Unfed Animals Research Articles

Postprandial Lipid Response Following a High Fat Meal in Rats Adapted to Dietary Fiber

Rats were adapted to diets containing 5 g/100 g cellulose (CL), 5 g/100 g oat bran fiber (OB) or 5 g/100 g psyllium husk (Psy) for 4 wk. Following a 12-h fast, animals were either killed at 0 h (baseline) or fed 4.5 g of a test meal that provided 50% energy from fat, then killed at 1, 4 or 6 h postprandially. Fasting plasma and HDL cholesterol concentrations were lower in Psy-fed animals than in rats fed either CL or OB. Plasma triglycerides increased significantly from baseline (0 h) in all groups but did not differ among diet treatments. Increases in triglyceride content of the chylomicron/VLDL fraction occurred in the CL- and OB-fed groups and in the HDL fraction of the Psy-fed group during the postprandial period. In unfed animals the hepatic and intestinal levels of apolipoprotein A-IV mRNA were higher in the CL-fed group than in the groups fed OB and Psy. Apolipoprotein B mRNA was higher in the intestine of the OB-fed group than in the groups fed CL and Psy and had a significant gradient along the small intestine, increasing in the distal third. The results suggest that chronic consumption of fiber is less likely to modify the acute plasma triglyceride response to a fat-containing test meal than if a fiber supplement is incorporated into the meal.

The Influence of the Toxin-Producing Dinoflagellate, Alexandrium catenella (1119/27), on the Survival and Reproduction of the Marine Copepod, Acartia tonsa, During Prolonged Exposure

Copepods can feed on, and may regulate, the blooms of harmful algae (HA), and may also facilitate dinoflagellate blooms by inducing toxin production and through selective grazing. However, exposure to HA may also cause mortality and reproductive impairment in copepods, with detrimental effects at the population-scale. Here we present the toxin profile of the dinoflagellate, Alexandrium catenella (formerly Alexandrium tamarense), and examine how it affects the survival and reproduction of the cosmopolitan marine copepod, Acartia tonsa. Healthy adult copepods were exposed to mono-specific diets of toxic and non-toxic strains of A. catenella (1119/27 and 1119/19, respectively) and non-toxic Rhodomonas sp. for 10 days alongside unfed controls to examine how their survival was influenced by likely HA bloom conditions. Additional 2-day experiments examined how their egg production rate and hatching success were affected by food deprivation, toxic A. catenella, a non-toxic alternative and a mixture of toxic and non-toxic prey, at high and low concentrations. Survival of A. tonsa declined over the 10-day experiment in all treatments but was not significantly lower in the toxic A. catenella treatment; mortality was only significantly enhanced in the unfed animals, which showed 100% mortality after 9 days. Egg production rates and hatching success from females in the unfed and toxic A. catenella treatments were all significantly lower than values observed in females fed Rhodomonas sp. or non-toxic A. catenella. Animals offered 1,000 μg C L–1 of Rhodomonas sp. and a 50:50 mixture of toxic A. catenella and Rhodomonas sp. produced significantly more eggs than animals fed toxic A. catenella alone. These results were not apparent at prey concentrations of 100 μg C L–1. The percentages of eggs to successfully hatch from females offered mono-specific diets of toxic A. catenella were always close to zero. Collectively, our results indicate that adult female A. tonsa can acquire sufficient energy from toxic A. catenella to survive, but suffer reproductive impairment when feeding on this prey alone.

Mass Spectrometry Quantification, Localization, and Discovery of Feeding-Related Neuropeptides in Cancer borealis.

