An examination of the role of the digestive gland of two loliginid squids,with respect to lipid: storage or excretion?

The role of the digestive gland, with respect to non-structural lipid, was examined using proximal analysis, histochemistry and quantitative histological techniques in the tropical loliginid squids Sepioteuthis lessoniana (Lesson) and Photololigo sp. The digestive gland of both species was characterized by large and numerous lipid droplets in the apical portion of the digestive cells and very few in the basal portion. The apical lipid droplets were released into the lumen of the gland and subsequently rapidly removed. Despite the numerous large apical lipid droplets, the lipid concentration in the digestive glands of S. lessoniana and Photololigo sp. was lower than that reported for most squid species. There was no relationship between lipid concentration and stage of digestion, suggesting that lipid is not stored in the gland after a meal. There was also no relationship between lipid concentration and the sex of an individual or stage of reproductive maturity, suggesting that these squids are not storing lipid in the digestive gland for use in fuelling reproductive maturation or providing an energy source for oocytes. I believe this study is the first to combine proximal analysis and quantitative histological techniques to examine the role of the squid digestive gland with respect to non-structural lipids. The results indicate that the digestive gland of these tropical loliginid squids is excreting, not storing, excess dietary lipid.     

Fine structure and absorption of ferritin in the digestive organs of Loligo vulgaris and L. Forbesi (Cephalopoda,Teuthoidea)

The digestive organs possibly involved in food absorption in Loligo vulgaris and L. forbesi are the caecum, the intestine, the digestive gland, and the digestive duct appendages. The histology and the fine structure showed that the ciliated organ, the caecal sac, and the intestine are lined with a ciliated epithelium. The ciliary rootlets are particularly well developed in the ciliated organ, apparently in relation to its function of particle collection. Mucous cells are present in the ciliated organ and the intestine. Histologically, the digestive gland appears rather different from that of other cephalopods. However, the fine structure of individual types of squid digestive cell is actually similar to that of comparable organs in other species, and the squid cells undergo the same stages of activity. Digestive cells have a brush border of microvilli, and numerous vacuoles, which sometimes contain “brown bodies.” However, no “boules” (conspicuous protein inclusions of digestive cells in other species) could be identified in their cytoplasm; instead only secretory granules are present. In the digestive duct appendages, numerous membrane infoldings associated with mitochondria are characteristic features of the epithelial cells in all cephalopods. Two unusual features were observed in Loligo: first, the large size of the lipid inclusions in the digestive gland, in the caecal sac, and in the digestive duct appendages; and second, the large number of conspicuous mitochondria with well-developed tubular cristae. When injected into the caecal sac, ferritin molecules can reach the digestive gland and the digestive duct appendages via the digestive ducts, and they are taken up by endocytosis in the digestive cells. Thus, it appears that the digestive gland of Loligo can act as an absorptive organ as it does in other cephalopods.     

Absorption of ferritin by cells of the digestive gland in Nassarius tegula (Gastropoda : Prosobranchia)

Within 12 min after the prosobranch snail, Nassarius, begins feeding on ferritin-labeled food, ferritin reaches the lumen of the digestive gland and is absorbed by the digestive cells lining the gland. Within the digestive cells, the ferritin is present in coated pinocytotic vesicles, in microvesicles and in macrovesicles. It is probable that ferritin (and components of the food as well) progresses rapidly in order from the gland lumen to the pinocytotic vesicles to the microvesicles to the macrovesicles. The macro vesicles are presumably an important site of intracellular digestion.     

