Preliminary investigation of the digestive enzymes in Pagellus erythrinus (Linneo 1758) larvae

To gain a better understanding of the role of the olfactory system in sexual selection, we conducted a series of behavioral assays to correlate the number of aesthetascs with pre-copulatory behavior and male mating success in a protandrous simultaneous hermaphroditic shrimp, Lysmata wurdemanni. We demonstrated that male pre-copulatory behavior and mating success were closely associated with the number of aesthetascs. Individuals with a higher numbers of aesthetascs started approaching/searching females earlier and had higher mating success than ones with lower numbers. Sexual selection theory predicts that small males in a pure searching mating system have advantages to mate with females because they are cryptic and agile. However, our results show that aesthetasc number, rather than shrimp size, may be the major factor in determining male mating success.     

Optimization of cell lysis and protein digestion protocols for the analysis of HeLa S3 cells by LC-MS/MS

In order to maximize the number of proteins identified from Hela S3 cell lysate we tested various cell lysis, protein precipitation and digestion protocols. First, we compared three different lysis buffers, two mechanical cell disruption methods and two precipitation methods. Then, we tested six different in-solution digestion protocols, three different in-gel digestion protocols and ten different peptide extraction protocols. The result is a proposal for an optimized protocol to prepare the whole cell lysate samples from HeLa S3 cells.     

Retention of different-sized particles and derived gut fill estimate in tammar wallabies (Macropus eugenii): physiological and methodological considerations

The capacity of the digestive tract is an important parameter in understanding digestive adaptations, particularly in herbivores. Measures of this capacity (‘gut fill’) are commonly performed in killed animals, which has ethical and logistical implications. Alternatively, dry matter gut contents (DMC) can be estimated in live animals from food intake, digesta retention and digestibility, based on physical principles (Holleman and White, Can. J. Zool. 67, 488-494, 1989). Although this method has been used to some extent, it still awaits thorough validation. Here we estimated DMC in seven tammar wallabies during 5-day feeding trials and compared the results to those gained from dissections immediately after the trials. Calculated DMC exceeded that actually measured by 29 ± 22%. A closer inspection of the data suggested that this was partly due to the fact that DMC as measured by dissection is susceptible to short-term influences such as daily variation in food intake, whereas the calculated DMC represents an integrative measure over the whole period of the feeding trial. Correlations between both the measured digesta retention times, and the calculated DMC, with the measured wet contents mass suggest that it is particularly the DMC determined via dissection that needs to be measured with care. For a comparison of gut capacities, the calculated DMC therefore can be considered adequate, but should for a more widespread use be validated in further studies including more species and experimental regimes controlling food intake variation. Additionally, we tested whether very small (100-500 μm) and small (500-1000 μm) particles were retained differently in the tammar wallabies. There was no indication of such a difference. Whether the macropod forestomach selectively passes a certain particle fraction (that represents microbes) with the generally faster-passing fluids remains to be investigated with even smaller markers, e.g. labelled bacteria.     

Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins

Identification of proteolytically resistant proteins with compact molecular structure and/or poor water solubility is a challenge in current proteomic study. In this study, sodium deoxycholate (SDC)-assisted tryptic digestion and identification of proteolytically resistant myoglobin and integral membrane proteins were systematically investigated. When the effect of SDC up to 10% on trypsin activity was investigated, little decrease in the trypsin activity was observed in 1% SDC solution, 2-5% SDC decreased the enzyme activity only by about 13.6%, and even in the presence of 10% SDC trypsin still retained 77.4% of its activity. Matrix-assisted laser desorption ionization time of flight mass spectrometry analysis showed that SDC could be removed from sample solution with acid treatment followed by centrifugation, and the remaining SDC, if any, had little effect on mass spectrometry analysis with regard to the number and signal/noise ratio of ions in the mass spectra. Compared with urea and methanol, two other commonly used additives in addition to SDS in proteomic analysis, SDC improved more efficiently the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides and the sequence coverage of matched proteins.     

Hysteresis of heart rate and heat exchange of fasting and postprandial savannah monitor lizards (Varanus exanthematicus)

Reptiles are ectothermic, but regulate body temperatures (T(b)) by behavioural and physiological means. Body temperature has profound effects on virtually all physiological functions. It is well known that heating occurs faster than cooling, which seems to correlate with changes in cutaneous perfusion. Increased cutaneous perfusion, and hence elevated cardiac output, during heating is reflected in an increased heart rate (f(H)), and f(H), at a given T(b), is normally higher during heating compared to cooling ('hysteresis of heart rate'). Digestion is associated with an increased metabolic rate. This is associated with an elevated f(H) and many species of reptiles also exhibited a behavioural selection of higher T(b) during digestion. Here, we examine whether digestion affects the rate of heating and cooling as well as the hysteresis of heart rate in savannah monitor lizards (Varanus exanthematicus). Fasting lizards were studied after 5 days of food deprivation while digesting lizards were studied approximately 24 h after ingesting dead mice that equalled 10% of their body mass. Heart rate was measured while T(b) increased from 28 to 38 degrees C under a heat lamp and while T(b) decreased during a subsequent cooling phase. The lizards exhibited hysteresis of heart rate, and heating occurred faster than cooling. Feeding led to an increased f(H) (approximately 20 min(-1) irrespective of T(b)), but did not affect the rate of temperature change during heating or cooling. Therefore, it is likely that the increased blood flows during digestion are distributed exclusively to visceral organs and that the thermal conductance remains unaffected by the elevated metabolic rate during digestion.     