The crab Cancer borealis nervous system is an important model for understanding neural circuit dynamics and modulation, but the identity of neuromodulatory substances and their influence on circuit dynamics in this system remains incomplete, particularly with respect to behavioral state-dependent modulation. Therefore, we used a multifaceted mass spectrometry (MS) method to identify neuropeptides that differentiate the unfed and fed states. Duplex stable isotope labeling revealed that the abundance of 80 of 278 identified neuropeptides was distinct in ganglia and/or neurohemal tissue from fed vs unfed animals. MS imaging revealed that an additional 7 and 11 neuropeptides exhibited altered spatial distributions in the brain and the neuroendocrine pericardial organs (POs), respectively, during these two feeding states. Furthermore, de novo sequencing yielded 69 newly identified putative neuropeptides that may influence feeding state-related neuromodulation. Two of these latter neuropeptides were determined to be upregulated in PO tissue from fed crabs, and one of these two peptides influenced heartbeat in ex vivo preparations. Overall, the results presented here identify a cohort of neuropeptides that are poised to influence feeding-related behaviors, providing valuable opportunities for future functional studies.

Temperature and food availability alters the physiology and aerobic capacity of tambaqui (Colossoma macropomum)

Fish of the Amazon experience both daily and seasonal variation in temperature and food availability. In the present work, we investigated the influence of nutrient status changes resulting from feeding Colossoma macropomum five flooded forest fruits on aerobic and swimming performance. To assess the effects of diet, three groups of fish were provided different types of food. One group of was provided with commercial feed, another was given fruit feed, and the last group was not fed. The effects of temperature were tested by keeping half of each group of animals at either 26 °C or 33°C for 30 days. After this period, the animals were transferred to either a respiratory chamber or swimming tunnel. Then, tissues were collected for relevant analyses. Results showed that C. macropomum depresses aerobic metabolism when food is withheld (27% reduction in ṀO2), and observed effects were intensified at the highest temperature (40% reduction in ṀO2 at 33 °C). Further, increased temperature negatively influenced hematological (Ht, Hb, VCM), biochemical (glucose, triglycerides, cholesterol), hepatossomatic index and enzymatic (MDH and CS in white muscle) parameters. The animals feeding commercial and fruits enriched diets, have a physiological and biological advantage when compared to the unfed animals at both temperatures. Metabolic suppression is an important strategy for maintaining swimming capacity in C. macropomum. It is suggested that even after a long period of fasting, carbohydrates are important energetic substrates.

Quantification of Mg2+, Ca2+ and H+ transport by the gastrointestinal tract of the goldfish, Carassius auratus, using the Scanning Ion-selective Electrode Technique (SIET).

An in vitro gut-sac technique and the scanning ion-selective electrode technique (SIET) were used to characterize Mg2+, Ca2+, and H+ transport at both the mucosal and serosal surfaces of non-everted and everted gastrointestinal tissues obtained from Carassius auratus. As part of the study, two magnesium ionophores were compared (II vs. VI). Unfed animals displayed uniform transport of all ions along the intestine. Feeding resulted in elevated Mg2+ and Ca2+ transport when the gut lumen contained chyme however, under symmetrical conditions this increased transport rate was absent. Furthermore, zonation of divalent cation transport was present for both Ca2+ and Mg2+ under non-symmetrical conditions while the zonation remained for Ca2+ alone under symmetrical conditions. High dietary Mg2+ decreased absorption and induced secretion of Mg2+ in the posterior intestine. Uptake kinetics in the esophagus suggest large diffusive and/or convective components based on a linear relationship between Mg2+ transport and concentration and lack of inhibition by ouabain, an inhibitor of Na+-K+-ATPase. In contrast, kinetics in the rectum were suggestive of a low affinity, saturable carrier-mediated pathway. A decrease in Mg2+ and Ca2+ transport was observed in the posterior intestine (both at the mucosal and serosal surfaces) in response to ouabain. This impact was greatest for Ca2+ transport and when applied to the mucosal fluid and measured in everted preparations. In contrast a putative Mg2+ transport inhibitor, cobalt(III)hexamine-chloride, did not affect Mg2+ transport. This is the first study to use SIET approaches to study ion transport in the gut of teleost fish. This is also the first study to provide characterization of Mg2+ transport in the gut of C. auratus. Due to the limited selectivity of Magnesium ionophore II, subsequent studies of tissues bathed in physiological saline should be made using Magnesium Ionophore VI.

