Bull Mar Sci. 90(4):903–919. 2014 research paper http://dx.doi.org/10.5343/bms.2013.1073
X-radiographic observations of food passage and nutrient absorption along the alimentary tract of archerfish, Toxotes jaculatrix
1,2 * 1 School of Environmental Simon K Das and Natural Resource Mazlan A Ghaffar 2 Sciences, Faculty of Science 1 and Technology, Universiti Yosni Bakar Kebangsaan Malaysia, 43600 Marcelo FG Brito 3 UKM Bangi, Selangor D.E., Sharifah Mastura SA 4 Malaysia. Shelby E Temple 5 2 Marine Ecosystem Research Centre, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 ABSTRACT.—X-radiography can be used to estimate UKM Bangi, Selangor D.E., gastric emptying time, which is valuable in calculating food Malaysia. consumption and growth rates in fishes. The movement 3 Universidade Federal de of food through the alimentary tract (gastric motility) of Sergipe, Programa de Pós- banded archerfish, Toxotes jaculatrix (Pallas, 1767), was Graduação em Ecologia e observed in the laboratory under natural conditions of Conservação, Av. Marechal temperature (27 °C) and salinity (28) using X-radiography, Rondon s/n, 49100-000, Rosa Elze, São Cristóvão, Brazil. with barium sulphate (BaSO4) as an inert food marker. Nutrient absorption along the alimentary tract (expressed as 4 Institute of Climate Change, relative percentage absorption gradient) was calculated using Universiti Kebangsaan Malaysia. the apparent digestibility coefficient (ADC) based on ash 5 School of Biological Sciences, contents of adjacent samples. We found that initial voiding University of Bristol, United of fecal matter began 6–12 hrs after feeding commenced, Kingdom. and that alimentary tracts were completely emptied within * Corresponding author email: 96 hrs. Considerable proportions of all macronutrients
Bulletin of Marine Science 903 © 2014 Rosenstiel School of Marine & Atmospheric Science of the University of Miami 904 Bulletin of Marine Science. Vol 90, No 4. 2014
The banded archerfish, Toxotes jaculatrix (Pallas, 1767), along with all members of the family Toxotidae, are known for their feeding technique wherein they use jets of water to knock aerial prey into the water where they can be eaten (Gill 1909, Allen 2004). Toxotes jaculatrix is euryhaline and can be found in both fresh waters and in more saline coastal waters (Froese and Pauly 2007). It has been reported that T. jacu- latrix is insectivorous (Gill 1909), however, recent longterm gut content analysis of natural populations have shown that their diets are more varied than previously sug- gested and include a large proportion of aquatic invertebrates and some small fishes (Simon et al. 2009, Simon and Mazlan 2010, Goutham-Bharathi et al. 2013). To date, nothing is known about archerfish gastric evacuation time and nutrient absorption along the alimentary tract. Rates of digestion and passage of food through the alimentary tracts of fishes have been studied in many other species because of their value in estimating food con- sumption and biomass production rates in fish populations (Windell 1978, Fänge and Grove 1979, Barbieri et al. 1998, Tekinay 2001, Heng et al. 2007). To develop tro- phic models that describe energy flow in marine food webs, important factors such as consumption, evacuation, and absorption of food need to be considered (Talbot 1985). Studies on food passage through the alimentary tract have also played an im- portant role in the rationale for development of efficient food supply and dietary formulation for captive and cultured fish (Fänge and Grove 1979). In general, digestion studies measure the rate and time for complete gastric evacu- ation, and examine where different nutrients are absorbed, as well as how different feeding rates affect nutrient uptake and digestive efficiency (Smith 1980, Wetherbee et al. 1987). Observation of the entire alimentary tract is technically difficult and digestion rates depend on several factors including, among others: fish size, water temperature, oxygen availability, meal size, feeding frequency, and food quality (Edwards 1973, Storebakken et al. 1999, Boyce et al. 2000, Temming and Herrmann 2001, Specziár 2002, Wuenschel and Werner 2004). Most estimates of gastric evacu- ation rate have examined large, fast-growing species of aquaculture importance, es- pecially temperate species (Jobling and Davies 1979, dos Santos and Jobling 1992, Mazlan and Grove 2003, Sweka et al. 2004, Andersen and Beyer 2008, Miegel et al. 2010), but little is known about digestion rates in smaller, slower-growing species and animals inhabiting tropical waters. A variety of techniques are available to measure gut passage time. Observations of the disappearance of food from the alimentary tract (serial slaughter, X-radiography) or observation of the production of fecal matter at regular intervals (Barbieri et al. 1998) provide simple and effective approaches. Serial slaughter is commonly used; however, it does not allow for the examination of variation in food passage within an individual and requires the sacrifice of potentially valuable or difficult-to-obtain -ani mals. Molnar and Tolg (1960) developed an X-radiographic method for monitoring the disappearance of bony and other hardparts from fish stomachs, without sacrify- ing the fishes. Since the development of this methodology, X-ray observations have been used to quantify the movement of food through the alimentary tracts of various fishes (Edwards 1971, Grove et al. 1978, Heng et al. 2007). Our study investigated the transit time of food within the alimentary tract of T. jaculatrix fed with live mealworms. We also collected data for nutrient absorption along the alimentary tract of T. jaculatrix that were either laboratory fed or col- lected from the wild. Since nothing has been previously reported about archerfish Simon et al.: Gastric digestion in archerfish 905 digestion, this investigation provides the first account of some basic parameters of digestion in T. jaculatrix.
