THE EFFECT OF A PRODUCE BASED GUT LOATNG DIET ON MINERAL, VITAMIN AND CAROTENOID CONTENT OF ADULT CRICKETS {ACHETA DOMESTICA)

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1 THE EFFECT OF A PRODUCE BASED GUT LOATNG DIET ON MINERAL, VITAMIN AND CAROTENOID CONTENT OF ADULT CRICKETS {ACHETA DOMESTICA) Amy S. Hunt, MS, 1 * Michael Birt, a Ann M. Ward, MS 1 ^ort Worth Zoological Gardens, 1989 Colonial Parkway, Fort Worth, TX MilfordDrive, Fort Worth, TX Abstract Adequate supplementation of calcium a other nutrients including vitamin A is integral to successfully holding insectivorous amphibians a reptiles in captivity. Dusting a gut loading insects are two commonly used methods of supplementation. A produce based gut loading diet may be more palatable a thus may gut load more successfully a for an exteed period of time. Adult crickets {Acheta domestica) were held on four produce based high calcium diets or a calcium carbonate diet for four days. Intake a survivability were recorded a insects were analyzed for mineral, vitamin, a carotenoid content. Survivability was high, only three out of approximately 9 crickets died over the study period. Intake did not change significantly within diet over time. The desired 1:1 calcium:phosphorus ratio was not achieved, however, the ratio was significantly higher in the apple, canot, a sweet potato treatments over that of the calcium carbonate a papaya treatments. A very wet diet (13-1% dry matter) may have precluded the ability of the crickets to consume enough to achieve the high calcium content. Retinol concentrations stayed consistently low throughout the study period (no detection or < 333 iu/kg, dry matter basis), however, there was a significant increase in the level of beta carotene of adult crickets consuming the canot a sweet potato diets. More research is needed to determine if beta carotene can be used as a source of vitamin A for insectivorous amphibians a reptiles a also to determine if a dry produce based diet would lead to successful gut loading. Introduction Invertebrate insects lack vital nutrients including calcium 1, a vitamin A. Several studies have attempted to supplement insects in order to reach a 1:1 calcium:phosphorus (Ca:P) ratio. 1 ' ' " n,i3,i5 j3 us ting the insect with a calcium-based powder a gut loading with a high (8%) calcium diet are two sporadically successful methods of calcium supplementation. 1 ' 9 ' ' 15 Numerous factors affect the ability to achieve the desired 1:1 Ca:P ratio by gut loading 1 ' 9 a, in general, dusting is greatly inconsistent. 15 Previous research has investigated factors such as age, stocking density, time on diet, water source, temperature, a particle size of the diet on the success of gut-loading. 7 " 11 It is possible that palatability of the diet may also have an effect on successful gut-loading. Survivability of the insect may be compromised when feeding a non-palatable high calcium diet. Gut loading is most effective between days two a four of feeding. 1 Considering the fact that many insects are held for up to seven days, a diet that supports a readily available 1:1 Ca:P ratio would be most practical for keeper management. The objective of this study was to create a

2 palatable gut loading diet a to test its effect on intake a survivability along with mineral, vitamin, a carotenoid concentrations of crickets. Methods Crickets {Acheta domestica) were obtained from the Armstrong Cricket farm, West Monroe, LA. Upon arrival, approximately g of crickets were removed, fasted for hours, a frozen at - 8 C for subsequent baseline a control dry matter, mineral, vitamin, a carotenoid analysis. Remaining crickets were divided into 15 tenariums (11.5" x 5.5" x 8.5"), with ventilated plastic tops (Penn-Plax, Incorporated, Hauppauge, NY), 3 grams per tenarium. Several squares of egg cartons provided shelter a distilled water was offered fresh daily in ** boats with marbles to prevent drowning. Crickets were fasted for hours to evacuate their gut contents a subsequently raomly assigned to one of five dietary treatments. The experiment consisted of three replicates of each of the following treatments: apple, papaya, canot, sweet potato, or calcium carbonate. Calcium carbonate was offered as is, however, each of the other food items was bleed with calcium carbonate to create an approximately eight % calcium (dry matter basis) slurry diet. All diets were formulated using the Animal Nutritionist (N-squared, Inc. Durango Software, Silverton, OR, USA). The diet was pre-made in bulk a a gram sample removed a frozen at -8 C for subsequent baseline dry matter, mineral, vitamin, a carotenoid analysis. The remaining slurry diet was carefully weighed as 15 gram portions into two*two*two labeled a tared weigh boats. Each day the remaining diet was removed a a fresh 15 gram portion offered. The remaining diet was dried at 6 C a weighed in order to calculate daily dry matter intake. The experiment lasted four days. A 15 gram sample of crickets was removed at days two a four a frozen at - 8 C for subsequent analysis. Over the four day period temperature a deaths were recorded. Laboratory analysis Insect a diet samples were pulverized (size = one mm) using liquid nitrogen a a mortar a pestle. All samples were split into two portions, one to be dried for mineral analysis a the other frozen at -8 C for vitamin a carotenoid analysis. Mineral analysis Samples were dried in a Fisher Isotemp Oven at 6 C to determine dry matter content. Once dry, they were regrou using a coffee grier (Mr. Coffee coffee mill, Model 316, Clevela, OH) into a homogenous mixture (size = one mm). Samples of approximately a half gram were dried at 8 to a constant weight for determination of a dry matter conection factor. Samples of approximately a half gram were digested using nitric acid in a CEM MARS-X (Microwave Accelerated Reaction System). Digested samples were analyzed for calcium, phosphoms, magnesium, sodium, a iron using a Varian Iuctively Coupled Plasma atomic emission spectrometer according to the Association of Analytical Chemists (AOAC) Official Methods of Analysis. 3