Maximum standard metabolic rate corresponds with the salinity of maximum growth in hatchlings of the estuarine northern diamondback terrapin (Malaclemys terrapin terrapin): Implications for habitat conservation

I evaluated standard metabolic rates (SMR) of hatchling northern diamondback terrapins (Malaclemys terrapin terrapin) across a range of salinities (salinity = 1.5, 4, 8, 12, and 16 psu) that they may encounter in brackish habitats such as those in the Maryland portion of the Chesapeake Bay, U.S.A. Consumption of O2 and production of CO2 by resting, unfed animals served as estimates of SMR. A peak in SMR occurred at 8 psu which corresponds closely with the salinity at which hatchling growth was previously shown to be maximized (salinity ∼ 9 psu). It appears that SMR is influenced by growth, perhaps reflecting investments in catabolic pathways that fuel anabolism. This ecophysiological information can inform environmental conservation and management activities by identifying portions of the estuary that are bioenergetically optimal for growth of hatchling terrapins. I suggest that conservation and restoration efforts to protect terrapin populations in oligo-to mesohaline habitats should prioritize protection or creation of habitats in regions where average salinity is near 8 psu and energetic investments in growth appear to be maximized.

Satiation level affects anti-predatory decisions in foraging juvenile crayfish.

Moving shadows signify imminent threat to foraging juvenile crayfish, and the animals respond with one of two discrete anti-predatory behaviors: They either freeze in place or rapidly flex their tails, which quickly propels them away from the approaching danger signal. Although a freeze might be the more risky choice, it keeps the animal near the expected food reward, while a tail-flip is effective in avoiding the shadow, but puts critical distance between the animal and its next meal. We manipulated the satiation level of juvenile crayfish to determine whether their behavioral choices are affected by internal energy states. When facing the same visual danger signal, animals fed to satiation produced more tail-flips and fewer freezes than unfed animals, indicating that intrinsic physiological conditions shape value-based behavioral decisions. Escape tail-flip latencies, however, were unaffected by satiation level, and an increase in food quality only produced a minor behavioral shift toward more freezing in both fed and unfed animals. Thus, satiation level appears to be the dominant factor in regulating decision making and behavioral choices of crayfish.

Be ready at any time: postprandial synthesis of salivary proteins in salivary gland cells of the haematophagous leech Hirudo verbana.

Sanguivorous leeches are ectoparasites having access to body fluids of potential hosts only infrequently. During feeding, salivary proteins are released from unicellular salivary glands into the wound. These substances, among them anti-coagulants, anti-inflammatory or anti-microbial agents, allow these animals proper feeding and long-term storage of host blood in their crops for several months. Using histological, protein biochemical and molecular techniques, we investigated whether synthesis of salivary proteins and refilling of salivary gland cells occur immediately after feeding or later when stored nutrients in the crop are getting scarce. The results of the histological analyses showed that gland cell area was significantly smaller right after feeding when compared with those in unfed animals. This parameter recovered quickly and reached the control level at 1 week after feeding. 2D gel electrophoresis and analysis of the abundance of individual proteins in extracts of leech tissues revealed that a subset of proteins that had been present in extracts of unfed animals virtually disappeared during feeding, but re-appeared within 1 week of feeding (most probably secretory proteins) while another subset did not change during the experimental period (most probably housekeeping proteins). Semi-quantitative PCR analysis of hirudin cDNA prepared from leech RNA samples revealed that the amount of hirudin transcripts increased immediately after feeding, peaked at 5 days after feeding and declined to control values thereafter. Our results indicate that bloodsucking leeches synthesize salivary proteins and refill their salivary gland cell reservoirs within a week of a blood meal to be prepared for another feeding opportunity.