Extracellular lipid droplets in Idiosepius notoides,the southern pygmy squid

Cephalopod metabolism typically involves carbohydrates and proteins, so that the lipid content of the mantle and all internal organs except the digestive gland is very low. Despite clear evidence of nonlipoid metabolic trends in cephalopods, we observed extracellular spheres, or droplets, in the cecum and digestive gland of newly collected juvenile, male, and female individuals of Idiosepius notoides, the southern pygmy squid. Prior to staining, the droplets were various shades of yellow and were often large enough to detect at 7 x magnification. The droplets were less dense than water, hydrophobic, and sudanophilic, staining positively with Sudan III, Sudan IV, and Sudan Black B. We conclude that these spheres are lipid and that they derive from the squid's normal field diet. When newly collected squid were starved in the laboratory, the droplets disappeared in 7-8 d and then reappeared in the cecum about 3 h after feeding.     

Characterization of the Bacterial Diversity in Indo-West Pacific Loliginid and Sepiolid Squid Light Organs

Loliginid and sepiolid squid light organs are known to host a variety of bacterial species from the family Vibrionaceae, yet little is known about the species diversity and characteristics among different host squids. Here we present a broad-ranging molecular and physiological analysis of the bacteria colonizing light organs in loliginid and sepiolid squids from various field locations of the Indo-West Pacific (Australia and Thailand). Our PCR-RFLP analysis, physiological characterization, carbon utilization profiling, and electron microscopy data indicate that loliginid squid in the Indo-West Pacific carry a consortium of bacterial species from the families Vibrionaceae and Photobacteriaceae. This research also confirms our previous report of the presence of Vibrio harveyi as a member of the bacterial population colonizing light organs in loliginid squid. pyrH sequence data were used to confirm isolate identity, and indicates that Vibrio and Photobacterium comprise most of the light organ colonizers of squids from Australia, confirming previous reports for Australian loliginid and sepiolid squids. In addition, combined phylogenetic analysis of PCR-RFLP and 16S rDNA data from Australian and Thai isolates associated both Photobacterium and Vibrio clades with both loliginid and sepiolid strains, providing support that geographical origin does not correlate with their relatedness. These results indicate that both loliginid and sepiolid squids demonstrate symbiont specificity (Vibrionaceae), but their distribution is more likely due to environmental factors that are present during the infection process. This study adds significantly to the growing evidence for complex and dynamic associations in nature and highlights the importance of exploring symbiotic relationships in which non-virulent strains of pathogenic Vibrio species could establish associations with marine invertebrates.     

The digestive gland of the Southern Dumpling Squid (Euprymna tasmanica): structure and function

The cephalopod digestive gland plays an important role in the efficient assimilation of nutrients and therefore the fast growth of the animal. The histological and enzymatic structure of Euprymna tasmanica was studied and used in this experiment to determine the dynamics of the gland in response to feeding. The major roles of the digestive gland were secretion of digestive enzymes in spherical inclusions (boules) and excretion of metabolic wastes in brown body vacuoles. High levels of trypsin, chymotrypsin and α-amylase, low levels of α-glucosidase and negligible carboxypeptidase activity were produced by the gland. There was no evidence of secretion of digestive enzymes in other organs of the digestive tract. Within 60 min of a feeding event, the gland produced increasing numbers of boules to replace those lost from the stomach during the feeding event. Initially, small boules were seen in the digestive cells, they increased in size until they are released into the lumen of the gland where they are transported to the stomach. There was no evidence of an increase in activity of digestive enzymes following a feeding event, despite structural changes in the gland. However, there was large variation among individuals in the level of digestive enzyme activity. A negative correlation between boule and brown body vacuole density suggested that the large variation in enzyme activity may be due to the digestive gland alternating between enzyme production and excretion.     

Multiple sources of esterase enzymes in the crop juice ofCepaea (Mollusca: Helicidae)

Summary Previous workers have (a) compared pulmonate crop juice and digestive gland extracts and found a close similarity in the enzymic complements from these two sources, and (b) located specific enzymes within the various cell types of the digestive gland. The digestive gland seems to be the major source of extracellular enzymes but what is not clear is which of the enzymes associated with particular intracellular structures are actively secreted into the crop juice. The present study has used polyacrylamide disc gel electrophoresis to investigate the digestive gland and crop juice esterases ofCepaea nemoralis andC. hortensis. It appears that only some of the digestive gland esterases are specifically secreted. The variation shown in crop juice esterases suggests three independent sources in the digestive gland. Less detailed studies ofHelix aspersa andArianta arbustorum also indicate multiple sources of extracellular esterases.     