Methods
Field Sampling and Laboratory Preparation.—In total, 168 fish (12–23 cm TL and 35–276 g body weight) were collected from coastal waters of Johor (01°24´53˝N, 104°09´44˝E) off the southern part of Peninsular Malaysia from July 2008 to June 2009. Of the 168 fish, 110 fish were captured using trammel nets (wild fed group) and were frozen at −20 °C in the laboratory. These wild-fed fish were packed in styrofoam box containing multiple layers of ice flakes to avoid extrusion of rectal content due to rigormortis. Upon examination it was found that all 110 wild fish had retained some or all of their gut contents and none were empty. The alimentary tract of wild-fed fish were removed and cut into four sections (Fig. 1) and their contents (e.g., crabs, shrimps, and insects) completely extruded. Due to the small size of the alimentary tract sections and the various analyses to be performed, the contents from each sec- tion of the alimentary tract—(A) stomach, (B) anterior intestine, (C) mid-intestine, and (D) rectum—of all wild fed fish were pooled into small plastic bottles (60 ml), homogenized, frozen (at −16 °C), and then freeze dried. The nutrient analyses was performed on 2–3 samples (replicates) from the pooled digesta.
Figure 1. X-radiography and schematic of banded archerfish Toxotes( jaculatrix) alimentary tract (A) stomach, (B) anterior intestine (black arrow indicates pyloric caeca), (C) mid intestine, and (D) rectum (posterior intestine). 906 Bulletin of Marine Science. Vol 90, No 4. 2014
The remaining 58 fish, which were captured using cages, were kept alive and trans- ported to the laboratory and housed in 150-L holding tanks. These fish (the laborato- ry-fed group) were maintained at ambient temperature (27 °C) and salinity (28) in the laboratory for a minimum of 2 wks prior to feeding experiments. These fish were fed to satiation three times weekly with live mealworms (laboratory fed group: 48). The 10 remaining fish (14–22 cm TL, 45–275 g body weight) from the group of 58 that fed actively were selected for an X-radiography experiment and the contrast agent barium sulphate (BaSO4) was added to their diet (BaSO4 group, for details see below). Food Passage Through the Alimentary Tract.—The laboratory-fed fish were transferred to eight experimental tanks (20 L), six fish per tank, and the BaSO4 group was placed in five experimental tanks (3 L), two fish per tank, and deprived of food for 120 hrs. When fed, the laboratory fed group were provided with live mealworms [22
(SE 1) mm] and the BaSO4 group was provided BaSO4 containing mealworm (meal- worm was frozen and freeze dried and injected BaSO4 paste at the ratio of 1 g BaSO4 to 5 ml distilled water prior to feeding). Both groups were fed to satiation (6%–8% body wet weight) at different predetermined time sequences (i.e., 6, 12, 24, 36, 48,
60, 72, and 96 hrs). The BaSO4 group was then anesthetized with α-methyl quinoline (Transmore®; Nika Trading, Puchong, Malaysia) (0.22 ml L−1 in 3 L of sea water as a anasthetic medium for 10–15 min) and X-rayed at predetermined times post feed- ing in a microradiographic unit (M60, Softex, Tokyo, Japan), and images acquired to trace the movement of food along the gut passage. The BaSO4 fish were allowed to recover and then were returned to their holding tank at the end of each round of X-rays between 3–4 min. All anesthetized fish were carefully handled to minimize physiological stress by placing them on blotted wet tissues with Stress Coat® liquid (APITM Aquarium Pharmaceutical, USA).
The laboratory fed group were fed fresh mealworms without BaSO4 marker and were then killed by a blow to the head. The brain was destroyed by pithing and the alimentary tract was dissceted out without disturbing the contents of different sec- tions of the gut. Samples from all fish were pooled by gut section and frozen at −16 °C. The frozen alimentary tracts were treated similarly to the wild samples. The feces of laboratory-fed fish were collected twice daily by siphoning, they were then sieved and soaked in distilled water and frozen at −16 °C and kept in plastic bottles for fur- ther chemical analysis. Nutrient Digestibility.—The diet and gastrointestinal contents of laboratory- fed fish were weighed to the nearest milligram, and moisture content was determined by measurement of weight loss after drying the samples in a freeze dryer (Christ Alpha 1–2 L Dplus, Shropshire, UK) at −40 to −60 °C for 3–4 d depending on the size of the samples. The dried samples were ground to a homogenous powder using a mortar and pestle and kept in small glass vials in desiccators for further biochemi- cal analyses. The inorganic matter or ash content of each part of gut samples was determined using a furnace (WiseTherm® FM Digital Muffle Furnaces, Nabertherm, Germany) at 450 °C for 3.5 hrs. The ash content was calculated by the difference be- tween initial and final dry weight of the sample. Total nitrogen content of diet and gut content was measured using a CHNS analyzer (EAGER 300, Thermo Finnigan, Italy). Protein was calculated from nitrogen content multiplied by the factor of 6.25 (Jones 1931). The lipid content of diet and gut content was measured by petroleum Simon et al.: Gastric digestion in archerfish 907 ether extraction using a Soxtec System (2055 Soxtec Avanti; Foss Teacator, Höganäs, Sweden). Carbohydrate levels of dry weight were calculated by the formula:
Percent carbohydrate = 100 − (percent protein + percent lipid + percent ash) following Xin et al. (2008). Energy content (kJ g−1) (gross energy, GE) of the samples was determined by bomb calorimeter (IKA® C200 Calorimeter System; IKA® Werke GmbH & Co. KG, Staufen, Germany). Nutrient absorption along the alimentary tract was determined using the apparent digestibility coefficient, ADC (%) = 100 × [1 − (Asha : Nutrienta ÷ Ashb : Nutrientb)], based on ash content of samples collected from adjacent segments of the gastrointes- tinal tract (Conover 1966, Kolb and Luckey 1972, Maynard et al. 1979). Statistical Analysis.—All nutrient data, except experimental meals and feces, were tested for normality (Anderson-Darling test) and homogeneity of variances (Levene test) using Minitab statistical software v14. Data were found to be normally distributed, but the variances were unequal; therefore, ANOVA and Tamhane’s (post hoc test) tests were employed for multiple comparisons of means using SPSS statisti- cal software (v15). To compare nutrient concentrations in experimental meals and feces, a Mann-Whitney test was used to account for the small sample size. The level of significance used was P < 0.05.