3 Vitamin a carotenoid analysis Approximately two grams of each sample was homogenized with a % pyrogallol methanol solution a then alloquoted at 1.5 grams each into 3 test tubes. Samples were saponified in a 6% v/v potassium hydroxide solution a a 7 C water bath for 6 minutes. Following saponification, samples were extracted according to methods previously described 1 a analyzed for retinol, alpha- a gamma-tocopherol, beta carotene, lutein, canthaxanthin, echinenone, beta-cryptoxanthin, a zeaxanthin using a Waters 695 separations module HPLC, with a Grace Vydac 1TP5 column a a Waters 87 dual wavelength absorbance detector. Statistical analysis One-way ANOVA's for iepeent samples were coucted to determine if there were differences in the calcium:phosphorous ratio a/or mineral, vitamin or carotenoid concentrations as a result of different dietary treatments. A one-way ANOVA for iepeent samples was coucted to see any significant differences in intake between treatments (diets) a a one-way ANOVA for conelated samples was coucted to see if there was a change in intake over time within treatment. Data are presented as mean + staard enor (SEM). Level of significance was set at five percent. (VasserStats: Website for Statistical Computation Richard Lowry, , Poughkeepsie, NY). Results Adult crickets on the calcium carbonate diet treatment consumed significantly less food over the period of four days than those on the apple, canot, sweet potato a papaya diet treatments (Table ). There were no statistically significant differences in daily intake within diet treatments over time (Table 3). Crickets did not reach the optimal 1:1 Ca:P ratio within the four day study period. The Ca:P ratio, however, was significantly higher at days two in the apple a canot treatments a at day four in the apple, canot a sweet potato treatments than that of the calcium carbonate a papaya treatments. There was a significant decrease in the Ca:P ratio from day zero (baseline) to day two in the calcium carbonate, papaya a sweet potato treatments, however, by day four there was not a significant difference in the sweet potato treatment Ca:P ratio. Mineral concentrations varied over the four day period, most decreasing significantly from day zero to days two a four (Table ). Retinol concentrations stayed consistently low throughout the study period (no detection or < 333 iu/kg cricket dry matter basis). There was a significant decrease in the level of alpha tocopherol from day zero (baseline) to two in the papaya treatment, however the papaya treatment at day two was the only treatment that did not have a significant decrease in the level of gamma tocopherol. All other treatments had a significant decrease in the level of gamma tocopherol by day two. Significant increases occuned in the beta carotene levels of adult crickets consuming the canot a sweet potato diets. The concentration of beta cryptoxanthin significantly decreased in all treatments with the exception of papaya a there was a significant increase in the level of lutein solely in the canot treatment (Table 5).

