Zoological Journal of the Linnean Society, 63: 75-97. With 4 plates and 4 figures
MaylJune 1978
Digestion in pycnogonids: a study of some
polar forms
PETER R. RICHARDS
Department of Mechanical Engineering,
University of Surrey, Guildford, Surrey
AND
WILLIAM G . FRY
Department of Science, Luton College,
Park Square, Luton, Bedfordshire
The process of digestion was studied in some polar pycnogonids, mainly the Antarctic forms
Nymphon australe and Nymphon orcadense. Our findings did not support the theory of the
digestive process proposed by Schlottke (1933) and this led to a re-examination of his ideas. We
propose a new scheme for digestion in pycnogonids and fit Schlottke’s observations into this
framework. I t is suggested that Antarctic pycnogonids may possess behavioural adaptations
peculiar to their supply of food. Some directions for further study are indicated.
KEY WORDS : Pycnogonida -polar species -digestion -pinocytosis-microvilli
endogenous fragmentation-phagosomes-inclusion granules-starvation.
-enzymes
-
CONTENTS
Morphology of the alimentary system . . . . . .
The proboscis . . . . . . . . . . . .
Midgut and hindgut. . . . . . . . . .
The digestive process . . . . . . . . . . . .
Introduction
The scheme for digestion proposed by Schlotrke
Wandering cells, mass transport and reconstruction.
Method of food uptake by cells . . . . . .
The midgut cells. . . . . . . . . . .
Enzymes of the midgut cells . . . . . . .
Proteases, carbohydrases and nucleases
Acid phosphatase
Alkaline phosphatase
Transmission electron microscope studies
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0024-4082/78/006 3-oO75/!$02.OO/0
@ 1978 The Linnean Society of London
P. R. RlCHARDS AND W. G . FRY
76
Proposed scheme for digestion in pycnogonids .
A new interpretation of Schlortke’s scheme
Discussion . . . . . . . . . . . .
Acknowledgements. . . . . . . . . .
References . . . . . . . . . . . .
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MORPHOLOGY OF THE ALIMENTARY SYSTEM
The digestive tract of pycnogonids is divisible into three distinct regions.
A foregut, which extends through the proboscis, a hindgut which extends
through the reduced abdomen and a midgut which occupies most of the rest
of the animal in between.
Probably the most peculiar morphological feature of the pycnogonid digestive system is that diverticula (caeca) of the midgut extend into the walking
legs beyond the leg bases. Table 1 lists species and the segment of the leg into
which their diverticula are reported to extend. There are insufficient data to
be able to attach any taxonomic significance to the variations. In addition to
the midgut extending into the walking legs, those species which possess chelicerae often have diverticula extending into these. Again, the extent of penetration varies with species, sometimes it is as far as the immoveable finger (e.g.
Nyrnphon australe). Yet other species have diverticula extending forwards into
the proboscis to lie alongside the foregut (see e.g. Fry, 1965). There are no
reports of the midgut penetrating into the ovigers. Figure 1 shows the
distribution of the digestive system in the Antarctic pycnogonid N . australe.
Table 1. Distribution and extent of midgut caeca in some pycnogonids
Caeca extending forwards into the proboscis:
Colossendeir spp.
Phoxichilus spp. (= Endeis)
Nymphon brachyrhynchum
Nymphon gracile
Phoxichilidium femoratum
Endeis spinosa
Extending into the second walking leg segment:
Rh yn rho th orax mediterraneus
Extending into the fourth segment of the walking legs:
Pantopipe t f a spp.
Extending into the sixth segment of the walking legs:
Nymphopsis
Phoxichilidium femoratum
Pycnogonum littorale
Extending to the end of the propodus:
Pallenopsis vanhoffeni
N ~ * m p h o hirtipes
r:
Acheliu echinata
Nymph on o rcade nse
Endeis spinosa
Nymphon rubrum
Ammothea carolinensis
Nymph on aitstrale
Nymphon gracile
Colossendeis proboscidea
Colossendeis wilsoni
Decolopoda austrulis
The proboscis
The external shape of this organ is generally described as “cylindrical” or
“ovate” but this term has to cover a multitude of configurations. Fry & Hedg-
PYCNOGONID DIGESTION
77
A
Figure 1. Dorsal (A) and lateral (B) views of the distribution of the midgut diverticula in
Nymphon australe. o.c., Ocular tubercle; p, proboscis; a, abdomen; e, cut ends of walking
legs.
peth (1969: 20-21), therefore, defined several types of proboscis shape for
taxonomic purposes.
The proboscis extends longitudinally from the mouth opening at its distal
end (tip) to a ring of flexible cuticle (arthrodial membrane) where it joins the
cephalic somite of the trunk. In transverse section the proboscis is tri-radial,
consisting of a single dorsal antimere and paired ventro-lateral antimeres.
Sanchez (1959) found that embryologically the dorsal antimere was an outgrowth of the cephdic somite whilst the two ventro-lateral antimeres arose
from a slightly posterior region. Hedgpeth (1954) has summarized and criti-
78
P. R. RICHARDS A N D W. G. FRY
cally discussed the arthropod/pycnogonid segmental homologies adduced by
earlier workers.