Results
Food Passage Through the Alimentary Tract.—Passage of food marked with BaSO4 through the alimentary tracts of T. jaculatrix could be observed clearly (Fig. 2). The X-radiographic images showed the stomach was full and a small portion of food entered the proximal and distal arms of the intestine loop and partly reached the rectum within 6–12 hrs after feeding (Fig. 2B). After 24, 36, and 48 hrs post feed- ing, approximately 10%–20% of the original meal remained in the stomach (Fig. 2), whereas after 60 and 72 hrs post feeding, <5% of the original meal remained in the stomach. Finally, within 96 hrs post feeding, the food items had completely left the stomach and were concentrated in the posterior part of the intestine and rectum, ready to be defecated (Fig. 2). Nutrient Digestibility.—Experimental food (mealworms) contained signifi- cantly less ash [38.93% (SE 0.12)] and moisture content [75.29% (SE 0.23)] than fe- ces [ash 76.93% (SE 0.31), moisture (80.01% (SE 0.52)] (Table 1). Stomach contents of both wild-fed [39.39% (SE 0.25)] and laboratory-fed [37.92% (SE 0.51)] fish contained less ash content than anterior [wild = 43.69% (SE 0.57), lab = 49.21% (SE 1.48)] and posterior intestines [wild = 57.91% (SE 0.26), lab = 74.36% (SE 0.51)] (Table 2). In the wild-fed samples, the ash content rose from 39.39% to 43.69% of the sample dry mass by the time the food reached the anterior intestine (Table 2A). Similarly, ash con- tent in lab samples rose progressively toward posterior regions of the gut to approxi- mately 74.36% (Table 2B). Moisture content in laboratory fed fish decreased along the gut from 72.03% to 45.02% (Table 2B). A similar trend was observed in wild-fed fish, where moisture content decreased from 78.05% to 42.04% along the gut (Table 2A). However, the concentrations of ash and moisture at various times after feeding 908 Bulletin of Marine Science. Vol 90, No 4. 2014
Figure 2. Passage of radio-opaque barium sulphate (BaSO4) through the alimentary tract of banded archerfish Toxotes( jaculatrix). Each panel shows the passage of ingested BaSO4 treated food.
mealworms in laboratory-fed fish confirm that these were changed between each gut region (Fig. 3). Protein was by far the largest fraction of the organic matter present in dried sam- ples of mealworms [49.35% (SE 0.86)], which contained less lipids [11.12 (SE 0.56)] and low carbohydrate levels [0.60% (SE 0.01)]. In feces, the values were reduced; how- ever, statistical differences were observed only in the concentration of protein and lipid (Table 1). Simon et al.: Gastric digestion in archerfish 909
Table 1. Nutrient analysis of experimental meals [% dry weight (SE)] for food (meal worms) and feces (for laboratory-fed fish). Mean values within the same column having the same superscript arenot significantly different P( > 0.05). For all analyses here, n = 6.
Ash Moisture Protein Total lipid Carbohydrate Energy (kJ g−1) Food 38.93a (0.12) 75.29a (0.23) 49.35a (0.86) 11.12a (0.56) 0.60a (0.01) 13.23a (0.47) Feces 76.93b (0.31) 80.01b (0.52) 18.98b (0.34) 3.17b (0.13) 0.93a (0.04) 4.89b (0.56)
Stomach contents of both wild-fed and laboratory-fed fish contained higher pro- tein and lipid content than other parts of the alimentary tract (Table 2). In laborato- ry-fed fish, carbohydrate fractions were normally <1% of dry mass for all portions of the alimentary tract, while it was relatively high in the anterior [1.20% (SE 0.02)] and posterior [0.90% (SE 0.01)] intestine of wild-fed fish (Table 2). No significant differences over time were found in the concentrations of each -nu trient (except the carbohydrate in anterior gut section) within a gut section (Fig. 3). However, nutrient contents were found to decrease progressively along the gut at all times (Table 2B). These declines were significant for protein, total lipids, and digestible energy content (Table 2B). For protein, contents of adjacent sections of the gut were not significantly different at specific time periods since feeding; however, decline in protien was significant between gut sections. Similar results were also observed for total lipids and digestible energy content (Fig. 4B). Carbohydrate content changed in a different manner along the gut. A significant increase occurred between stomach and anterior intestines, followed by a clear decline except for at 36 hrs. A significant declining trend of CHO was evident from mid intestine to rectum (Fig. 3E). The mean energy content for whole mealworms and feces was statistically different (Table 1), as was energy content for different portions of the gut in captive-fed fish (Table 2B). This trend was also observed in the wild-fed fish (Table 2A). For wild-fed fish, whatever the original diet mix, approximately 17.3% protein, 5.0% total lipids, and 27.3% carbohydrate appeared to have been absorbed in passage be- tween the stomach and the anterior intestine (including the pyloric caeca). An appar- ent negative absorption between samples from the stomach and anterior-intestine was observed for carbohydrate analyzed (Fig. 4C). In the passage of the remaining nutrients from mid-intestine to rectum, approximately 20.40% protein, 41.00% total lipids, and 2.00% carbohydrate were absorbed. Laboratory-fed fish, fed on a mealworm diet, showed similar patterns of ADC for the major nutrients when compared with wild-fed fish fed on a natural diet (Fig. 4A–D). The difference from wild-fed fish was that active absorption of protein, lipids, and energy was detected in all parts of the intestine, including the region between the anterior intestine and mid-intestine. In both groups of fish, net absorption had begun as food passed from the stomach into the anterior intestine. The pattern for carbohydrates, however, was again different. The apparent net addition of carbo- hydrates to gastrointestinal contents occurred in the stomach due to the presence of stomach mucus (carbohydrate), which also persisted in the anterior intestine for laboratory-fed fish (Fig. 4C). Between the stomach and rectum in laboratory-fed fish, the overall effect of absorption in the different zones meant that 75% of protein, 96% of lipids, 50% of carbohydrate, and 87% of energy had been removed. Comparison of food with feces in these fish suggested that a further small absorption of all nutrients occurred in the rectum. However, it is equally likely that this apparent increase in ADC could have been caused at least in part by leaching of nutrients from the faeces prior to collection (Table 3). 