4 Only three crickets of approximately 9 crickets died over the study period, all on the calcium carbonate diet treatment. Temperature remained constant at 76 F over three days, dropping slightly to 7 F on day four. Discussion The desired 1:1 Ca:P ratio was not achieved in this study. It is likely that this occurred because of the high water content of the diets. A typical gut loading diet has a dry matter content of approximately 9% whereas the fruit a vegetable diets used in this study had dry matters varying between 13% a 1%. There was, however, no decrease in intake over time a therefore the diets were most likely palatable. It is likely that the crickets' guts became full with water a, though the diets were formulated for approximately eight percent calcium on a dry matter basis, the crickets never consumed enough dry matter to achieve the high calcium levels. The palatability of a fruit or vegetable based diet may be an integral part of keeping crickets alive a gut loaded over an exteed period of time. Very few deaths occuned during this four day period in contrast to those on a typical cricket feed. 9 The three deaths that did occur were crickets on the calcium carbonate treatment. It would be interesting to see if a dry fruit or vegetable diet would be as palatable as a wet diet, a if so, if the crickets would achieve the 1:1 Ca:P ratio. The analyzed mineral concentrations of feeder crickets in this study that met minimum carnivore requirements were phosphorus (.6%), magnesium (.%), a sodium (.68%). Neither calcium (.9%), 1 iron (8 ppm), 1 nor vitamin E (1 iu/g) 1 met minimum requirement levels. The concentration of vitamin A in feeder crickets in this study as well as others ' 5 did not meet 19 minimum carnivore requirements (5-9 iu/kg, maintenance, growth/reproduction). It is therefore necessary to supplement not just calcium but also vitamins A a E. No significant changes in the concentration of vitamin A occuned between treatments in this study; however there was a significant increase in beta carotene in the canot a sweet potato treatments over that of the other treatments. It is unknown, however, whether amphibians a reptiles can utilize beta carotene as a source of vitamin A. Additional research is necessary in this area. In conclusion, neither a calcium carbonate diet nor four wet fruit a vegetable based diets successfully gut loaded adult crickets. More research is needed to determine the best way to create a palatable diet that will increase survivability of the cricket while successfully gut loading calcium a vitamin A. Acknowledgements The authors would like to thank Roy McClements for his help in the design of this study.

5 LITERATURE CITED 1. Allen, M.E., a O.T. Oftedal Dietary manipulation of the calcium content of feed crickets. J. Zoo Wildl. Med. : Aerson, S J.. Increasing calcium levels in cultured insects. Zoo Biology. 19: AOAC Official Methods of Analysis of the Association of Analytical Chemists International. 16 ed. Published by AOAC International, Gaithersburg, MD. Volumes I a n.. Barker, D., Fitzpatrick, M.P., Dierenfeld, E.S Nutrient composition of selected whole invertebrates. Zoo Biology. 17: Finke, M.D.. Complete nutrient composition of commercially raised invertebrates used as food for insectivores. Zoo Biology. 1: Finke, M.D. 3. Gut loading to enhance the nutrient content of insects as food for reptiles: a mathematical approach. Zoo Biology. : Finke, M.D., Dunham, S.U., Cole, J.S.. Evaluation of various calcium-fortified high moisture commercial products for improving the calcium content of crickets, Acheta domesticus. Journal ofherpetological Medicine a Surgery. 1: Finke, M.D., Dunham, S.U., Kwabi, CA. 5. Evaluation of four commercial gut loading products for improving the calcium content of crickets, Acheta domesticus. Journal of Herpetological Medicine a Surgery. 15: Hunt, A.S., McClements, R.D., Ward, A.M. 5. The effect of tenarium size, egg crate area, stocking density, a time on cricket mortality a the ability to achieve a 1:1 calcium to phosphorus ratio. Proc. 6 Conference on Zoo a Wildlife Nutrition, AZA Nutrition Advisory Group Hunt, A.S., Ward, A.W., Ferguson, G. 1. Effects of a high calcium diet on gut loading in varying ages of crickets (Acheta domesticus) a mealworms (Tenebrio molitor). Proc. th Conference on Zoo a Wildlife Nutrition, AZA Nutrition Advisory Group McClements, R.D., Lintzenich, B.A. a Slifka, K.A. 3. Calcium a insect gut loading: The development of a protocol for achieving the best Ca:P ratio for insectivorous animals. Proc. 5 Conference on Zoo a Wildlife Nutrition, AZA Nutrition Advisory Group National Research Council. Nutrient Requirements of Cats. 6. Washington, DC: National Academy Press Schlegal, M.L., Renjifo, A., Valdes E.V. 5. Evaluating gut-loading diets a dusting to improve the calcium concentration of pin-head a adult crickets (Acheta domesticus). Proc. 6 th Conference on Zoo a Wildlife Nutrition, AZA Nutrition Advisory Group Stacewicz-Sapuntzakis, M., Bowen, P.E., Kikeall, J.W., Burgess, M Journal of Micronutrient Analysis 3: Trusk, A.M., a S. Crissey Comparison of calcium a phosphorus levels in crickets fed a high calcium diet versus those dusted with supplement. Proceedings of the 6 th a 7 Dr. Scholl Conferences on the Nutrition of Captive Wild Animals