The result of the tri-radial symmetry is that the mouth is Y shaped, becoming a triangular shaped aperture when opened (Snodgrass, 1952). The mouth
leads directly into the cavity of a large sac which occupies about two thirds of
the length of the proboscis. This sac has a triangular to circular cross-section
depending upon its dilatation. Most authors (e.g. Helfer & Schlottke, 1935;
Sanchez, 1959; King, 1973) have called this sac a “pharynx”. D’Arcy Thompson (1909) called it the “oesophageal cavity”. The pharynx leads into the socalled “oesophagus”. This is a narrow tube with a Y cross-section to its lumen
when constricted and a triangular to circular one when expanded. In the
anterior region of the oesophagus are the bases of numerous setae, which
protrude forwards into the proximal lumen of the pharynx and form what
has been termed an “oyster basket” sieve (Schlottke, 1933). The pharynx
and oesophagus together form the foregut and are lined with cuticle as far as
the most posterior setal origins (see Dencker, 1974 for an excellent threedimensional cutaway diagram of proboscis structure). Amongst recent authors,
Fry (1965) and Wyer (1972) have discussed the functional morphology of
the proboscis and foregut.
Midgut and hindgut
The oesophagus extends into the cephalic somite and opens via a tripartite
valve into the midgut. In N . orcadense and N . australe this junction of foregut
with midgut is located in a region of the somite just posterior to the ocular
tubercle. The midgut then extends (with the diverticula already noted) through
the rest of the trunk and opens via another tripartite valve into the hindgut.
This latter valve is similar in gross cross-section to that between foregut and
midgut but differs from it in cellular detail. The hindgut opens to the exterior
via an anus.
THE DIGESTIVE PROCESS
Introduction
An investigation of the literature of feeding and digestion in pycnogonids
reveals that most work has been concerned with feeding, and comparatively
little with digestion. Helfer & Schlottke (1935), Crapp (1968), King (1973)
and Professor Stock (this Volume, p. 59) have listed possible food substrates
of pycnogonids. We have found N . australe and N. orcadense, which seem to
be general detritus feeders, additionally feeding on Crustacea (dead amphipods) and Nemerteans (Tetrastema validum). I t would seem that pycnogonid
tastes are catholic.
The most recent published work concerned solely with the mechanism of
digestion is that of Schlottke (1933), which also summarizes all work to that
dare. Since then, Sanchez (1959) has examined the structure of the digestive
system in certain embryos and Wyer (1972) has investigated digestion in certain
British species. Helfer & Schlottke’s compendium (1935) contains a section on
digestion which is basically Schlottke’s paper of 1933 modified for review
purposes. Fage (1949) and Nicol (1967) have written reviews which are not
reports of research by their authors.
PYCNOGONID DIGESTION
79
The scheme for digestion proposed by Schlottke
Though it might be thought that a forty-year old scheme could be dismissed
without consideration, this action is avoided for a number of reasons:
(a) The scheme has become deeply entrenched in the literature, particularly in general texts.
(b) The scheme has aspects which improved techniques do not confirm.
(c) It brings together most previous work to that date.
(d) The new scheme (presented below) reinterprets Schlottke’s findings.
(e) The original (German) paper is not readily available to the English
reader.
Schlottke’s scheme (summarized in Fig. 2) is as follows:
The proboscis acts as a scraping or piercing sucking organ and food which
enters the pharynx, after maceration and filtration by the setae, passes into the
midgut via the tripartite valve. No cellular structure is visible in the food pulp
entering the midgut. Peristalsis moves the pulp along the trunk and into the
limb diverticula.
The midgut epithelium consists of three types of cell. From lumen towards
basement membrane these are: Absorption cells (Resorptionzellen), Gland
cells (Drusenzellen), and Embryo cells (Embryozellen). Digestion is intracellular, but in addition, the gland cells discharge their acidophilic globules
(containing enzymes) into the lumen at the start of food uptake. As a result of
ingestion, the midgut lumen expands, the epithelium is stretched and basally
situated young absorption cells come into contact with the food. These bulge
Excretion
Figure 2. Main events in the pycnogonid midgut during digestion according to the scheme
proposed by Schlottke (1933). Broken lines indicate cell movements. See text for discussion.
5
80
P. R . RlCHARDS AND W. G . F R Y
into the lumen, growing considerably in length and width as food is taken into
their small distal vacuoles. Some 1% hours after feeding the number of vacuoles
has increased and they seem to be surrounded by mitochondrion-like granules.
The vacuole contents precipitate and are deposited around the vacuole wall.
At this stage the contents are basophilic. Basophilia gradually extends into the
interior of the vacuole and its contents then become distinctly acidophilic. In
the final stage, vacuoles become compacted and weakly basophilic.
One day after feeding, the whole epithelium is seen to be crammed with
distinct granules, the larger ones being of protein, the smaller ones of fat. Four
to eight days after feeding, gland cells are seen to be in an enlarged state and
completely surrounded by absorption cells, so that no part of any gland cell
borders the gut lumen. After a period of starvation, vacuoles in all stages of
digestion still exist in the epithelial cells.