910 Bulletin of Marine Science. Vol 90, No 4. 2014 n 2 2 3 2 3 3 2 2 ) −1 (0.24) (0.25) (0.25) (0.25) b c a d 3.24 11.28 13.28 15.73 23.77 (0.35) 22.76 (0.25) 20.98 (0.36) 21.20 (0.51) > 0.05). n = number of Total energy (kJ g energy Total ) for (A) wild-fed and (B) 3 2 2 2 3 n 3 2 2 (0.02) (0.02) (0.02) (0.02) c b d b Total CHO Total 0.85 (0.01) 1.20 (0.02) 0.90 (0.01) 1.00 (0.01) 0.78 0.67 0.74 0.66 2 2 3 2 2 n 3 2 2 (0.25) (0.01) (0.25) (0.25) a b c d Total lipid Total 9.84 (0.52) 8.97 1.12 6.98 9.32 (0.25) 7.95 (0.26) 5.30 (0.36) 12.63 Organic compound Organic 3 2 2 2 2 n 3 2 2 (0.16) (0.25) (0.25) (0.25) a c b d Protein 49.92 (0.25) 45.79 (0.25) 39.81 (0.44) 35.79 (0.25) 48.78 33.54 41.04 23.86 2 2 2 2 n 3 3 2 2 (0.36) (0.25) (0.30) (0.51) c e b d Moisture 42.04 (0.11) 78.05 (0.14) 63.32 (0.21) 59.07 (0.15) 59.70 45.02 72.03 52.97 3 2 2 n 2 2 3 2 2 Inorganic compound Inorganic (1.48) (0.51) (0.51) (0.25) a c b d Ash 39.39 (0.25) 43.69 (0.57) 51.34 (0.36) 57.91 (0.26) 58.73 37.92 74.36 49.21 Ant int = Anterior intestine, Mid Int = Posterior int intestine. Ant int = Stomach Intestine (Mid int) Rectum (Posterior int) Pyloric caeca (Ant int) Pyloric caeca (Ant int) Intestine (Mid int) Rectum (Posterior int) Stomach (A) Wild-fed fish Wild-fed (A) replicates analyzed. laboratory-fed fish. For laboratory fish, mean values within the same column having the same superscript are not significantly different ( P different significantly not are superscript same the having column same the within values mean fish, laboratory For fish. laboratory-fed Table 2. Nutrient analysis of Table digesta [% dry weight (SE)] from four jaculatrix alimentary tract sections of banded archerfish ( Toxotes (B) Laboratory-fed fish Simon et al.: Gastric digestion in archerfish 911
Figure 3. Macronutrients in percent dry weight (A: ash, B: moisture, C: total protein, D: total lipid, E: total carbohydrate, and F: total energy) concentrations in digesta from different gut sections of laboratory fed banded archerfish Toxotes( jaculatrix) at different times after feeding whole mealworms. Asterisks indicate significant differences at P < 0.05 level. (Ant int = Anterior intestine, Mid int = Mid intestine). Number of replicates in each time sequence = 6.
Discussion
Based on X-radiographic observations using BaSO4 as an inert marker, T. jaculatrix take approximately 96 hrs to pass food from the stomach to the rectum. Comparable rates of food passage were observed with and without BaSO4, demonstrating that at concentrations of up to 25% of volume of digesta BaSO4 does not alter digestion rate, consistent with previous findings (Edwards 1971, Jobling et al. 1977, Ross and Jauncey 1981). This is the first report of food passage rates in T. jaculatrix, or any 912 Bulletin of Marine Science. Vol 90, No 4. 2014
Figure 4. Apparent digestibility coefficient (ADC, percent dry weigth) for (A) protein, (B) lipid, (C) carbohydrate, (D) energy between adjacent regions of the banded archerfish Toxotes( jacu- latrix). Gray bars are values for wild-fed fish, while white bars are for laboratory-fed fish with-
out BaSO4 in food. Bars to the right of the vertical dashed line are values for non-adjacent gut sections. [Fd = food, St = stomach (section a; for description of sections of the digestive tract see Fig. 1), Ant int = Anterior intestine with pyloric caeca (section b), Mid int = Mid intestine (section c), Rect = rectum posterior intestine/rectum (section d), Fec = Feces). archerfish for that matter, and the 96-hr time period to completely eliminate chitin- ous mealworm food from the alimentary tract is longer reported for other carnivo- rous fishes, e.g., brook trout Salvelinus[ fontinalis (Mitchill, 1814) = approximately 70 hrs; Sweka et al. 2004]; whiting [Merlangius merlangus (Linnaeus, 1758) = 72 hrs; Mazlan and Grove 2003]. The differences are likely due to differences in type of- ex perimental food and feeding regime used in the study (Flowerdew and Grove 1979, Gutowska et al. 2004, Fines and Holt 2010). There were relative changes in the nutrient content of digesta as it passed through the system, which may have been attributable to the addition of mucus and enzymes during digestion. We found that the concentrations of proteins and lipids, as well as energy content, decreased along the gut. The decrease of these nutrients is related to absorption, higher in the initial gut (degradation in stomach) and lower in the distal region of the gut. Similar findings have been reported for other carnivorous fish- es, namely, rainbow trout (Onchorhynchus mykiss Walbaum, 1792 Austreng 1978), Atlantic cod [Gadus morhua (Linnaeus, 1758); Lied et al. 1982], and whiting (M. mer- langus; Mazlan and Grove 2003). There was no indication that lipids or proteins were Simon et al.: Gastric digestion in archerfish 913
References
Lied and Lambertsen 1985
Gaylord and Gatlin 1996 Fernández et al. 1998 Nengas et al. 1997 1998 Gomes Da Silva and Olivia-Teles Mazlan and Grove 2003 Present study Hossain 1998 dos Santos and Jobling 1988 Lied et al. 1982, Grisdale-Helland and Helland 1998 et al. 2000 Tibbetts Cho et al. 1985 Dimes and Haard 1994 Cowey et al. 1974 Espe et al. 1999 Røsjø et al. 2000 Sveier et al. 1999 Olsen et al. 1998 – – – – – – – – – 95.0 91.0 95.8 81.4 90.0 88.4 90.3 91.0 85.0–95.0 Gross energy – – – – – – – – – – – – – – 82.0 80.5 22.0 82.7 Carbohydrates Nutrient digestibility – – – – – – – 88.0 86.0 96.6 85.6 82.0 97.5 97.0 97.9 94.0 94.0 88.0 Lipids – – – – 77.0 90.8 83.0 93.0 94.1 80.6 91.0 84.1 90.7 92.0 70.0 91.0 82.0 82.5 Proteins Food – feces Food – feces Food – feces Food – feces Food – feces Food – feces Food – feces Food – feces Food – feces Food – feces Food – feces Food – feces Food – feces Food – feces Food – hindgut Food – stomach Ant int – Mid Stomach – Ant int Stomach – Sample acquisitions Diets Brown fish meal Fish meal Fresh sprats Meal worm Fish meal (menhaden) Fish meal Fish meal Fish meal (herring) Fish meal (Saithe) Fish meal (herring) Fish meal (herring) Meat + bone meal Fish meal (herring) Fish meal Fish meal diet Isoenergetic Cod muscle Fish meal (Linnaeus, 1758) (Linnaeus 1766) (Linnaeus, 1758) (Pallas, 1767) Sparus aurata (Linnaeus, 1758) S. aurata Merlangius merlangus (Linnaeus, 1758) jaculatrix Toxotes Table Table 3. Comparison of the nutrient apparent digestibility coefficients for protein, lipids, carbohydrate, grossintestine, Mid Int = intestine. energy (% dry weight) in various carnivorous fish species. Ant int = Anterior Species Sciaenops ocellatus labrax (Linnaeus, 1758) Dicentrarchus Clarias gariepinus (Burchell, 1822) (Linnaeus, 1758) Gadus morhua G. morhua (Lesueur, 1817) (Lesueur, Anguilla rostrata 1792) mykiss (Walbaum, Oncorhynchus O. mykiss platessa Pleuronectes Salmo salar (Linnaeus, 1758) S. salar S. salar Salvelinus alpinus Hippoglossus hippoglossus (Linnaeus, 1758) 914 Bulletin of Marine Science. Vol 90, No 4. 