formation and
storage of large quantities of glycogen (Jennings & Mettrick, 1968). The diet is essentially
the same as in free-living species, consisting largely of protozoa common in the habitat,
but includes a proportion of host tissue in the form of coelomocytes so that this rhabdocoel
may be regarded as semi-parasitic rather than wholly entocommensal. The digestive physi-
ology shows no variation from that of free-living species.
The ectocommensal rhabdocoels (suborder Temnocephalida) live on the gills or general
body surface of freshwater crustaceans or, more rarely, on gastropod molluscs or turtles.
The diet, so far as is known, resembles that offree-living species (Gonzales, 1949; Jennings,
1967) but nothing is known of their digestive processes or food reserves other than a
report of glycogen storage in species from Ceylon (Fernando, 1945). Accordingly, using
histological and histochemical techniques, two temnocephalids have been examined to
determine the basic pattern of their nutrition. This is compared with that of entocommensal
and free-living species.

Material and methods

The two species of temnocephalids studied were Temnocephala brenesi Jennings 1967 and
T. novae-zealandiae Haswell, 1888.
T. brenesi was obtained from the gills of the shrimp Macrobrachium americanum Bate, collected
from the Rio Virilla and its tributaries the Quebrada Honda and Quebrada El Cacao in Alajuela
Province, Costa Rica, Central America. Living and preserved specimens of T. novae-zealandiae,
from the gills of Paranephrops planifrons White living in small streams near Palmerston North,
New Zealand, were kindly supplied by Mr P. S. Dale of Massey University.
Both species survived for over two months in vitro and observations were made on their feeding
mechanisms and dietary preferences by providing them with appropriately sized representatives
of the fauna of their hosts' natural habitat.
Gut structure and the site and sequence of digestive processes were studied in serial sections
prepared after fixation in Bouin or Susa at progressive intervals after an observed meal of one of
the natural foods. Sections were stained by the bromo-phenol blue technique of Mazia, Brewer
& Alfert (1953), or by Ehrlich's haematoxylin and eosin, Mallory's trichrome or Feulgen's
methods. Fat reserves in well fed and starved animals were studied from frozen sections of forma-
lin fixed specimens stained in Oil Red 0 or Sudan IV, and carbohydrate reserves from serial sec-
tions prepared after fixation in Bouin or 90 % alcohol and subsequently stained in Best's carmine
or periodic acid-Schiff (PAS).
Enzymes associated with digestion were studied histochemically in frozen and paraffin wax
sections of individuals fixed at intervals after an observed meal. Fixation was in 10% formalin
buffered to pH 7·0, for 6-12 h at 1°C, and for preparation of the paraffin sections this was followed
by rapid dehydration in absolute acetone at the same temperature, clearing in xylol at room tem-
perature and in vacuo embedding in wax melting at 45°C.
Proteolytic enzymes were demonstrated by the Hess & Pearse (1958) method for endopeptidases
 FEEDING AND DIGESTION IN TEMNOCEPHALID FLATWORMS 3

and by the Burstone & Folk (1956) method for exopeptidases of the leucine aminopeptidase
(LAP) type. Lipolytic activity was detected by the Tween 80 method of Gomori (1952), alkaline
phosphatase by Gomori's (1952) lead salt method, and acid phosphatase by Burstone's (1958)
azo dye method. Incubation times, pH conditions, the performance of control experiments using
selective activators or inhibitors and the basis for the interpretation of the results were as described
for similar studies on the digestive enzymes of other turbellarian and rhynchocoelan worms (Jen-
nings, I 962a,b).
Changes in the pH of the gut contents during digestion were followed by feeding small pieces
of crustacean muscle stained in 0·1 % aqueous solutions of bromo-cresol green (pH range 3,6-
5'2), chloro-phenol red (4·6-7·0) and phenol red (6'8-8'4).