Concomitant with these intracellular changes, changes in the general structure of the epithelium occur. The height of the epithelium varies with the
region of the midgut and nutritional state. I t is found that after food ingestion,
a considerable increase in the number of embryonic cells takes place by some
form of amitotic division. About one day after feeding the absorption cells
start to detach themselves from the epithelium. These cells may either detach
singly, or, after being formed into villi, break off as clumps of cells. Digestion
continues within them as they circulate in the gut, eventually re-attaching t o
the epithelium (never touching the basement membrane) and yielding up their
digestive products. Those epidermal cells which contain only wast products or
little useful material have less tendency to re-attach and eventually leave in the
faeces. Schlottke described these cells as divided into two regions; a grey,
unstructured region rich in protein globules, which are initially contained in
vacuoles. The other region consists of lighter staining, refractile vacuoles
containing chromatin. The two regions, he claimed, show that digestion of
protein and nucleoprotein are separated from each other within the phagocytic
cell.
Wandering cells, mass transport and reconstruction
A peculiar feature of the above scheme is the process by which the absorption cells detach themselves from the epithelium, circulate in the lumen (whilst
digestion continues within them) and eventually re-attach to the epithelium.
Schlottke claimed this to be similar to the system he thought existed in the
endoderm of Hydra and cited McConnell (19311, who called the process
“endogenous fragmentation ”. Schlottke claimed his findings for Hydra to be
similar to those of McConnell, who believed that the diploblastic nature of this
animal and its consequent lack of mesodermal tissue from which to elaborate
transport structures necessitated such a mechanism. His views of the problem
of the diploblastic state are contradicted by Hyman (1940) and Pantin (1960)
and were proposed with no knowledge of mass transport in Coelenterates. I t
is only as recently as 1972 that Chapman & Pardy have been able to apply
Fick’s law to nutrient transport in a hydroid (and then not Hydra). McConnell’s endogenous fragmentation scheme does not appear in the recent coelenterate digestion literature known to the authors. Campbell (1967) found that
Hydra’s cells move as sheets within the epithelium, rather than individually.
PYCNOGONID DIGESTION
81
Schlottke (193 3 : 652) presented the following case: “During food intake
its uniform distribution is maintained by peristalsis which continues until the
food is absorbed. During starvation periods, an unequal use of nutrients by
different organs occurs and its even distribution is lost. To rectify this, as in
Hydra, absorbed foodstuffs are transported via the endoderm to where they
are needed”. We have found that gut peristalsis continues even under starvation conditions. As indicated, only a little is known of mass transport in
Hydra, but it would appear that a considerable knowledge of this subject and
of mass transport in pycnogonids would be required t o verify Schlottke’s
hypothesis.
In our study of some 15,000 sections of pycnogonids in all states of nutrition, free cells in the midgut lumen were found only very rarely. Most of the
sections were serial and to study them, three dimensional reconstruction
techniques similar to those described by Jordan & Saunders (1976) were used.
A
+I
C
Figure 3. Some results of sectioning a gut model constructed from silicone rubber and with
lumen filed with Plasticene for support. The villi (v) were all of the same size, randomly
positioned and orientated radially. A, Cut-away illustration of the gut model; B, outline tracing
of a section cut from A in the plane of the vertical broken line (= vertical longitudinal). Note
the false “wandering cells”. C, Outline tracing of a section of a cut at 45” to the long axis of the
tube. Note false “wandering cells” and changes of thickness of layering (T).
82
P. R . RICHARDS A N D W. G. F R Y
Schlottke’s classic paper contains no photomicrographs and his figures show a
few cells, not complete sections. This makes it impossible to ascertain precisely
the orientation of his sections.
With our own material we found that, unless sections were orientated
exactly, misleading interpretations could result. Thus, oblique sections may
show isolated cells in the lumen. If a series of sections has been made, however, these free cells usually prove to be the distal ends of cells which remain
part of the epithelium, but which protrude into the lumen to a greater degree
than their fellows. Without access to Schlottke’s material it is suggested that
misinterpretation could have arisen if oblique sections were studied singly,
rather than in a series.
By the same model it is found that section orientation must be exact to
enable comparisons of epithelium thickness between different nutritional
states. We found no easily detectable difference in thickness with varying states
of nutrition.
Similarly, the model can be used to show that different section orientations
will give different cell positions. Thus, whilst Schlottke found gland cells
always nearer the basement membrane than absorption cells, we found no
such constancy. Figure 3 illustrates the foregoing discussion.
Method of food uptake by cells
Early authors claimed that food particles enter the cells of the midgut by
phagocytosis. An examination of their evidence reveals, however, that their
case was weak. The most important evidence was that of Dogie1 (1913), who
observed diatoms in the midgut cells of protonymphon larvae in which the
“oyster basket’’ filter was undeveloped. He claimed that such diatoms must
have been taken up phagocytically. The only other evidence seems to be that
of Schlottke who presented it as follows: “Breakdown of a substance for
intake is important. Proteins must be utilized as amino-acids, poiysaccharides
as monosaccharides . . . These are not present in the gut lumen, therefore
proteins and polysaccharides must be broken down in the cells . . . thus phagocytosis must occur!” Schlottke used histological techniques which would not
favour the retention of small molecules in the lumen during processing; he did
not use stains specific for amino-acids or monosaccharides.