Predatory strategies and behaviours in cephalopods are altered by elevated CO2

There is increasing evidence that projected near‐future carbon dioxide (CO₂) levels can alter predator avoidance behaviour in marine invertebrates, yet little is known about the possible effects on predatory behaviours. Here we tested the effects of elevated CO₂ on the predatory behaviours of two ecologically distinct cephalopod species, the pygmy squid, Idiosepius pygmaeus, and the bigfin reef squid, Sepioteuthis lessoniana. Both species exhibited an increased latency to attack and altered body pattern choice during the attack sequence at elevated CO₂. I. pygmaeus also exhibited a 20% decrease in predation rate, an increased striking distance, and reduced preference for attacking the posterior end of prey at elevated CO₂. Elevated CO₂ increased activity levels of S. lessoniana comparable to those previously shown in I. pygmaeus, which could adversely affect their energy budget and increase their potential to be preyed upon. The effects of elevated CO₂ on predatory behaviours, predation strategies and activity levels of cephalopods reported here could have far‐reaching consequences in marine ecosystems due to the ecological importance of cephalopods in the marine food web.     

The tidal rhythm of feeding and digestion in the pacific oyster, Crassostrea gigas (Thunberg)

A study has been made of the processes of extra- and intracellular digestion in the Pacific oyster, Crassostrea gigas (Thunberg) over two 24-h cycles in winter and summer.The results show that the digestive processes are discontinuous and related to the tidal cycle. Variations in tidal height resulting from a diurnal inequality of the tide affect both the relative dissolution of the crystalline style and the relative lengths of the constituent phases of the intracellular digestive process in the digestive diverticula. On a seasonal basis the style is present for a greater length of time in winter and, conversely, remains dissolved longer in summer. A seasonal variation in the structure of the digestive tubules has also been found.The results confirm conclusions reached earlier that the processes of extracellular digestion in the stomach and intracellular digestion in the digestive diverticula of intertidal bivalves are both discontinuous, alternate, and irrevocably interrelated since breakdown of the digestive cells of the digestive diverticula following intracellular digestion results in the dissolution of the crystalline style. The arrival of food in the stomach has a minimal effect upon the style. Moreover, the cytological structure of the digestive diverticula of C. gigas undergoes a series of synchronized changes related to the tidal cycle.     

The Ultrastructure of the Gastrodermal Gland Cells in the Freshwater Planarian Dugesia gonocephala s.l.

Light and transmission electron microscopy have been used to study the gastrodermal gland cells of the triclad Dugesia gonocephala s.l. The events involved in the ultrastructural transformation and the secretion process in these cells were followed at four different stages in both fasted and fed animals. During the feeding stage their secretory granules are directly discharged into the intestinal lumen by means of a secretion process of the holocrine type that is described in this paper. It is suggested that such secretions contribute to extracellular digestion and that disintegration of the gland cells is accompanied by a differentiation of neoblasts into new gland cells, reflecting a turnover of gland cells during the triclad digestive stages.     

Observations on the feeding mechanism,structure of the gut,and digestive physiology of the european lobster Homarus gammarus (L.) (Decapoda: Nephropidae)