Digestion of maize and sunflower pollen by the spotted maize beetle Astylus atromaculatus (Melyridae): is there a role for osmotic shock?

We investigated the mechanism and efficiency of digestion of two types of pollen, maize, Zea mays, and sunflower, Helianthus annuus, by the spotted maize beetle, Astylus atromaculatus (Melyridae). We found similar and high extraction efficiencies, but different mechanisms of digestion. Osmotic shock was apparently involved in digestion of the large and thin-walled maize pollen grains. In the anterior midgut most maize pollen grains were already ruptured, in contrast with the intact exines of sunflower pollen, which suggests another mechanism of digestion for the latter, such as enzymatic action. We investigated the effect of osmotic shock on maize pollen in vitro by looking at the behavior of pollen grains in varying osmotic concentrations. Maize pollen grains burst in both distilled water and sugar solutions of various concentrations, and the amount of rupturing decreased with an increase in sugar concentration. Digestion of maize pollen was much slower in honeybees than in spotted maize beetles. Maize pollen bursts early in the midgut of maize beetles, but remains intact in honeybees: this suggests that osmotic shock may not be as important for honeybees as previously suggested.     

Sugar digestion in mosquitoes: identification and characterization of three midgut alpha-glucosidases of the neo-tropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

Dietary carbohydrates provide an important source of energy for flight, and contribute to longevity and fecundity of mosquitoes. The most common sugar mosquitoes ingest is sucrose, and digestion of this substance is carried out mainly by alpha-glucosidases. In the current work, we tested the efficiency of sucrose on Anopheles aquasalis female diet. The best longevity (days) was reached when sugar was available in the diet, whereas most only blood fed females were dead 6 days after emergence. Three alpha-glucosidase isoforms were detected in the adult female midgut, named alphaGlu1, alphaGlu2 and alphaGlu3. These are acidic alpha-glucosidases with optima pH around pH 5.5. alphaGlu1 and alphaGlu2 are present in both secreted and membrane-bound forms, whereas alpha-Glu3 only in anchored to membranes. The alpha-glucosidase activity is concentrated mainly in the posterior midgut (70%), both in non-fed or 10% sucrose fed females. The single form of these alpha-glucosidases seemed to be approximately 70 kDa polypeptides, although alphaGlu2 is presented in >or=600 kDa self-aggregates. Km values of alphaGlu1, alphaGlu2 and alphaGlu3 differed significantly from each other, supporting the statement that three alpha-glucosidases are produced in the female midgut. Together, all data suggest that sugar is an essential component of A. aquasalis female diet. In addition, alpha-glucosidases are synthesized in the same place where sucrose is digested and absorbed, the midgut.     

Cholecystokinin (CCK) in Atlantic herring (Clupea harengus L.) - ontogeny and effects of feeding and diurnal rhythms

Neural and alimentary cholecystokinin (CCK) levels in Atlantic herring, Clupea harengus, were analyzed from hatching to 40days after hatching (DAH). The head compartment representing the neural pool was quantitatively dominant (>80% of the total CCK content) while the digestive tract pool represented 6-10%. During ontogeny the CCK level in whole larvae increased almost 15-fold from 0 to 40 DAH, being particularly marked from 14 to 20 DAH. Larvae of 24 to 26 DAH were examined for potential occurrence of a circadian rhythm and to analyze the effects of feeding. Fed and fasted larvae were significantly different, where fed larvae showed higher CCK levels. There were large fluctuations in CCK levels analyzed at 3h intervals without an apparent diurnal pattern. Shorter sampling intervals of 1h in the morning when lights were switched gradually on and food was offered to the larvae demonstrated a marked drop in the relative gut CCK levels and a concurrent increase in the CCK carcass to gut ratio, 1h after introduction of food followed by a return to prefeeding levels after 2h. This response probably results from a release and re-synthesis of CCK in the gut after initiation of feeding. Taken together, these results support earlier reports that CCK participates in the regulation of digestive processes in herring larvae, but CCK does not seem to have a circadian rhythm independent of feeding.     

Designing Food Structure to Control Stability, Digestion, Release and Absorption of Lipophilic Food Components

The bioavailability of dietary lipophilic components may be either increased or decreased by manipulating the microstructure and/or physicochemical properties of the foods that contain them. This article stresses how knowledge of the molecular, physicochemical, and physiological processes that occur during lipid ingestion, digestion, and absorption can be used to rationally design food structures to control these processes and therefore impact the rate or extent of lipid digestion and/or absorption. These approaches include controlling the molecular characteristics of the lipid molecules, altering lipid droplet size or interfacial properties, and manipulating food matrix structure and composition. Improved knowledge of the molecular, physicochemical, and physiological processes that occur during lipid ingestion, digestion, and absorption will facilitate the rational design and fabrication of functional foods for improved health and wellness.