Demonstration of phagocytic uptake in the present work has proved difficult. Pycnogonids seem to prevent unwanted matter entering their midguts by
keeping the foregut/midgut valve firmly closed. Attempts to introduce traceable particles or dyes through the mouth invariably met with failure. In vitro
studies of particle uptake in isolated midgut cells showed no apparent uptake.
I t was found, however, that such cells are able to ingest highly dispersed basic
(cationic) dyes (e.g. Neutral red, Azure B and Acridine orange) and initially
concentrate these in vacuoles. Koenig (1962, 1965) found that such vital dyes
stain lysosomes. We found that there is no uptake of anionic vital dyes. Experiments on the uptake of fluorescence labelled proteins and horseradish peroxidase/acid phosphatase incubations to identify phagosomes (Straus, 1967)
were unsuccessful, but were limited by lack of material and so are inconclusive.
Although brush borders do not occur, Hetherington (pers. comm.) has
found microvilli in transmission electron micrographs of the gut epithelium/
PYCNOGONID DIGESTION
83
lumen interface. Scanning electron micrographs (Plate 1) confirm the lack of
brush borders and show such microvilli. Ruffle membranes, which might be
associated with phagocytosis, were not found.
It is suggested that uptake into the midgut cells is not by phagocytosis, but
by “pinocytosis”. This process, though discovered by Lewis in 1931, was
apparently ignored for many years (Oberling, 1959). It appears highly unlikely
that Schlottke was aware of it when he performed his researches, i.e. before
1933. The uptake of cationic dyes, such as we have shown to occur, is thought
to be characteristic of pinocytosis. The uptake of anionic colloidal dyes, which
we have not been able to demonstrate, is characteristic of phagocytosis.
As previously mentioned, the pulp of food in the lumen contains no recognizable cellular structures. It would appear that the symmetry of the proboscis
and the oyster basket apparatus form a very efficient crushing and sieving
apparatus. A similar tripartite symmetry is found in the proboscis of nematodes (see Albertson & Thomson, 1976) and in this phylum the symmetry
supports a mechanism which is quite efficient in crushing bacteria. It is suggested that the production of particles small enough to be pinocytosed in the
pycnogonid midgut is no problem. In addition, gut peristalsis, a very slight
amount of enzyme secretion into the midgut and a long digestion period (see
below) would aid cellular disintegration.
The midgut cells
As indicated above, Schlottke (1933) reported three types of cell in the
midgut. Sanchez (1959) found that in protonymphon larvae only two types of
midgut cell could be distinguished. These she described as (a) “digestive cells”
and (b) much less numerous “secretory cells”. Dohrn (1881) also found only
two cell types. We have found it difficult to distinguish between the glandular
and the absorptive cells described by Schlottke and, like Sanchez and Dohrn,
subscribe to the theory of an epithelium composed of two cell types. The
following classification of the cell types found in N. orcadense and N . australe
is based on their morphology and does not presume function:
(i) Small non-vacuolated cells with a distinct nucleus. These are situated
near, and sometimes in contact with, the basement membrane. They are
approximately ovoid in shape, typical dimensions being 10 to 12 pm along
the longer axis by 5 to 6 pm along the shorter axis. The nucleus stains obviously and is about 3 pm in diameter. The cytoplasm has a tendency to take
up nuclear stains, but always less intensely than the nucleus; it therefore has a
tendency towards basophilia. These cells never border the gut lumen. I t is
thought that they correspond to the “embryo” cells of Schlottke’s terminology and the “secretory cells” of Sanchez’.
(ii) Large vacuolated cells, which in some places border the gut lumen.
These cells are of variable size, but are always several times the size of the cells
mentioned in (i). Nuclei are not obvious. There seem to be boundaries between
the cells which do not, therefore, form a syncytium. Frequently, the region
near the distal (luminal) border of the cell is granular and has small vacuoles
which take up cytoplasmic stain. Basally the vacuoles are much larger and do
not take up stain. The basal region has the appearance of miniature pancreatic
acini but this glandular morphology is not thought to indicate glandular func-
P. R. RICHARDS AND W. G. FRY
84
Plate 1. A , B. Scanning electron micrographs of the luminal face of the midgut epithelium. m,
microvilli. N . orcadense. C. Transmission electron micrograph of the rnidgut epithelium/lurnen
interface in N . hirtipes. 1, Lumen; m, microvilli. Photograph by permission of A. M. Hetherington.
PYCNOGONID DIGESTION
85
tion. Occasionally, these large cells are either solely granular or solely vacuolated.
From examination of several thousand sections, it is concluded that these
large cells are different stages of the same cell type. It is proposed that this
single type of cell constitutes both the gland and absorption cells of Schlottke’s
scheme (= the digestive cells of Sanchez’ scheme).