2014 selectively retained in, or expelled from, the stomach relative to other nutrients dur- ing the 96-hr period. Although the carbohydrate level in the mealworm diet was low, this component increased in the anterior intestine, possibly reflecting secretion of mucus as has been reported previously (Kapoor et al. 1975, Mazlan and Grove 2003). Estimates of carbohydrate content are derived from modelled calculations that are based on values for other nutrients and may be subject to limitations of how well existing models fit this species. Additionally, estimates of moisture content may have been affected by the necessity to thaw and refreeze our samples prior to measure- ment of moisture content. For protein, the small negative ADC between original food and stomach contents is thought to be due to release of enzymes from the secretory “oxynticopeptic” gland cells of the stomach wall, and similar findings were reported for whiting (Mazlan and Grove 2003). Approximately 40% of the protein leaving the stomach was absorbed in the short anterior intestine where numerous pyloric caeca are located. These results are consistent with those for rainbow trout (O. mykiss) (Austreng 1978, Dabrowski et al. 1986), and Atlantic salmon [Salmo salar (Linnaeus, 1758)], in which 40%–50% of amino acid absorption took place in the pyloric region (Krogdahl et al. 1999). In T. jaculatrix, about 75% of the protein had been removed between the stomach and rectum, and the apparent further removal based on the fecal analysis measurement (81%) may include nutrient leaching into the experimental tank water. Protein ADCs are typically in the range of 82%–93%, although occasionally values as low as 70% have been reported (Table 3). There was no indication of total lipid digestion after food entered the stomach. Contents in distal parts of the guts probably come from different meals because of the prolonged digestion time. After reaching the anterior intestine, 45% of the lipid content had been absorbed. Lipid digestion and absorption take place mainly in the anterior intestine, where the pyloric caeca are situated and pancreatic lipases are secreted (Borlongan 1990). In T. jaculatrix, a considerable amount of lipid was ab- sorbed between the mid-intestine (35%) and rectal sections (88%), as has been re- ported for other carnivorous fishes (Ferraris and Ahearn 1984, Krogdahl et al. 1999). The differences may result when food enters the stomach, additive effects of protein- acous enzymes and acid may increase the energy content. Typical ADCs for lipid are in the range 86%–97%, although values as low as 82% have been reported (Table 3). The patterns for energy removal between the gut sections closely followed those of the high-energy lipids. Greater than 85% of food energy was removed upon reaching the rectum, and fecal contents suggested a maximum ADC of 81%. In theory, the ADC value between food and fecal materials should have been higher due to com- plete assimilation, however, in the present experiment, the form of feces excreted was more fluid than solid, and was thus potentially subject to considerable dilution in the experimental tanks. Typical expected values are in the 85%–95% range (Table 3). Absorption of carbohydrates was relatively low in comparison with proteins and lipids, which probably reflects relatively low carbohydrate content in the mealworm diet, thus accounting for the lower total energy value as well. ADC values indicated that considerable addition of carbohydrates occurred in the stomach as well as in the anterior intestine, probably in the form of mucus (Kapoor et al. 1975). Absorption continued in the posterior regions of the gut, leading to overall ADC values of 50% (rectum) or 22% (feces) (Table 3). Simon et al.: Gastric digestion in archerfish 915
The ADC values obtained here are likely to vary under different conditions. Although the data from wild-fed fish give generally similar patterns to those of labo- ratory-fed fish, the unknown diet (e.g., the quantity/composition) eaten in the wild as well as timing of last meal in wild-caught fish may have led to the observed differenc- es (e.g., negative ADC values in the stomach and anterior intestine). However, inde- pendent sampling of common prey items in the present study enabled us to quantify the proximate contents of the wild-fed group of T. jaculatrix. Also, variable results among similar studies of other carnivorous fishes indicate that many variables such as diet types, meal size, ration energy, water temperature, and type of marker need to be carefully considered for comparability (Cui et al. 1994, Pääkkönen and Marjomäki 1997, Andersen 1998, Boyce et al. 2000, Temming and Herrmann 2001, Specziár 2002, Wuenschel and Werner 2004). A slow rate of food passage prolongs digestion and absorption time and may reflect the increased challenge of digesting prey with hard exoskeletons (e.g., mealworms; archerfish are considered as opportunistic feeder see detail in Simon et al. 2009, Goutham-Bharathi et al. 2013), or a reduced comsumption rate owing to determi- nant growth in this species. Banded archerfish have never been reported to exceed 30–40 cm in length, either in the wild or in captivity. Longer gastric emptying time as procured in the present study are consistent with other similar studies using the chitinous feeds (Gutowska et al. 2004, Fines and Holt 2010). Prolonged stomach emp- tying may reflect a high degree of resiliency for archerfish species living in limited food resource environments.