Subchronic Effects of Ectoine on Survival, Growth and Physiological Parameters of Daphnia Magna

Ectoine (ECT) is an osmoprotectant produced and accumulated by halophilic bateria under hyperosmotic stress. There is a lack of knowledge on the effect induced by this amino acid during subchronic animal exposure to ECT in vivo. Therefore, the aim the present study was to determine influence of ECT on survival, motility, physiological activity (heart rate, thoracic limb activity, and mandible movement rate), growth rate and moulting of unfed and fed with normal diet Daphnia magna exposed for 10 days to 2.5, 4, 20 and 25 mg/L ECT. The results showed that survival of unfed animals exposed to 25 mgL ECT was decreased; however it was increased at lower concentrations of the amino acid when compared to the control. 20 and 25 mg/L of ECT significantly decreased swimming velocity, heart rate and thoracic limb activity and mandible movement rate. On the other hand, the osmoprotectant did’nt cause mortality of fed daphnids at any concentration but stimulated swimming velocity and the physiological parameters, however no differences were found in mandible movement rate. We found that ECT increased growth rate both in starving and fed daphnids, however the stimulatory effect was not concentation-dependent. The most significant increase of growth rate both in unfed and fed dapnids was found at 4 mg/L ECT. Moulting frequency was increases only in unfed daphnids exposed to the highest concentrations of ECT. Fed daphnids showed slighlty elevated moulting frequency only at 25 mg/L ECT. Our results indicate ECT is a stimulator of growth rate and physiological indices in fed Daphnia magna, however the elucidation of its mechanisms requires further studies.

Sources and range of long-term variability of rhythmic motor patterns in vivo.

The mechanisms of rhythmic motor pattern generation have been studied in detail in vitro, but the long-term stability and sources of variability in vivo are often not well described. The crab stomatogastric ganglion contains the well-characterized gastric mill (chewing) and pyloric (filtering of food) central pattern generators. In vitro, the pyloric rhythm is stereotyped with little variation, but inter-circuit interactions and neuromodulation can alter both rhythm cycle frequency and structure. The range of variation of activity in vivo is, with few exceptions, unknown. Curiously, although the pattern-generating circuits in vivo are constantly exposed to hormonal and neural modulation, the majority of published data show only the unperturbed canonical motor patterns typically observed in vitro. Using long-term extracellular recordings (N=27 animals), we identified the range and sources of variability of the pyloric and gastric mill rhythms recorded continuously over 4 days in freely behaving Jonah crabs (Cancer borealis). Although there was no evidence of innate daily rhythmicity, a 12 h light-driven cycle did manifest. The frequency of both rhythms increased modestly, albeit consistently, during the 3 h before and 3 h after the lights changed. This cycle was occluded by sensory stimulation (feeding), which significantly influenced both pyloric cycle frequency and structure. This was the only instance where the structure of the rhythm changed. In unfed animals the structure remained stable, even when the frequency varied substantially. So, although central pattern generating circuits are capable of producing many patterns, in vivo outputs typically remain stable in the absence of sensory stimulation.

Change in excitability of a putative decision-making neuron in Aplysia serves as a mechanism in the decision not to feed following food satiation

Although decision making is a ubiquitous function, the understanding of its underlying mechanisms remains limited, particularly at the single-cell level. In this study, we used the decision not to feed that follows satiation in the marine mollusk Aplysia to examine the role of putative decision-making neuron B51 in this process. B51 is a neuron in the feeding neural circuit that exhibits decision-making characteristics in vitro, which bias the circuit toward producing the motor programs responsible for biting behavior. Once satiated, Aplysia decided not to bite for a prolonged period of time (≥24h) when presented with a food stimulus that normally elicits feeding in non-satiated animals. Twenty-four hours after satiation, suppressed feeding was accompanied by a significant decrease of B51 excitability compared to the control group of unfed animals. No differences were measured in B51 resting membrane properties or synaptic input to B51 between the satiated and control groups. When B51 properties were measured at a time point in which feeding had recovered from the suppressive effects of satiation (i.e., 96h after satiation), no difference in B51 excitability was observed between satiated and control groups. These findings indicate that B51 excitability changes in a manner that is coherent with the modifications in biting resulting from food satiation, thus implicating this neuron as a site of plasticity underlying the decision not to bite following food satiation in Aplysia.