Schlottke (1933) stated that investigations of the gut wall show that cells
at the base of the epithelium have numerous intertwining branches. These
form a meshwork, whose fibres are fragile and resemble smooth muscle fibres.
He stated that in sections they are “not as clearly recognizable’’ as the musculoepithelial cells of Hydra or Ascaris. Dohrn (1881) thought that there were
muscle fibres on the outside of the gut in Pallene (= Callipallene) but Schlottke
considered this to be part of the leg musculature which Dohrn had misinterpreted. In our wax, methacrylate or frozen sections cut thicker than 4 pm
and treated with various histological stains (e.g. Mallory’s Triple Stain, Heidenhain’s iron haematoxylin, Masson’s trichrome), a picture of the gut wall was
built up which was not unlike that described by Schlottke. There appears to
be a fibrous meshwork near the basement membrane and it is not unreasonable
to interpret the cells in this region as musculo-epithelial.
In material prepared by glutaraldehyde fixation, osmium tetroxide post
fixation, Araldite embedding, sectioning at one or two microns on an ultramicrotome and viewed with phase-contrast or Nomarski interference optics,
quite a different picture emerged. The fibrous basal meshwork was seen to be
due to the complex interdigitation of cell bases. Muscle fibres were not observed within the cells. However, there was a fibrous, possibly muscular, region
within the basement membrane (Plates 2 and 3A). These fibres were not present in all regions of the midgut and their function requires further study.
In isolated cells in vitro (Plate 3B) the projections of the cell base reported
by Helfer & Schlottke (1935) have not been observed, most cells rounding
themselves off after a period. Whatever are the mechanisms that bring about
gut movement, it appears that in the Antarctic pycnogonids studied a musculoepithelial r6le is not applicable to midgut epithelial cells.
Enzymes of the midgut cells
In his 1933 paper, Schlottke noted a similarity in appearance and, he therefore assumed function, between globules in pycnogonid midgut gland cells
and those of the gland cells of the gastroderm of Hydra. However, as the function of these cells in Hydra (see e.g. Burnett, 1959; Lentz & Barrnett, 1961)
is held in doubt in more recent works, the functional analogy that Schlottke
drew appears to be an unlikely one.
Wyer ( 1972) examined acid phosphatase and leucine aminopeptidase activity
in British species by histochemical means. Leucine aminopeptidase was detected but not localized in the midgut cells. Acid phosphatase he found to be
localized in the basal region of midgut cells (no distinction was made as to the
cell type) and to be particulate in distribution.
In our investigation of Antarctic forms, acid phosphatase distribution was
investigated using the technique of Gomori (also used by Wyer, 1972), the
standard azodye coupling technique (both in Pearse, 1968) and the naphthol
86
P. R. RICHARDS AND W . G . FRY
Piate 2. A , B. N.orcadense. Two arrangements of fibres within the midgut epithelium basement
membrane. 2 &mlight microscope sections. H, Haemocoel; F, fibres;M, midgut epithelium.
AS phosphate method of Burstone (Pearse, 1968). Several types of fixative were
used and sections were prepared both as cryostat frozen sections and after
embedding in a cold-polymerized polyethylene glycol methyl methacrylate
medium (Richards, 1976). As will be discussed, our results differed from
those of Wyer.
An attempt was made to study protease activity by the substrate film tech-
PYCNOGONID DIGESTION
87
Plate 3. A. Transmission electron micrograph of the basement membrane region of midgut
epithelium in N. orcadense. F, Fibres; L.M., part of leg musculature. B. Isolated living midgut
cells in vitro. Projections of the cell bases are absent. N. orcadense.
nique of Fratello (1968) (see also Hasegawa & Hasegawa, 1977 for a difficulty
of this technique). Other substrate film techniques were used to investigate
amylase, RNA-ase and DNA-ase activity. Carbohydrase activity was investigated by using the technique of Evans (1956). Time and the limited amount of
fresh material available unfortunately prevented the application of other
biochemical techniques.
P. R. RICHARDS AND W. G. F R Y
88
Proteases, carbohydrases and nucleases
Results of the digestion of unexposed, processed colour film soaked in
buffer in the technique of Fratello are given in Table 2. From these it can be
seen that N. orcadense exhibits midgut proteolytic activity between pH 5 and
8.
There seems to be a subtle difference between the proteases found in the
midgut of N. orcadense and those found in another southern species,
Decolopodu uitstraiis. The available evidence indicates that the enzyme(s) of N .
ovcadensc is trypsin-like, whilst in Decofopodu australis there is evidence of two
enzymes or enzyme groups, one active at pH 5.5 to 6.5 and one at pH 7 .
However, further biochemical experiments are required to verify this.
Amylase, DNA-ase and RNA-ase substrate film tests gave unclear results.