Investigations have been made on the feeding mechanism, structure of the gut, and digestive physiology of the European lobster Homarus gammarus (L.).Ciné-photography has shown that the mandibles do not possess a masticatory function, merely serving to grip food morsels during the tearing process effected by the pulling action of the third maxillipeds. The remaining maxillipeds, together with the maxillae, then direct food fragments to the mouth for ingestion.Ingestion is facilitated by mucoid secretions discharged from the oesophageal tegumental glands; the glands do not appear to produce any enzymes which directly contribute to the digestive processes.The hepatopancreas is the principal organ concerned with digestion. It possesses a complex tubular organization in which sequential cellular differentiation culminates in the discharge of enzymes from the B-cells for extracellular digestion in the cardiac stomach. The enzymes are synthesized within vacuoles contained in the B-cell precursors (F-cells) and are secreted in three bursts of activity at 0–15 min, 1–2 h, and 3.5–5 h after a meal. The initial secretory phase is holocrine. Extracellular digestion involves esterases, arylamidases, and lipases; endopeptidases have not been positively identified by histochemical means despite the fact that Homarus is a carnivore. There is an intracellular digestive phase, not previously described in decapod crustaceans, at the 7–9 h post-ingestive stage in the hepatopancreatic R-cells which is effected by arylamidases and lipases.Various phosphatase enzymes have been identified in the hepatopancreatic cells. Acid and alkaline phosphatases are apparently concerned with several stages in the digestive cycle, including enzyme synthesis and secretion, and the absorption of digestive products. Adenosine triphosphatase activity is primarily associated with granules located in the distal R-cell cytoplasm; the possible significance of these granules in the elimination of metabolic wastes is discussed. Acid phosphatases and esterases are present in the midgut epithelium. The possibility of a passive uptake of material from the midgut lumen is considered.Faecal material in the hindgut is bound by mucoid secretions derived from the tegumental glands of this alimentary region; the mucus may also assist in defaecation.A complete digestive cycle in Homarus occupies ≈ 12 h.Food reserves in the gut consist principally of fat deposits in the R-cells, but minute amounts of glycogen can also be detected.No evidence of calcium, copper or ferric iron deposition in any part of the alimentary tract was found.     

Histochemical and ultrastructural observations on digestion in Tetrameres fissispina Diesing, 1861 (Nematoda: Spiruridea) with special reference to intracellular digestion

A large proportion of the blood ingested by Tetrameres fissispina is digested extracellularly to haematin. The probable site of extracellular haemoglobin degradation is the glycocalyx of the microvilli which may carry adsorbed enzymes functional in contact digestion. A smaller proportion of the haemoglobin released from haemolysed erythrocytes is endocytosed in an unchanged state by isolated groups of absorptive cells. In the latter, haemoglobin-containing phagosomes apparently fuse with primary lysosomes ultimately to produce large, heterogeneous, multiple phagolysosomes (digestive complexes). Lipid droplets produced during digestion are extruded from these at intervals. Haemosiderin is the end-product of intracellular haemoglobin breakdown—the differences in residues of the extracellular and intracellular processes (haematin and haemosiderin) reflecting differences in the two enzyme systems employed. Haemosiderin is accumulated as sphaerocrystals in dilated cisternae of the ER. It is suggested that the purpose of intracellular digestion is to provide a source of ferric ions (in the form of haemosiderin) for the biosynthesis of endogenous haemoglobin which the extracellular degradation of haemoglobin cannot supply.     

The effects of tidal currents on the rhythm of feeding and digestion in Pecten maximus L.