Acknowledgments
We would like to thank the three anonymous reviewers and JE Serafy for their very useful comments that greatly improved the quality of the manuscript. We also would like to thank the dedicated laboratory technicians of the Marine Science laboratory, UKM for their coop- eration in collecting samples. This study was supported by Ministry of Science Technology and Innovation, Malaysia (MOSTI) through the Science fund grant # 04-01-02-SF0124, and Institute of Climate Change, Universiti Kebangsaan Malaysia through the Young Researcher Incentive Grant # GGPM-2011-057 and University Research Grant GUP-2013-006. A pre- liminary review of this manuscript was provided by J Sweka (United States Fish & Wildlife Service). All field sampling and laboratory protocols followed and complied with the current laws of Malaysia.
Literature Cited
Allen GR. 2004. Toxotes kimberleyensis, a new species of archerfish (Pisces: Toxotidae) from fresh waters of Western Australia. Rec West Aust Mus. 56:225–230. http://dx.doi.org/10.3 853/j.0067-1975.56.2004.1423 Andersen NG, Beyer JE. 2008. Precision of ingestion time and evacuation predictors for indi- vidual prey in stomachs of predatory fishes. Fish Res. 92:11–22. http://dx.doi.org/10.1016/j. fishres.2007.12.007 Andersen NG. 1998. The effect of meal size on gastric evacuation in whiting. J Fish Biol. 52:743–755. http://dx.doi.org/10.1111/j.1095-8649.1998.tb00817.x Austreng E. 1978. Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture. 13:265–272. http://dx.doi.org/10.1016/0044-8486(78)90008-X 916 Bulletin of Marine Science. Vol 90, No 4. 2014
Barbieri RL, Leite RG, Stermann FA, Hernandez-Blazquez FJ. 1998. Food passage time through the alimentary tract of brazilian teleost fish, Prochilodus scrofa (Steindachner, 1881) using radiography. Braz J Vet Res Anim Sci. 35(1):32–36. http://dx.doi.org/10.1590/ S1413-95961998000100006 Borlongan IG. 1990. Studies on the digestive lipases of milkfish, Chanos chanos. Aquaculture. 89:315–325. http://dx.doi.org/10.1016/0044-8486(90)90135-A Boyce SJ, Murray AWA, Peck LS. 2000. Digestion rate, gut passage time and absorp- tion efficiency in the Antarctic spiny plunderfish. J Fish Biol. 57:908–929. http://dx.doi. org/10.1111/j.1095-8649.2000.tb02201.x Cho CY, Cowey CB, Watanabe T. 1985. Finfish Nutrition in Asia: methodological approaches to research and development. Ottawa, Ontario, IDRC. 154 p. Conover RJ. 1966. Assimilation of organic matter by zooplankton. Limnol Oceanogr. 11:338– 345. http://dx.doi.org/10.4319/lo.1966.11.3.0338 Cowey CB, Adron J, Blair A. 1974. Studies on the nutrition of marine flatfish. Utilization of various dietary proteins by plaice (Pleuronectes platessa). Br J Nutr. 31:297–306. http:// dx.doi.org/10.1079/BJN19740038 Cui Y, Chen S, Shaomei W. 1994. Effect of ration size on the growth and energy budget of the grass carp Ctenopharyngodon idella Val. Aquaculture. 123:95–107. http://dx.doi. org/10.1016/0044-8486(94)90122-8 Dabrowski K, Leray C, Nonnotte G, Colin DA. 1986. Protein digestion and ion concentra- tions in rainbow trout (Salmo gairdneri Rich.) digestive tract in sea- and fresh water. Comp Biochem Physiol A. 83:27–39. http://dx.doi.org/10.1016/0300-9629(86)90084-8 Dimes LE, Haard NF. 1994. Estimation of protein digestibility-I. Development of an in vitro method for estimating protein digestibility in salmonids (Salmo gairdneri). Comp Biochem Physiol A. 108:349–362. http://dx.doi.org/10.1016/0300-9629(94)90106-6 dos Santos J, Jobling M. 1988 Gastric emptying in cod, Gadus morhua L.: effects of food particle size and dietary energy content. J Fish Biol. 33:511–516. http://dx.doi. org/10.1111/j.1095-8649.1988.tb05495.x dos Santos J, Jobling M. 1992. A model to describe gastric evacuation in cod (Gadus morhua L.), fed single-meals of natural prey. ICES J Mar Sci. 49:145–154. http://dx.doi.org/10.1093/ icesjms/49.2.145 Edwards DJ. 1971. Effect of temperature on rate of food passage through the alimenta- ry canal of the plaice, Pleuronectes platessa (L.) J Fish Biol. 3:433–439. http://dx.doi. org/10.1111/j.1095-8649.1971.tb05915.x Edwards DJ. 1973. The effects of drugs and nerve section on the rate of passage of food through the gut of the plaice, Pleuronectes platessa (L.). J Fish Biol. 5:441–446. http://dx.doi. org/10.1111/j.1095-8649.1973.tb04473.x Espe M, Sveier H, Hoegoey I, Lied E. 1999. Nutrient absorption and growth of Atlantic salm- on (Salmo salar L.) fed fish protein concentrate. Aquaculture. 174:119–137. http://dx.doi. org/10.1016/S0044-8486(98)00502-X Fänge R, Grove DJ. 1979. Fish digestion. In: Hoare W, editor. Fish physiology. New York: Academic Press. p. 161–260. Fernández F, Miquel AG, Guinea J, Martinez R. 1998. Digestion and digestibility in gilthead sea bream (Sparatus aurata): the effect of diet composition and ration size. Aquaculture. 