This was considered to be due to difficulties of technique. Dauost (1965)
recommended consideration of centrifugation techniques for spreading substrate films and cited the paper of Koehler et al. (1963). Absence of amylase
is, however, in agreement with our findings when using the methods of Evans
(1956), by which no carbohydrases were found. Another explanation for the
negative results is that enzymes are not absent, but that they are at too low a
concentration to be detected. Support is given to this by the finding that,
with Fratello’s method, incubation at 37°C normally required from 4 to 8
hours to produce results, whereas Fratello has stated that only 30 minutes
incubation is required to achieve positive results with low protease concentrations in vertebrates. When incubated at 0°C (the environmental temperature of Antarctic pycnogonids) positives were not produced even after incubations of two weeks. Further support for low concentration is given by the
extended time that these polar forms take to digest their meals. Paradoxically, with the tests for acid phosphatase, positives were obtained after the
normal incubation periods for mammal tissues.
Table 2. Proteolytic activity of the pycnogonid midgut
PH
2
3
4
5
5.5
6
6.5
7
7.5
8
8.5
N. orcadense
Decolopoda australis
+
-
+++
+++
+++
+++
+++
+ ++
+++
-
9
Inhibited by
Ovomucoid
Formaldehyde
caw
Sodium cyanide
Soyabean extract
Formaldehyde
PYCNOGONID DIGESTION
89
Acid phosphatase
All the methods used indicated the presence of acid phosphatase in the large
cells. In animals fixed in the act of feeding, distribution within the large cells
was distal and cytoplasmic. No acid phosphatase was detected in the small cells,
As digestion proceeded, distribution in the large cells became vacuolar in addition to cytoplasmic. The “glandular” regions of these cells did not stain but
“enigmosome” granules (see below), contained within the vacuoles, did stain.
Acid phosphatase was not found in the foregut, or the hindgut epithelia, or in
the midgut lumen, except when contained in free enigmosomes.
Wyer (1972), working on some British species, used the Gomori method
with frozen sections for his acid phosphatase studies and found distribution to
be particulate and localized in the basal region of the midgut cells. Whilst we
agree with particulate localization when the Gomori technique is used, we
always found the main localization to be distal (lumenal).
With our technique we found that ice damage in frozen sections presented
difficulties (“histologically adequate, cytologically inadequate”, David &
Brown, 1967). Hence our use of several staining and embedding methods.
From the consistent results we have no doubt of the distal localization of
acid phosphatase.
Wyer also found acid phosphatase in the lumen of the pharynx and suggested, therefore that extra-oral digestion takes place in pycnogonids. Although we found acid phosphatase in the anterior lumen of the pharynx of
one specimen fixed as it was feeding, we cannot support Wyer’s conclusion.
Our specimen had been sectioned vertically longitudinally in the midline and
showed positive staining elsewhere only in the midgut epithelium. We conclude that, if extra-oral digestion had occurred, then acid phosphatase would
have been detected in the midgut lumen and the foregut epithelium. It is our
belief that acid phosphatase detected in the foregut probably came from the
Prey.
Alkaline phosp ha tase
Alkaline phosphatase was investigated using the Standard azo-dye coupling
method (Pearse, 1968) and frozen formaldehydeheawater fixed sections.
Results, although positive, were variable and indicated a change in concentration with nutritional state. Cytological localization tended to correspond with
that found for acid phosphatase, although at a slightly later stage in the digestive process. Once again the enigmosome granules were positive, and no staining
occurred in the foregut or hindgut epithelium.
Transmission electron microscope studies
For transmission electron microscopy, specimens were fixed in cacodylate
sucrose buffered glutaraldehyde, post-fixed in osmium tetroxide and embedded in Araldite after alcohol dehydration. Sections were cut on a LKB ultramicrotome, stained with uranyl acetate and lead citrate and viewed with a
Hitachi Hu 11ES electron microscope. There are two striking features of the
midgut prepared in this manner. First, cells appeared to have large empty
areas. Second was the occurrence of bodies which we originally termed “ enigmosomes” (see Plate 4). The appearance of the latter varied. Some consisted
90
P. R. RICHARDS AND W. G . FRY
Plate 4. Transmission electron micrographs of various “enigmosomes”. In B and C the enigmos o m e lie within membrane-boundedvacuoles. In A, one lies free in the cytoplasm.