Observations
The food and feeding mechanisms
Histological examination of the gut contents of specimens fixed immediately or soon
after removal from the respective hosts showed that both T. brenesi and T. novae-zealandiae
feed on protozoa, rotifers, small oligochaetes, insect larvae and larval or adult copepod
and ostracod crustaceans. The gut often also contained large quantities of diatoms and
algal filaments, the cellular contents of which were partially or wholly digested. These
materials, in similar condition, were common in the guts of many of the food organisms
and it is concluded that those found in the temnocephalid gut originated from this source
and had not been ingested directly. This conclusion is supported by laboratory observa-
tions that both species take only animal food and consistently refuse any form of plant
material.
The distal parts of the gills in M. americanum, the host of T. brenesi, often carry consider-
able numbers of the temnocephalid's eggs, ranged in rows of five to ten, and the empty
egg capsules remain attached to the gill lamellae after the eggs have hatched. Small
particles of detritus gather between and around the capsules as soon as they are laid,
resulting in the formation of brownish patches easily visible to the naked eye (Plate lea))
and providing a ready indication of a past or present infestation by the flatworm. The
accumulated material forms a substratum on which diatoms, protozoa and rotifers flourish
(Plate l(b)) and these organisms appear to form the principal food of the young T. brenesi,
judging from their preponderance in the gut contents of specimens less than 1·5 mm in
length (adult individuals are 3·0 mm long and 1·4-1·6 mm broad).
Gills of P. planifrons, host of T. novae-zealandiae, were not available for examination
and it is not known whether in this instance, also, the egg-laying activities of the temno-
cephalid create conditions favouring establishment and multiplication of organisms suit-
able for ingestion by the newly hatched and juvenile flatworms.
No evidence suggesting ingestion of host tissues was found in either species, and the
gills of M. americanum showed no traces of damage attributable to the presence or feeding
activities of T. brenesi, other than the accumulations of material around the egg capsules.
Laboratory observations show that the feeding mechanism is virtually identical in both
species. Hungry flatworms adhere to the substratum by the posterior pedunculate sucker
and extend the body to its fullest length. Occasional sweeping movements are made, with
the body pivoting upon the sucker, but for the most part the flatworms remain quite
still. Suitably sized organisms moving into the vicinity are seized by the tentacles which
4 1. B. JENNINGS

then immediately shorten and draw the food back towards the anterior margin of the
flatworm (Plate I(c),(d)). The entire body then contracts and curls ventrally, the tentacles
also curl ventrally and the food is pushed into the subterminal mouth (Plate lee)). The
body and tentacles are then extended again and the flatworm remains quiescent whilst
digestion progresses (Plate I(f)). Small organisms are swallowed within 14 seconds of
being seized and undergo considerable disruption as they pass through the pharynx into
the intestine. Animals too large for ingestion intact are held to the mouth by the tentacles.
The pharynx, which is capable of slight protrusion, is applied to the integument and this
eventually ruptures and the body contents are rapidly ingested. Insect larvae are often
dealt with in this way, the entire process occupying only two to three minutes.

Structure of the gut

In both T. brenesi and T. novae-zealandiae the alimentary system is of the typical
temnocephalid form and consists of mouth, small buccal cavity, doliiform pharynx and
saccate intestine. The structure of the pharynx in T. novae-zealandiae has been described
in detail by Fyfe (1942) and that of T. brenesi shows no significant differences. In both
instances the radial musculature is extremely well developed and the pharyngeal lumen
is lined by a strongly acidophilic, cuticle-like structure which probably plays an important
part in the crushing of the food during ingestion. The pharynx in both species is slightly
protrusible, contraction of the general body musculature during feeding causing it to
move anteriorly into the buccal cavity so that its tip protrudes through the mouth for
application to food organisms too large for ingestion intact.
The intestinal wall or gastrodermis (Plate I(g)) consists of a single layer of cells, the
great majority of which are columnar phagocytes, 50-60 JL tall and 20-30 JL wide, with
basal vesicular nuclei and the cytoplasm generally containing a number of food vacuoles.
About 5 % of the cells, however, are glandular and of an elongate pyriform shape, of the
same height as the phagocytes but containing numerous acidophilic, proteinaceous spheres,
1-2 JL in diameter. Similar cells occur below the basement membrane of the gastrodermis,
lying parallel with the membrane and, therefore, at right-angles to the long axis of the
gastrodermal cells. Each of these subgastrodermal cells communicates with the gut
lumen via a long slender neck which runs through the gastrodermis between the phago-
cytic cells. The proteinaceous spheres secreted by the cells pass up the necks and are
discharged into the gut lumen, as also are those from the cells sited within the gastrodermis.
The spheres give an intense positive reaction to the Hess & Pearse (1958) method for
endopeptidases of the cathepsin C type (Plate I(g)), using o-acetyl bromoindoxyl acetate
as substrate. The reaction is resistant to 1O-5M E-600 (diethyl-p-nitrophenyl phosphate),
is intensified by preincubation of sections in 10-3M cysteine and significantly inhibited
by 1O-3M lead nitrate, confirming the endopeptidase nature of the spheres.