The orientation of individuals in two populations of Pecten maximus L., from the west coast of Ireland, shows that they have a marked preference to face directly into a tidal flow. In both localities examined there was a reversal of tides and the members of the populations were divided equally for flood and ebb tides. Twenty-four hour in situ studies of the animals were made and at all stages of the tide individuals facing either due east or west were observed. A cyclical feeding pattern imposed by the reversal of tidal flow is proposed.The pH of various parts of the digestive tract was investigated and showed a wide range of values. The most acid region was that of the stomach. The variations in pH of all of the regions of the gut examined throughout a 24-h period closely followed the pattern of the tidal cycle.Histological analysis of the stomach and digestive diverticula of representative samples of scallops taken at regular intervals over the 24-h periods clearly indicated a diphasic pattern of digestion within the tubules of the digestive diverticula. A close correlation between the phases of intracellular digestion, the pH variations in different regions of the gut, and the tidal cycle indicate distinct feeding cycles in Pecten. Those scallops facing into the ebb current show the same diphasic patterns as those individuals facing the flood current, but are 6 h out of phase. The cycle is considered as a duplication of a diphasic feeding pattern. The tubules themselves undergo a digestive process similar to that in Lasaea rubra (Montagu). The digestive cells phagocytose food material, begin intracellular digestion, and increase in size until they obscure the lumina. The dispersal of waste material and residual bodies is accomplished dramatically by a dehiscence of the tubule cells together with a loss of both digestive and crypt cells. New tubules are regenerated from the apices of those tubules breaking down. In any section of the diverticula tubules in two different conditions are found. The cycle of digestion takes 24 h and in order to facilitate feeding at each 12-h tidal cycle the tubules are equally divided into two phases with one 12 h behind the other.     

Salt soluble without jelly-like component from the oviducal gland induces chorionic expansion in the ova of the Japanese common squid Todarodes pacificus

Abstract

In vitro fertilization of squid requires the jelly substance found in the female oviducal gland; yet, the active component of this substance that facilitates fertilization remains unknown. Here, we used biochemical methods to separate the jelly substance of Todarodes pacificus (Oegopsida) and Sepioteuthis lessoniana (Myopsida family) into four fractions; specifically, two water soluble fractions (Molecular weight > 10,000 and < 10,000), one salt soluble fraction, and one insoluble fraction. The salt soluble fraction of T. pacificus induced chorionic expansion (perivitelline space formation), which precedes the normal embryonic development of ova. In contrast, the salt soluble fraction of S. lessoniana elicited insufficient expansion of the ova, only producing embryos with high abnormality rates. These results suggest that the salt soluble component(s) (not the jelly-like substances) in the oviducal gland induce chorionic expansion and hatching in Oegopsida, and that these components may be similar in Myopsida.     

Molecular evidence for co-occurring cryptic lineages within the Sepioteuthis cf. lessoniana species complex in the Indian and Indo-West Pacific Oceans

The big-fin reef squid, Sepioteuthis cf. lessoniana (Lesson 1930), is an important commodity species within artisanal and near-shore fisheries in the Indian and Indo-Pacific regions. While there has been some genetic and physical evidence that supports the existence of a species complex within S. cf. lessoniana, these studies have been extremely limited in scope geographically. To clarify the extent of cryptic diversity within S. cf. lessoniana, this study examines phylogenetic relationships using mitochondrial genes (cytochrome oxidase c, 16s ribosomal RNA) and nuclear genes (rhodopsin, octopine dehydrogenase) from nearly 400 individuals sampled from throughout the Indian, Indo-Pacific, and Pacific Ocean portions of the range of this species. Phylogenetic analyses using maximum likelihood methods and Bayesian inference identified three distinct lineages with no clear geographic delineations or morphological discriminations. Phylogeographic structure analysis showed high levels of genetic connectivity in the most widespread lineage, lineage C and low levels of connectivity in lineage B. This study provides significant phylogenetic evidence for cryptic lineages within this complex and confirms that cryptic lineages of S. cf. lessoniana occur in sympatry at both small and large spatial scales. Furthermore, it suggests that two closely related co-occurring cryptic lineages have pronounced differences in population structure, implying that underlying differences in ecology and/or life history may facilitate co-occurrence. Further studies are needed to assess the range and extent of cryptic speciation throughout the distribution of this complex. This information is extremely useful as a starting point for future studies exploring the evolution of diversity within Sepioteuthis and can be used to guide fisheries management efforts.     