Change in food ingestion induces rapid shifts in the diversity of microbiota associated with cutaneous mucus of Atlantic salmon Salmo salar

To test the hypothesis that a switch in diet might cause changes in the abundance and composition of mucous-dwelling microorganisms, a short-term experiment was conducted with Atlantic salmon Salmo salar. Fish were fed on three different diets: pelleted S. salar feed, macroinvertebrates or pellets supplemented with an antibiotic. A fourth group of fish was deprived of food throughout the trial. Seven days after manipulating diets, significant differences were found in microbial density and community composition (quantified by different morphologically distinct colonies), particularly between fed and unfed animals. Moreover, food deprivation caused a rapid decrease in the number of epidermal mucous cells of the lateral skin, which may indicate a decrease in mucous secretion and explain differences in the diversity of mucous-dwelling microbiota observed in the fish. This is the first report of an effect of feeding regime on the abundance of microbial communities associated with cutaneous mucus of fishes.

Control of the release of digestive enzymes in the caeca of the cricket Gryllus bimaculatus

Abstract In Gryllus bimaculatus, more digestive enzymes (amylase, trypsin, aminopeptidase) are secreted in the caecum of fed crickets than in unfed crickets, but the enzymes are released continuously at a basal rate in unfed animals. The rate of synthesis of the enzymes appears to parallel their rate of release. Digestive enzymes are released in response to a specific ratio of nutrients, although a high nutrient component in the food does not necessarily induce a high digestive enzyme release for that component. Rinsed flat‐sheet preparations of the caecum are incubated with specific nutrients (carbohydrates and proteins) and various concentrations of a neuropeptide (type‐A allatostatin), which affects generally the basal rates of secretion. Both maltose and glucose increase the release of amylase in vitro, but starch produces an inhibition of amylase release at lower concentrations. Bovine serum albumin (BSA), peptone and a mixture of amino acids have almost no effect on the release of aminopeptidase or carboxypeptidase, and only low concentrations of peptone increase trypsin release. High concentrations of both BSA and peptone strongly inhibit trypsin activity, perhaps by excess substrate binding to the trypsin active site. The allatostatin Grybi‐AST 5 elevates the release of amylase in vitro, but not of trypsin or aminopeptidase, in 2‐day‐old fed females. In the caeca from 1‐day‐old unfed crickets, both amylase and the trypsin release are stimulated in the presence of AST 5. The paracrine AST 5 is probably released from the gut endocrine cells and binds to the enzyme‐producing caecal cells.

Wing‐spreading, wing‐drying and food‐warming in great cormorants Phalacrocorax carbo

Wing‐spreading of cormorants (Phalacrocoracidae) is a characteristic and enigmatic aspect of their behavioural repertoire. It has been suggested to have a range of functions including wing‐drying, food‐warming, and social signalling of foraging success. We investigated two of these putative roles by comparing the wing‐spreading behaviour of fed and unfed animals after they had been swimming and diving. The duration of wing‐spreading was correlated only with time spent on the water. The ingestion of food did not influence the duration of wing‐spreading, a finding that supports a wing‐drying, rather than a food‐warming, function.