Digestion
As food enters the intestine the gland cells discharge their spheres and within one hour
of feeding very few of these cells remain demonstrable by the Hess & Pearse technique.
The cells then gradually secrete more spheres and 24-36 hours after a meal have returned
to their original condition.
 J. Zool., Lond. (1968) 156

PLATE I. (a) Anterior region of Macrobrachium americanum, with the carapace removed. The dark areas at the
bases of the gills are caused by accumulation of detritus, and the subsequent growth of diatoms, protozoa and
rotifers, between and around the egg capsules of Temnocepltala brenesi. Life size.
(b) A gill filament from M. americanum, showing a newly hatched T. brenesi together with empty egg cases and
the materials which accumulate around these. Unstained. x 35.
(c), (d), (e), (f) Teninocephala novae-zealandiae capturing and ingesting a portion of Tubifex, Photographed by
electronic flash at intervals of three to four seconds. x 15.
(g) Transverse section of T. brenesi showing the gastrodermis with darker staining gland cells, one of which is
subgastroderrnal and at right-angles to the others. Hess & Pearse method for endopeptidases, counterstained
with nuclear fast red. .: 300.
(h) T. brenesi. Acid phosphatase activity in the gastroderrnis and wall of the vesicula resorbiens, S., Degenerat-
ing spermatozoa within the vesicula resorbiens. Burstone azo dye method, counterstained with Mayer's haernalum.
x300.
(i) Transverse section of the intestine in T. brenesi, 12 hours after a meal of Tubifex, showing exopeptidase activity
in the gastrodermis. The reaction is particularly intense in food vacuoles in the proximal half of the cells. Burstone
& Folk method. >< 200.

[To face page 4

J. Zool., Loud. (1968) 156

PLATE I
 FEEDING AND DIGESTION IN TEMNOCEPHALID FLATWORMS 5

Most of the observations on digestion in both species were made on temnocephalids

fed on living food, and gut contents, therefore, generally showed some endopeptidase
activity due to the food's intrinsic enzyme systems. Within three hours of feeding, however,
much of the material in the gut lumen was completely disorganized and tending in parts to
become homogeneous apart from fragments of exoskeletons, setae and diatom cases. This
breakdown of the food is accompanied by a significant increase in the amount of endo-
peptidase activity present, distributed uniformly through the gut contents and obviously
due to the material discharged by the gland cells. Optimum pH value for histochemical
demonstration of endopeptidases in both gut contents and gland cells was 4·0.
No other enzymic activity originating from the gastrodermis could be demonstrated in
gut contents. Small amounts of exopeptidase and phosphatase activity were often found,
but their level showed no significant increase with time and it was clear that the enzymes
responsible originated within the food. Thus it would appear that the fairly rapid extra-
cellular digestion occurring in both species is effected solely by endopeptidases.
The columnar gastrodermal cells phagocytose particles of food as the larger masses
in the lumen disintegrate. Food vacuoles appear distally, with contents showing the same
histological and histochemical reactions as material in the lumen, and move back deeper
within the cell whilst more form in their place. This process continues until the gut lumen
is emptied of all but indigestible residues.
The contents of the food vacuoles condense into homogeneous spheres which decrease
in size as intracellular digestion and absorption of the products proceed. The time for
complete digestion of a meal varies with the amount of food taken, but usually all vacuoles
have disappeared within four days of the meal.
Formation of food vacuoles at the distal border of the phagocytic cells is accompanied
by development of acid phosphatase activity in the surrounding cytoplasm, and as the
cells become loaded with vacuoles so a general, diffuse, phosphatase reaction spreads
throughout the gastrodermis (Plate l(h)). Endopeptidase activity in the vacuolar contents
is at first no stronger than in lumen contents, but the amount present increases rapidly
with time and eventually every vacuole in all parts of the gastrodermis shows an intense
positive reaction. This is due, presumably, to secretion of endopeptidases into the vacuoles
from the surrounding cytoplasm, but since no activity could be demonstrated in the cyto-
plasm the enzyme or enzymes must be present there in an inactive condition.
The vacuolar endopeptidase activity persists for up to 12 hours and then gradually
declines. As this occurs, a positive reaction to the Burstone & Folk technique for exo-
peptidases of the LAP type appears, first in the deeper vacuoles and then eventually
throughout all those present (Plate 10)). This persists until intracellular digestion is com-
pleted and all food vacuoles have disappeared.
The replacement of endopeptidases by exopeptidases in the vacuoles is accompanied by
a change-over from acid to alkaline phosphatase activity in the cytoplasm. Alkaline phos-
phatase also appears at this time in the vacuolar contents, and in parenchyma cells im-
mediately below the gastrodermis. At this latter site it is concerned, presumably, in the
movement of the products of digestion from the gastrodermal cells into the rest of the
body.
During these later stages of intracellular digestion occasional vacuoles show positive
reactions to the Gomori Tween 80 method for lipase, especially after a meal of insect
larvae or crustaceans which had contained oil or fat globules amongst their body tissues.
6 J. B. JENNINGS