PYCNOGONID DIGESTION
91
of concentric rings of electron opaque and electron transparent material; in
other cases the opaque and transparent regions were not arranged concentrically and their structure a peared more complicated. Enigmosomes were
usually found in membrane- ounded vacuoles, but could occur freely in the
cytoplasm itself. Similar bodies have been found in Arctic and British species
by Hetherington and by King respectively (pers. comrr..). After their initial
discovery, some time was spent searching the literature for reports of similar
structures. We now believe that such structures, with different names, have
been reported several times. Thus, Threadgold (1967) depicted “mineralized
granules from the Malpighian tubules of Rhodnius”. It seems likely that the
“formed bodies” found by Riegel (1966a, b) and the “crystallization nuclei”
reported by Wigglesworth & Salpeter (1962) are related structures. Recently
Coombs & George (in press) have reviewed “inclusion granules” in a variety
of marine invertebrates. The function of enigmosome-type particles is unknown although Coombs & George (in press) and Riegel (1972) believe that
they may play an important role in salt balance. We believe that in pycnogonids, enigmosomes are “residual bodies” formed from the phagosomal breakdown of food. They are not retained within cells but are released into the
lumen, their acid phosphatase and alkaline phosphatase contents contributing
to some slight extracellular digestion.*
E
PROPOSED SCHEME FOR DIGESTION IN PYCNOGONIDS
The proposed scheme for intracellular digestion in pycnogonids is as follows:
Uptake of nutrients is by micropinocytosis. This is concluded on morphological and vital staining evidence. Large particles of external (i.e. non pycnogonid) origin have not been observed in the midgut lumen of any of the animals
investigated, although a mush of very minute particles has been observed. In
sections, the distal borders of cells do not possess pseudopodia and appear
symmetrical. Scanning electron microscopy has not shown pseudopodia or
convincing “ruffle membranes” which would indicate phagocytosis. Vital
dyes taken up by the cells were basic (cationic) ones whose uptake is normally
associated with pinocytosis. Vital dyes whose uptake is associated with phagocytosis were not found in cells. Numerous attempts to induce phagocytotic
uptake were unsuccessful. Brush-borders were not observed in any region of the
gut.
Our observations indicate that pinocytotic vesicles are formed at the lumen
borders of midgut cells and gradually move in the direction of the basement
membrane. In the region of the lumen border primary lysosomes fuse with the
pinocytotic vesicles, which then become large secondary lysosomes. These are
the large distal vacuoles of the midgut cells. In movement towards the basement epithelium the vacuole contents become condensed, so that eventually
“residual bodies” are formed. The enigmosomes are a type of residual body.
* Since this article was accepted for publication we have discovered additional references to particles
which seem similar to those we have called enigmosomes. Becker et al. (1974) labelled electron
micrographs of what appear to be identical bodies as “type 111 granules”. Our more flamboyant name
cannot therefore, take precedence. Simkiss (pers. comm.) has noted the almost universal occurrence of
these granules in marine invertebrates and their large numbers in the midguts of some species, particularly
when digestion is nearing completion. We direct readers to his review article in addition to that of Coombs
e t a [ . The knowledge gained from Simkiss’ article does not alter our views on pycnogonid digestion.
92
P. R. RJCHARDS AND W. G. FRY
It seems likely that when residual bodies are formed, autophagy may also
occur in the region of the cell in which these are found (hence the cell’s vacuolar appearance, see Plate 4B, C). This would give rise to areas in the midgut
epithelium which are morphologically similar to the “gland cells” described
by earlier authors.
It is concluded, therefore, that the gland cells are incorrectly named and
are, in fact, regions of formation of autophagic vesicles and residual bodies
within absorption cells. I t is thought that two courses may be open to such
a region: either it may move distally and empty its contents into the lumen,
the rest of the cell remaining intact, or else the whole cell may become autophagic and break down into the lumen.
Enigmosomes are composed of excretory material, but they also contain
acid phosphatase and alkaline phosphatase. By introducing these enzymes into
the gut lumen, they contribute towards some extracellular digestion. The
release of residual bodies from the cell does not always occur in lysosomal
digestion and the occurrence of enigmosomes in the cytoplasm as well as in
vacuoles is in keeping with this.
In summary, the scheme proposed for intracellular digestion in pycnogonids is:
(a) Pinocytosis.
(b) Fusion of primary lysosomes with pinocytotic vesicles.
(c) Digestion of the contents of the secondary lysosomes formed in (b).
(d) Formation of residual bodies (enigmosomes or spherical bodies) and
autophagic vacuoles.
(e) Possible migration and release of residual bodies into the gut lumen.
This scheme is represented by Fig. 4.
A new interpretation of Schlottke s scheme
It is interesting tht it is possible to re-interpret most of Schlottke’s observations to fit the scheme proposed above:
When a pycnogonid fed, Schlottke noted large, heavily staining vacuoles
appearing in the midgut cells and after 1% hours these became surrounded by
mitochondrion-like granules. I t is suggested that these mitochondrion-like
granules are primary lysosomes.
Schlottke stated that “The contents of the vacuoles become basophil with
toluidine blue”. It has been noted that toluidine blue stains lysosomes vitally.
Again basophilia in the vacuoles could indicate an acid content, which would
be in agreement with the finding of acid phosphatase in vacuoles as well as in
the cytoplasm as digestion proceeds.
Schlottke reported that the gland cells become basal in the midgut epithelium as digestion progressed. If a reinterpretation of gland cells as autophagic regions of cells, or even autophagic whole cells, is accepted, then this
observation is in agreement with the formation of these regions by the basal
movement of vacuoles. He also reported that used gland cells are taken in
and broken down by absorption cells. I t is suggested that here he was observing
the movement of the autophagic regions of cells towards the lumen rather than
their uptake after “wandering” (for which we find no evidence). He noted that
at the start of the food uptake the gland cells discharge their weakly acidophilic
93
PYCNOGONID D I G E S T I O N
Food crushed
in foregut
,
I
'
Autophagy+
vacuolar digestion
of food particles
Interconvertible
Haernocoel
..