6 Table 1: Dry matter, mineral, a vitamin content of high calcium apple, canot, papaya, sweet potato slurry diets or calcium carbonate (CaC3) diet fed to adult crickets over a period of four days. Mineral a vitamin concentrations are on a dry matter basis. Apple Dry matter, % Calcium, % Phosphorous, % Potassium, %' Magnesium, % Sodium, % Iron, ppm Manganese, ppm Zinc, ppm Retinol, iu/kg Alpha Tocopherol, iu/kg Gamma Tocopherol, ppm Beta Carotene, ppm Lutein, ppm Beta Cryptoxanthin, ppm Zeaxanthin, ppm Apple <.1.95 <.1 Papaya < Sweet potato < Canot < CaC Table : Average intake (four days, dry matter basis) per gram of adult cricket fed a high calcium apple, canot, papaya, sweet potato slurry diet or calcium carbonate. Calcium carbonate Apples Canots Sweet Potato Papaya.113±.* a.317 ±.5 b.13 ±.16 b.38 ±.1 b.3 ±.18 b * Values expressed as mean ± staard enor of the mean (SEM). abc Means fou in rows with identical letters are not significantly different by single factor ANOVA (PO.5). Table 3: Average daily intake (n = three, grams ± staard enor of the mean) on a dry matter basis per gram of adult cricket fed a high calcium apple, canot, papaya, sweet potato slurry diet or calcium carbonate. There were no statistically significant differences within diet over time. Day 1 3 Calcium carbonate.1 ±.9.5 ±.9.8 ± ±.7 Apples.55 ±.19. ±.8.3 ±.1.17 ±.1 Canots.6 ±.3.38 ±.. ± ±. Sweet Potato.39 ±..31 ±..3 ±.7.35 ±.9 Papaya.6 ± ±.3.1 ±.3.38 ±.

7 Table : Mean (n = three) Calcium:Phosphorous ratio a mineral (dry matter basis) concentration of adult crickets fed a high calcium apple, canot, papaya, sweet potato slurry or calcium carbon ate diet at days zero ^baseline), two a four o f intake. Sweet Apples CaC3 Papaya potato Calcium: Phosphoms Calcium, % Phosphorous, % Magnesium, % Sodium, % Day.13 ±. ac.13 ±.3 a.7 ±.13 ab.7 ±.6 ac.57 ±.1 b.56 ±.1 ac.3 ±. b.3 ±.3 b.8 ±.3 bc.9 ±.6 bc.5 ±. b.6 ±.9 b.3 ±. b. ±.6 c.61 ±.1 b.6± 9abc. ±.3 b.3 ±.7 b.7 ±. c.7 ±.3 bc Canots.15 ± ±.13 ac.18 ±. a.11 ±.7.9 ±.7 a.11 ±.15 ab.75 ±.3.6 ±.18 a.66 ±.15 b.11 ±.7. ±.3 b. ±.3 b.37 ±.1.11 ±.6 b.1 ±.6 c ±.333. ±.6 b.7 ±.9 b.3 ±.3 b. ±.6 c.6 ±.35 b.56 ±. ac. ±.3 b. ±.3 b.6 ±.3 c.7 ±.6 b.7 ±. bc.11 ±.37 a. ±.13 ab.9 ±.3 ab.58 ±.1 b.56 ±.1 ac.3 ±.3 b.3 ±.3 b.8 ±.7 bc.9 ±. b Iron, ppm ±.3 bc 6.5 ± bc 5.37 ± ac 16.3 ± 8.1 b 3.97± 1.76 bc 37.1 ± 18.7 ± 5.5 ± ± ±.66 abd 3.31 c b ad 191abd * Values expres jsed as mean ± stanc ard enor oft le mean. a c Means fou in rows with identical letters are not significantly different by single factor ANOVA (P<.5).

8 Table 5: Mean (n = three) vitamin a carotenoid concentrations on dry matter basis of adult crickets fed an high calcium apple, canot, papaya, sweet potato slurry or calcium carbonate diet at days zero (bas«jline), 1 wo a four of h itake. Sweet Apples potato Retinol, iu/kg Alpha Tocopherol, iu/kg Gamma Tocopherol, ppm Beta Carotene, ppm Beta cryptoxanthin, ppm Lutein, ppm Zeaxanthin Canthaxanthin Echinenone Day no detection no detection node tection 3.9 ± I.999* 8.5 ± ab 6.19±.1 b 5.93 ±.15 b <.1 b <.1 b CaC3 Canots Papaya 9.99 ± ± 3.5 ± 3.6 ±.685 a.18 ab.53 b 3.31 ± ± 18. ± a.931 a.639 b 5.6 ±.99 b 5.96 ±. b <.1 b <.1 b 8.8 ± ±.95 b 6.13 ±.3 b < ± b.8 ±.665 b 1.5 ±.15.5 ±.7 c.68 ±.66 c <.1.75 ±.19 b 1.6 ±.35 b 7.11 ± 1.56 ab 6.3 ± 1.86 b 1.7±.39 a 1.65 ±.318 a.58 ± 1.91 ab.8 ± 1.76 b 5.9 ±.56 b 5.68 ±.569 b 1.83 ±.965 c 3.1 ±.315 b.5 ±.59 c.89 ±.9 c * Values expressed as mean ± staard enor of the mean. = not detectable a c Means fou in rows with identical letters are not significantly different by single factor ANOVA (PO.5).