Gastric processing in the Dungeness crab, Cancer magister, during hypoxia

The gastric physiology of the Dungeness crab, Cancer magister, was investigated over a range of oxygen tensions. Postprandial crabs reacted differently to hypoxia compared with unfed animals. The bradycardic response in postprandial animals was reduced, suggesting a summation of responses with feeding. A similar pattern was observed for ventilation rate. In unfed animals ventilation rate increased slightly as oxygen levels declined, but dropped significantly in oxygen tensions below 3.2 kPa, whereas in postprandial crabs it increased significantly in the lower oxygen regimes. Gastric processing of the meal was followed using a fluoroscope. Pyloric contraction rates were maintained during mild hypoxia, but decreased in 5.3 kPa oxygen tension and below. This led to an increase in clearance times of digesta from the foregut, midgut and hindgut regions. The slowing of gastric processing in the lower oxygen tensions suggested that the animals were unable to maintain their internal oxygen concentration. A significant reduction in efficiency of assimilation only occurred in the lowest oxygen regime tested (1.6 kPa). The range of hypoxia where gastric processing was affected corresponded closely to the levels of oxygen that modulate the foraging behaviour of C. magister. By using both physiological and behavioural mechanisms C. magister can maintain digestive processes, even in severely oxygen depleted environments.

Rates of fission, somatic growth and gonadal development of a fissiparous sea star, Allostichaster insignis, in New Zealand

Seasonal changes in population structure and incidence of fission were measured in intertidal and subtidal populations of Allostichaster insignis, a fissiparous sea star. Population size structure was stable over the course of the 1-year study. Sea stars in the subtidal zone attained greater maximum size (mean arm length, R = 35 mm) than those in the intertidal population (20 mm). Fission rates were greatest among small individuals (R < 20 mm). The frequency of fission ranged from 5 to 32% with peaks in early austral summer in the intertidal zone, and in autumn and winter in the subtidal zone. Sexual reproduction occurred in early spring in sea stars larger than 12 mm. The populations were heavily biased toward males. In the laboratory, A. insignis of three size classes (small, R = 9–13 mm; medium, 19–21 mm; and large, 29–31 mm) were fed mussels ad libitum or starved (not fed macroscopic food) for ∼1 year in a 3 × 2 factorial experiment. Small and medium-sized sea stars divided throughout the experiment and the ramets of most individuals regenerated sufficiently to divide again after 6–9 months. Unfed sea stars did not undergo fission (with one exception), had a higher mortality rate, and did not grow. Small, fed sea stars grew significantly faster than medium-sized or large individuals. At the end of the experiment, the pyloric caeca index (a measure of nutritional condition) was greater in fed than in unfed animals. Gonads (only testes were observed) developed in medium-sized and large, fed sea stars. Our field and laboratory results indicate that asexual reproduction in A. insignis predominantly occurs in small, well-nourished individuals. Ramets grow gradually through repeated fission and regeneration to a size (mean length of regenerating arms, Rr ∼ 20 mm) at which they begin to switch to sexual reproduction as the dominant reproductive mode.

CRABS PRIORITIZE DINNER

![Figure][1] In a body of water as large as the Pacific Ocean, you might think that salinity levels would be fairly constant; and they are in the ocean depths. But head closer in to shore, and it can be a very different matter. At stormy times of year, coastal freshwater run-off can

Postprandial increases in nitrogenous excretion and urea synthesis in the Chinese soft-shelled turtle, Pelodiscus sinensis