Development of the reaction only at this late stage confirms the endogenous origin of the
vacuolar lipase.
Direct observation of starved specimens fed crustacean muscle soaked in indicators
showed that the pH value of the food drops rapidly after ingestion and reaches a minimum
of 4·0 within 30 minutes. The source of the acid secretions bringing this about has not been
detected. As extracellular digestion progressed the colour of the indicator became more
diffuse but occasionally sufficient was present in phagocytosed material to be visible in
vacuoles seen in neutral saline squashes of the entire worm. This showed that the intra-
cellular endopeptidase activity, like that in the gut lumen, was carried on in acidic condi-
tions at pH 4'0-5·0. Insufficient colour persisted for detection of the pH conditions of
subsequent stages of intracellular digestion, but since the optimum pH values for histo-
chemical demonstration of the exopeptidases, lipase and alkaline phosphatase concerned
were 6·8, 7·2 and 8·5 respectively it is concluded that the later stages of digestion proceed
under alkaline conditions, as in most other animals.

Enzymes in the vesicula resorbiens

A characteristic feature of the temnocephalid reproductive system is the presence of a
relatively large vesicula resorbiens, or seminal bursa, which generally contains degenerat-
ing spermatozoa and yolk globules. In T. brenesi this structure is embedded within the
gastrodermis (Plate I(h)), and in T. novae-zealandiae it is pressed closely against the base-
ment membrane.
In each species the walls of both the vesicula resorbiens and the duct connecting it to
the rest of the reproductive system show at all times, in mature individuals, an intense
positive reaction for acid phosphatase and exopeptidases of the LAP type (Plate I(h)).
The contents of the vesicle, in contrast, show only slight positive reactions for these
enzymes and these would appear to be due to the intrinsic enzymes of the spermatozoa
present, since similar reactions were observed in the contents of the seminal and prostatic
vesicles.
The parenchyma surrounding the vesicula resorbiens and its duct, in T. novae-zealandiae,
and around the duct in T. brenesi, showed similar positive reactions for acid phosphatase
and LAP.

The food reserves

Neither T. brenesi nor T. novae-zealandiae form any significant reserves of fat or glycogen
in the parenchyma or gastrodermis, which are the usual sites of food storage in the Tur-
bellaria. Considerable amounts of fat occur in the vitelline glands, however, as droplets
1-1'5 I-'- in diameter, and eggs and embryos in the uterus similarly contain relatively large
amounts. The vitelline glands also show a positive reaction to the Tween 80 method for
lipase, and frozen sections show this to be localized around the fat droplets.
In both species large amounts of glycogen occur in the testes between the spermatocytes,
and this site shows intense acid phosphatase activity. Glycogen is also present in the mus-
culature of the pharynx and sucker, and a few scattered granules occur in the parenchyma
cells immediately adjacent to the vesicula resorbiens and its duct.
 FEEDING AND DIGESTION IN TEMNOCEPHALID FLATWORMS 7

The amounts of fat and glycogen present at the various sites diminished slowly with
starvation, but even after three weeks without food both substances were still present in
appreciable amounts.