Salivary digestive enzymes of the wheat bug, Eurygaster integriceps (Insecta: Hemiptera: Scutelleridae)

The digestive enzymes from salivary gland complexes (SGC) of Eurygaster integriceps, and their response to starvation and feeding were studied. Moreover, digestive amylases were partially purified and characterized by ammonium sulfate precipitation and gel filtration chromatography. The SGC are composed of two sections, the principal glands and accessory glands. The principal glands are further divided into the anterior lobes and posterior lobes. The SGC main enzyme was α-amylase, which hydrolyzed starch better than glycogen. The other carbohydrases were also present in the SGC complexes. Enzymatic activities toward mannose (α/β-mannosidases) were little in comparison to activities against glucose (α/β-glucosidases) and galactose (α/β-galactosidases), the latter being the greatest. Acid phosphatase showed higher activity than alkaline phosphatase. There was no measurable activity for lipase and aminopeptidase. Proteolytic activity was detected against general and specific protease substrates. Activities of all enzymes were increased in response to feeding in comparison to starved insects, revealing their induction and secretion in response to feeding pulse. The SGC amylases eluted in four major peaks and post-electrophoretic detection of the α-amylases demonstrated the existence of at least five isoamylases in the SGC. The physiological implication of these findings in pre-oral digestion of E. integriceps is discussed.     

Digestive enzymes present in crustacean feces as a tool for biochemical, physiological, and ecological studies

The effect of food composition on the digestive system of Penaeus vannamei shrimp was used to determine the suitability of feces for analysis of class, type, composition of digestive proteinases, and whether alterations in the digestive gland are mirrored in feces composition. Enzymes recovered from feces and the midgut gland of white shrimp P. vannamei were used for comparison purposes. Three groups of shrimp were assembled: two groups fed two different brands of commercial feeds (PI and SC) with different content of protein, and the last group fed 50% PI feed and 50% thawed giant squid. Composition of proteinases in the midgut gland and feces were identical, and trypsin and chymotrypsin paralogues were identified in both samples by substrate-electrophoresis. Total proteolytic, trypsin, and chymotrypsin enzyme activities were higher in both samples from organisms fed SC, than in the other two groups. In the hepatopancreas, trypsin activity was ∼30% higher in SC fed group. Final average weights of shrimp were close in three groups, but hepatopancreas weight was 20% higher in the SC group. The degree of protein hydrolysis (DH) in vitro for the SC and PI was evaluated by the pH-stat method, using enzymes from feces and hepatopancreas of each group. The DH of food was no different, but it was affected by enzyme source, hepatopancreas extract (HPE) or feces extract (FE). DH was always higher when FE was the enzyme source than when HPE was the source. The proposed methods for recovery of enzymes from shrimp feces can be applied to other crustaceans. Measurements were sufficiently sensitive to allow quantifying the effects of feed on digestion physiology and other ecological and physiological applications, without the necessity of killing specimens.     

The short life of the Hoyle organ of <Emphasis Type="Italic">Sepia officinalis</Emphasis>: formation,differentiation and degradation by programmed cell death

Cephalopods encapsulate their eggs in protective egg envelopes. To hatch from this enclosure, most cephalopod embryos release egg shell-digesting choriolytic enzymes produced by the Hoyle organ (HO). After hatching, this gland becomes inactive and rapidly degrades by programmed cell death. We aim to characterize morphologically the development, maturation and degradation of the gland throughout embryonic and first juvenile stages in Sepia officinalis. Special focus is laid on cell death mechanisms and the presence of nitric oxide synthase during gland degradation. Hatching enzyme has been examined in view of metallic contents, commonly amplifying enzyme effectiveness. HO gland cells are first visualized at embryonic stage 23; secretion is observed from stage 27 onwards. Degradation of the HO occurs after hatching within two days by the rarely observed autophagic process, recognized for the first time in cephalopods. Nitric oxide synthase immunopositivity was not found in the HO cells after hatching, suggesting a possible NO role in cell death signalling. Although the HO ‘life course’ chronology in S. officinalis is similar to other cephalopods, gland degradation occurs by autophagy instead of necrosis. Eggs that combine a large perivitelline space and multi-layered integument seem to require a more complex and large gland system.