The objective of this study was to determine the effects of feeding on the excretory nitrogen (N) metabolism of the aquatic Chinese soft-shelled turtle, Pelodiscus sinensis, with a special emphasis on the role of urea synthesis in ammonia detoxification. P. sinensis is ureogenic and possesses a full complement of ornithine-urea cycle enzymes in its liver. It is primarily ureotelic in water, and the estimated rate of urea synthesis in unfed animals was equivalent to only 1.5% of the maximal capacity of carbamoyl phosphate synthetase I (CPS I) in its liver. Approximately 72 h was required for P. sinensis to completely digest a meal of prawn meat. During this period, there were significant increases in ammonia contents in the stomach at hour 24 and in the intestine between hours 12 and 36, which could be a result of bacterial activities in the intestinal tract. However, ammonia contents in the liver, muscle, brain and plasma remained unchanged throughout the 72-h post-feeding. In contrast, at hour 24, urea contents in the stomach, intestine, liver, muscle, brain and plasma increased significantly by 2.9-, 3.5-, 2.6-, 2.9-, 3.4 and 3.0-fold, respectively. In addition, there was a 3.3- to 8.0-fold increase in the urea excretion rate between hours 0 and 36 post-feeding, which preceded the increase in ammonia excretion between hours 12 and 48. By hour 48, 68% of the assimilated N from the feed was excreted, 54% of which was excreted as urea-N. The rate of urea synthesis apparently increased sevenfold during the initial 24 h after feeding, which demanded only 10% of the maximal CPS I capacity in P. sinensis. The postprandial detoxification of ammonia to urea in P. sinensis effectively prevented postprandial surges in ammonia contents in the plasma and other tissues, as observed in other animals, during the 72-h period post-feeding. In addition, postprandial ammonia toxicity was ameliorated by increased transamination and synthesis of certain amino acids in the liver and muscle of P. sinensis. After feeding, a slight but significant increase in the glutamine content occurred in the brain at hour 24, indicating that the brain might experience a transient increase in ammonia and ammonia was detoxified to glutamine.

INFLUENCE OF FEEDING ON HEPATOPANCREAS STRUCTURE AND DIGESTIVE ENZYME ACTIVITIES IN PENAEUS MONODON

This study examines the influence of feeding on digestive enzyme activities in the black tiger prawn, Penaeus monodon, using histological and biochemical techniques. Juvenile P. monodon (>50 mm total length) were sacrificed after feeding at sequential time intervals, with unfed controls also sacrificed at the same time intervals. Resin histology revealed that there were no morphological changes in the digestive gland F-cells between fed and unfed P. monodon over time. There were no significant changes in α-amylase specific activities in fed animals over time post feeding, nor were there any changes in starved animals through time. Trypsin and α-glucosidase activities were significantly higher in unfed animals than fed animals. There was a significant peak in α-glucosidase activities at 0 min post feeding but this could not be conclusively determined as an influence from feeding because of high variability and the near negligible levels of α-glucosidase activities. A peak in lipase activity was observed at 60 min post feeding, but because there was no significant differences in lipase activities between fed and unfed animals it was therefore inconclusive. Results from histological and biochemical analyses failed to prove that feeding had a significant influence on digestive enzyme production of P. monodon and suggest that digestive enzyme production in P. monodon may be continuous and is not strongly influenced by feeding.

Role of Intestinal Epithelial Cells in Immune Effects Mediated by Gram-Positive Probiotic Bacteria: Involvement of Toll-Like Receptors

The mechanisms by which probiotic bacteria exert their effects on the immune system are not completely understood, but the epithelium may be a crucial player in the orchestration of the effects induced. In a previous work, we observed that some orally administered strains of lactic acid bacteria (LAB) increased the number of immunoglobulin A (IgA)-producing cells in the small intestine without a concomitant increase in the CD4(+) T-cell population, indicating that some LAB strains induce clonal expansion only of B cells triggered to produce IgA. The present work aimed to study the cytokines induced by the interaction of probiotic LAB with murine intestinal epithelial cells (IEC) in healthy animals. We focused our investigation mainly on the secretion of interleukin 6 (IL-6) necessary for the clonal expansion of B cells previously observed with probiotic bacteria. The role of Toll-like receptors (TLRs) in such interaction was also addressed. The cytokines released by primary cultures of IEC in animals fed with Lactobacillus casei CRL 431 or Lactobacillus helveticus R389 were determined. Cytokines were also determined in the supernatants of primary cultures of IEC of unfed animals challenged with different concentrations of viable or nonviable lactobacilli and Escherichia coli, previously blocked or not with anti-TLR2 and anti-TLR4. We concluded that the small intestine is the place where a major distinction would occur between probiotic LAB and pathogens. This distinction comprises the type of cytokines released and the magnitude of the response, cutting across the line that separates IL-6 necessary for B-cell differentiation, which was the case with probiotic lactobacilli, from inflammatory levels of IL-6 for pathogens.