.
0
.
'
'
Glandular'
.
cells
'
,
'@
'. . .
@
.
Enigrnosome release
+
Phosphatase sqcretibn
Diffusion
into hoemolymph of products
of intracellular digestion
+
Transport throughout rest of body
Figure 4. T h e proposed scheme for intracellular digestion in pycnogonids. T h e diagram illustrates the m a i n events in the rnidgut.
globules into the lumen. This, as well as contradicting his earlier claim that
gland cells never border the lumen, suggests that his description of gland cell
resorption could be based on observation of the stage prior to the release of
autophagic and residual bodies (enigmosomes) into the lumen. Schlottke noted
the globules of gland cell to be weakly acidophilic. This supports our
observation that there appears t o be a dominance of alkaline phosphatase over
acid phosphatase in the enigmosomes when they are released into the lumen.
We can find no evidence for Schlottke's claim that proteins are digested within
the cytoplasm and nucleoproteins digested in the vacuoles of circulating cells.
As indicated previously, we have not been able to find such cells. Furthermore,
the sequestration that he claimed is contrary to current views on the lysosome
concept and the digestion of bodies within cells. We believe that Schlottke's
investigative techniques were not powerful enough to make the above claim.
Indeed, even with present day techniques, schemes for the formation and
packing of enzymes within cells are still the subject of controversy (see e.g.
Rothman, 1975 versus Palade, 1975).
DISCUSSION
Complete verification of the scheme for digestion presented in this paper
requires further work. A chance observation during the investigations indicates one potentially fruitful line of additional research.
This observation was of the ability of polar nymphons (N. hirtipes from
94
P. R. RICHARDS A N D W. G. FRY
the Arctic and N. orcadense and N. australe from the Antarctic) to survive
long periods (approximately 18 months in each case) without exhibiting
their normal feeding behaviour. The animals do not appear abnormally quiescent, but neither do they appear to feed. Research is needed to find if nutrient uptake has actually ceased and whether the animals compensate for this
cessation by changes in metabolic rate, or metabolic pathways, or resorption
of certain tissues (e.g. reproductive), or by a combination of these stratagems.
Observations of specimens undergoing this apparent starvation showed that
it took two months for their guts to empty completely.
A number of authors, most recently Wyer (1972), have commented on the
occurrence of unusual pits within the pycnogonid cuticle. Although Stephens
(1972) doubts that arthropods can take up nutrients through their cuticles,
the long, starving survival of pycnogonids, which have such a high surface to
volume ratio and cuticular pits of unknown function, indicates that cuticular
uptake experiments would not be misplaced.
A final possible explanation of the apparent non-feeding phenomenon is
concerned with the fact that pycnogonids are essentially filter feeders. Although they may tear off large lumps of prey with their chelicerae, the teeth
and setae of the pharynx so grind and filter the food that what enters the
midgut no longer resembles the prey and is very finely particulate. It is presumed that it is possible for an animal, which normally crushes and filters
food in its pharynx, to save the energy required in searching out prey, carrying it or tearing it and crushing it, by filtering directly from a medium which
is rich in particles. Specimens of Colossendeis proboscidea from the Arctic
were seen opening and closing their mouths goldfish-fashion. Hedgpeth ( 1954)
has drawn parallels between the larval proboscis of the archiannelid Protodrilus,
which is an apparatus highly specialized for catching micro-plankton, and the
proboscis of pycnogonids. The polychaete worm favoured as food by the
Antarctic Nymphon orcadense was not available for most of the austral summer of 1973/74 and, although animals were seen feeding on other prey, these
observations were less frequent than were feeding observations when polychaetes were available.
It is proposed that N . orcadense may feed during some of the year in predatory or necrophilic style (Arnaud, 1970), and during other parts of the year
by remaining inactive and filtering the medium. In support of this it is noted
that there are other Antarctic benthic invertebrates for which seasonal change
from predatory to filtering behaviour is recorded. Thus, the starfish Odontaster
validus (M. G . White, pers. comm.) spends part of the season aboral side uppermost feeding on bivalves and the rest of the season oral side uppermost, using
its tube feet in a “ciliary” fashion to waft suspended particles to its mouth. I t
would seem that conversion of N. orcadense, for example, from predator/
scavenger to filter feeder would require a much less drastic change of behaviour
than in Odontaster.
ACKNOWLEDGEMENTS
We wish to thank the following bodies for providing facilities during the
course of this work: the Science Department of Luton College, British Antarctic Survey, the Lowestoft Laboratory of the Ministry of Agriculture, Food
PYCNOGONID DIGESTION
95
and Fisheries, the Zoology Department of Oxford University and the Structural Studies Unit of Surrey University. The individuals who provided help
and kindness during this work are too numerous to mention. We apologize
to them for the omission of their names and hope that this will not be taken
as a sign of ungratefulness. P.R.R. was in receipt of a Bedfordshire County
Council Postgraduate Studentship, W.G.F. was in receipt of Scientific Investigations Grants from the Royal Society.
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