166(1–2):67–84. http://dx.doi.org/10.1016/S0044-8486(98)00272-5 Ferraris RP, Ahearn GA. 1984. Sugar and amino acid transport in fish intestine. Comp Biochem Physiol A. 77:397–413. http://dx.doi.org/10.1016/0300-9629(84)90204-4 Flowerdew MW, Grove DJ. 1979. Some observations of the effects of body weight, tempera- ture, meal size and quality on gastric emptying time in the turbot, Scophthalmus maximus (L.) using radiography. J Fish Biol. 14:229–238. http://dx.doi.org/10.1111/j.1095-8649.1979. tb03514.x Froese R, Pauly D. 2006. FishBase. 7 June, 2007. Accessed 10 January, 2008. Available from: http://www.fishbase.org Simon et al.: Gastric digestion in archerfish 917
Gaylord TG, Gatlin DM. 1996. Determination of digestibility coefficients of various feed- stuffs for red drum (Sciaenops ocellatus). Aquaculture. 139:303–314. http://dx.doi. org/10.1016/0044-8486(95)01175-7 Gill Th. 1909. The archerfish and its feats. Smithsonian Miscellaneous Collections. 52:277–286. Gomes Da Silva J, Olivia-Teles A. 1998. Apparent digestibility coefficients of feedstuffs in seabass (Dicentrarchus labrax) juveniles. Aquat Liv Res. 11:187–191. http://dx.doi. org/10.1016/S0990-7440(98)80115-0 Goutham-Bharathi MP, Mohanraju R, Krishnan P, Sreeraj CR, Simon KD. 2013. Stomach con- tents of banded archerfish, Toxotes jaculatrix (Pallas, 1767) (Toxotidae) from brackish wa- ters of South Andaman, India. Asian Fish Sci. 26:243–250. Grisdale-Helland B, Helland SJ. 1998. Macronutrient utilization by Atlantic halibut (Hippoglossus hippoglossus): diet digestibility and growth of 1 kg fish. Aquaculture. 166:57– 65. http://dx.doi.org/10.1016/S0044-8486(98)00274-9 Grove DJ, Loizides LG, Nott J. 1978. Satiation amount, frequency of feeding and gastric emptying rate in Salmo gairdneri. J Fish Biol. 12:507–516. http://dx.doi.org/10.1111/j.1095-8649.1978. tb04195.x Heng HG, Ong TW, Hassan MD. 2007. Radiographic assessment of gastric emptying and gas- trointestinal transit time in hybrid tilapia (Oreochromis niloticus × O. mossambicus). Vet Radiol Ultrasound. 48(2):132–134. http://dx.doi.org/10.1111/j.1740-8261.2007.00218.x Hossain MA. 1998. Optimization of feeding and growth performance of African catfish (Clarias gariepinus Burchell, 1822) fingerlings. Dissertation, University of Stirling. Jobling M, Davies PS. 1979. Gastric evacuation in plaice, Pleuronestes platessa L.: effects of tem- perature and meal size. J Fish Biol. 14:539–546. http://dx.doi.org/10.1111/j.1095-8649.1979. tb03553.x Jobling M, Gwyther D, Grove DJ. 1977. Some effects of temperature, meal size and body weight on gastric evacuation time in the dab, Limanda limanda (L.). J Fish Biol. 10:291–298. http:// dx.doi.org/10.1111/j.1095-8649.1977.tb05134.x Jones DB. 1931. Factors for converting percentages of nitrogen in foods and feeds into percent- ages of protein. USDA Circ No. 183:1–21. Kapoor BG, Smit H, Verighina IA. 1975. The alimentary canal and digestion in teleosts. Adv Marine Biol. 13:109–239. http://dx.doi.org/10.1016/S0065-2881(08)60281-3 Kolb AR, Luckey TD. 1972. Markers in nutrition. Nutr Abstr Rev. 42:813. Krogdahl A, Nordrum S, Soerensen M, Brudeseth L, Røsjø C. 1999. The effects of diet composition on apparent nutrient absorption along the intestinal tract and of subsequent fasting on mu- cosal disaccharidase activities and plasma nutirent concentration in Atlantic salmon Salmo salar. Aquacultur Nutr. 5(2):121–133. http://dx.doi.org/10.1046/j.1365-2095.1999.00095.x Lied E, Julshamn K, Braekkan O. 1982. Determination of protein digestibility in Atlantic cod (Gadus morhua) with internal and external indicator. Can J Fish Aquat Sci. 39:854–861. http://dx.doi.org/10.1139/f82-116 Lied E, Lambertsen G. 1985. Apparent availability of fat and individual fatty acids in Atlantic cod (Gadus morhua). Fisk Dir Skr Ser Ernaering. 2:63–75. Maynard LA, Loosli JK, Hintz HF, Warner RG. 1979. Digestive processes in different species, in Animal Nutrition, New York, McGraw-Hill, inc. p. 21–46. Mazlan AG, Grove DJ. 2003. Gastric digestion and nutrient absorption along the alimentary tract of whiting (Merlangius merlangus L.) fed on natural prey. J Appl Ichthyol. 19:229–238. http://dx.doi.org/10.1046/j.1439-0426.2003.00471.x Miegel RP, Pain SJ, van Wettere WHEJ, Howarth GS, Stone DAJ. 2010. Effect of water tem- perature on gut transit time, digestive enzyme activity and nutrient digestibility in yel- lowtail kingfish (Seriola lalandi). Aquaculture. 308:145–151. http://dx.doi.org/10.1016/j. aquaculture.2010.07.036 Molnar G, Tolg I. 1960. Röntgenologic investigation of the duration of gastric digestion in the pike-perch (Lucioperca lucioperca L.) Acta Biologica Hungarica. 11:103–108. 918 Bulletin of Marine Science. Vol 90, No 4. 2014