Discussion
Although the two temnocephalid species studied come from widely separated geographic
localities they occupy the same type of microhabitat, on the gills of freshwater crustaceans,
and are very similar in both structure and general nutritional physiology.
The diet and feeding mechanisms are the same as in free-living rhabdocoels, and the
gastrodermis is of the typical turbellarian form, being differentiated into glandular and
phagocytic components as in other rhabdocoels, triclads and acotylean polyclads (Jennings,
1962b, 1968). A number of the gland cells, however, are subgastrodermal and the
functional significance of this is not clear, unless it is to allow the maximum number of
cells actually lining the gut and in contact with the food to be phagocytic.
The site and sequence of digestive processes, in both T. brenesi and T. novae-zealandiae
are also typically turbellarian, in that digestion occurs both extra- and intracellularly, in
acidic and alkaline phases, and endopeptidases, exopeptidases, lipases, and acid and
alkaline phosphatases are involved.
It would appear, therefore, that the ectocommensal habit has not caused any significant
departure from the general pattern of nutrition characteristic of the free-living flatworms.
This contrasts somewhat with the situation in the entocommensal rhabdocoel Syndesmis
jranciscana, where the diet includes a proportion of host tissue, in the form of coelomo-
cytes, and where considerable amounts of glycogen are stored, as in entoparasitic
organisms (Jennings & Mettrick, 1968). It is possible, then, that the entocommensal
habit, rather than the ectocommensal, is more likely to lead to adoption of a parasitic
mode of life. Certainly the temnocephalids show no resemblances in diet, feeding mechan-
isms, gut structure, digestive physiology or food reserves to monogenetic trematodes,
many of which occupy a similar microhabitat on fish gills but whose nutrition is consider-
ably modified as an adaptation to, or consequence of, a wholly ectoparasitic life (Halton
& Jennings, 1965; Halton, 1967).
The temnocephalid vesicula resorbiens, or seminal bursa, has long been considered to
be the site of degradation and absorption of excess sexual products (Hyman, 1951) and
the consistent presence of exopeptidases and acid phosphatase in the walls of the vesicle
and its duct would appear to confirm this. Since the contents of the vesicles show only
slight reactions for these enzymes, however, it seems likely that degeneration of yolk
globules and spermatozoa, often visible histologically, is due to gradual autolysis. This is
presumably followed by absorption of soluble substances which are then digested by the
enzymes present in the walls of the vesicle, its duct, and the surrounding parenchyma
cells. The source of the enzymes is unknown, but in view of the close association of the
vesicula resorbiens with the gastrodermis they may well originate in the gastrodermal
phagocytes and simply diffuse to the sites where they are to act.
The egg-laying activities of T. brenesi are of interest in that they create conditions suit-
able for growth of the small organisms upon which the newly hatched individuals feed.
This is probably of considerable importance since such food may be difficult to obtain,
otherwise, at the distal ends of the host gills, and may permit fairly rapid growth of the
8 1. B. JENN1NGS

young flatworms. A similar phenomenon has been observed on the gills of the crayfish
Atyaephyra desmaresti (Millet) from Yugoslavia, where the feeding activities of Branchi-
obdella hexadonta Gruber (Annelida: Branchiobdellida) cause lesions around which
diatoms and protozoa collect, and these organisms are frequently found amongst the
branchiobdellid's gut contents (S. R. Gelder, pers. comm.).

This work was supported by a travel grant from the Royal Society and Research Grant Al
06295-01 of the United States Public Health Service. My grateful thanks are also due to Mr P. S.
Dale, Department of Zoology, Massey University, for supplying living T. novae-zealandiae, and
to Dr Rodrigo R. Brenes, Departamento de Parasitologia, Facultad de Microbiologia, Universi-
dad de Costa Rica, for generous provision of laboratory facilities and invaluable assistance in the
field.

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