Nengas I, Foundoulaki E, Alexi MN, Papoutsi E. 1997. Digestibility of nutrients in diets of seabream (Sparus aurata) containing different levels of protein and lipids. Proc Hell Symp Oceanogr Fish Fisheries, Aquaculture, Inlands Waters. 2:161–164. Olsen RE, Henderson RJ, Ringo E. 1998. The digestion and selective absorption of dietary fatty acids in Arctic charr, Salvelinus alpinus. Aquacult Nutr. 4:13–21. http://dx.doi. org/10.1046/j.1365-2095.1998.00099.x Pääkkönen J-PJ, Marjomäki TJ. 1997. Gastric evacuation rate of burbot fed single-fish meals at different temperatures. J Fish Biol. 50:555–563. http://dx.doi.org/10.1111/j.1095-8649.1997. tb01949.x Røsjø C, Nordrum S, Olli JJ, Krogdahl A, Ruyter B, Holm H. 2000. Lipid digestibility and me- tabolism in Atlantic salmon (Salmo salar) fed medium-chain triglycerides. Aquaculture. 190:65–76. http://dx.doi.org/10.1016/S0044-8486(00)00395-1 Ross B, Jauncey K. 1981. A radiographic estimation of the effect of temperature on gastric emptying time in Sarotherodon niloticus (L.) × S. aureus (Steindachner) hybrids. J Fish Biol. 19:333–344. http://dx.doi.org/10.1111/j.1095-8649.1981.tb05836.x Simon KD, Bakar Y, Samat A, Zaidi CC, Aziz A, Mazlan AG. 2009. Population growth, tro- phic level, and reproductive biology of two congeneric archerfishes (Toxotes chatareus, Hamilton 1822 and Toxotes jaculatrix, Pallas 1767) inhabiting Malaysian coastal waters. J Zhejiang Univ Sci B. 10(12):902–911. http://dx.doi.org/10.1631/jzus.B0920173 Simon KD, Mazlan AG. 2010. Trophic position of archerfish species (Toxotes chatareus and Toxotes jaculatrix) in the Malaysian estuaries. J Appl Ichthyol. 26(1):84–88. http://dx.doi. org/10.1111/j.1439-0426.2009.01351.x Smith LS. 1980. Digestion in teleost fishes, in fish feed technology. FAO (Food and Agriculture Organization of the United Nations), United Nations Development Program, Rome. p. 3–18. Specziár A. 2002. In situ estimates of gut evacuation and its dependence on temperature in five cyprinids. J Fish Biol. 60:1222–1236. http://dx.doi.org/10.1111/j.1095-8649.2002.tb01716.x Storebakken T, Kvien IS, Shearer KD, Grisdale-Helland B, Helland SJ. 1999. Estimation of gas- trointestinal evacuation rate in Atlantic salmon (Salmo salar) using inert markers and col- lection of faeces by sieving: evacuation of diets with fish meal, soybean meal or bacterial meal. Aquaculture. 172:291–299. http://dx.doi.org/10.1016/S0044-8486(98)00501-8 Sveier H, Wathne E, Lied E. 1999. Growth, feed and nutrient utilization and gastrointestinal evacuation time in Atlantic salmon (Salmo salar): the effect of dietary fish meal particle size and protein concentration. Aquaculture. 180(3–4):265–282. http://dx.doi.org/10.1016/ S0044-8486(99)00196-9 Sweka JA, Cox MK, Hartman K. 2004. Gastric evacuation rates of Brook trout. Trans Amer Fish Soc. 133:204–210. http://dx.doi.org/10.1577/T02-064 Talbot C. 1985. Laboratory methods in fish feeding and nutritional studies. In: Tytler P, Calow P, editors. Fish Energetics New Perspectives. Croom Helm, London & Sydney. p. 125–154. http://dx.doi.org/10.1007/978-94-011-7918-8_5 Tekinay AA. 2001. The use of X-radiography for the return of appetite measurements in rain- bow trout (Oncorhynchus mykiss Walbaum, 1792). Turkish J Mar Sci. 7:31–39. Temming A, Herrmann J-P. 2001. Gastric evacuation in horse mackerel. I. The effects of meal size, temperature and predator weight. J Fish Biol. 58:1230–1245. http://dx.doi. org/10.1111/j.1095-8649.2001.tb02282.x Tibbetts SM, Lall SP, Anderson DM. 2000. Dietary protein requirement of juvenile American eel (Anguilla rostrata) fed practical diets. Aquaculture. 186:145–155. http://dx.doi. org/10.1016/S0044-8486(99)00363-4 Wetherbee BM, Gruber SH, Ramsey A. 1987. X-radiographic observations of food passage through digestive tracts of lemon sharks. Trans Amer Fish Soc. 116:763–767. http://dx.doi. org/10.1577/1548-8659(1987)116<763:XOOFPT>2.0.CO;2 Simon et al.: Gastric digestion in archerfish 919
Windell JT, Foltz JW, Sarokon JA. 1978. Effects of fish size, temperature and amount fed on nutrient digestibility of a pelleted diet by rainbow trout, Salmo gairdneri. Trans Amer Fish Soc. 107:613–616. http://dx.doi.org/10.1577/1548-8659(1978)107<613:EOFSTA>2.0.CO;2 Wuenschel MJ, Werner RG. 2004. Consumption and gut evacuation rate of laboratory-reared spotted seatrout (Sciaenidae) larvae and juveniles. J Fish Biol. 65:723–743. http://dx.doi. org/10.1111/j.0022-1112.2004.00479.x Xin Li, Xiaoyun H, Yunbo L, Guoying X, Xianbin J, Kunlun H. 2008. Comparative analysis of nutritional composition between herbicide-tolerant rice with bar gene and its non- transgenic counterpart. J Food Compos Anal. 21:535–539. http://dx.doi.org/10.1016/j. jfca.2008.06.001
B M S