• View in gallery

    CONSORT flow diagram for the Tenebrio molitor (mealworms) diet trial.

  • View in gallery

    CONSORT flow diagram for the Zophobas morio (superworms) diet trial.

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Effects of various diets on the calcium and phosphorus composition of mealworms (Tenebrio molitor larvae) and superworms (Zophobas morio larvae)

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  • 1 Department of Clinical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.
  • | 2 Department of Nutrition, Philadelphia Zoo, Philadelphia, PA 19104.
  • | 3 Zodiac Pet & Exotic Hospital G/F #4 Gordon Road, Tin Hau, Hong Kong.
  • | 4 Department of Clinical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.
  • | 5 Department of Clinical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104.
  • | 6 USDA, APHIS Inspection Service, Riverdale, MD 20737.

Abstract

OBJECTIVE To evaluate whether the nutritive quality of Tenebrio molitor larvae and Zophobas morio larvae, which are commonly cultured as live food sources, is influenced by 4 commercially available diets used as nutritional substrates; identify which diet best improved calcium content of larvae; and identify the feeding time interval that assured the highest calcium intake by larvae.

ANIMALS 2,000 Zophobas morio larvae (ie, superworms) and 7,500 Tenebrio molitor larvae (ie, mealworms).

PROCEDURES Larvae were placed in control and diet treatment groups for 2-, 7-, and 10-day intervals. Treatment diets were as follows: wheat millings, avian hand feeding formula, organic avian mash diet, and a high-calcium cricket feed. Control groups received water only. After treatment, larvae were flash-frozen live with liquid nitrogen in preparation for complete proximate and mineral analyses. Analyses for the 2-day treatment group were performed in triplicate.

RESULTS The nutrient composition of the high-calcium cricket feed groups had significant changes in calcium content, phosphorus content, and metabolizable energy at the 2-day interval, compared with other treatment groups, for both mealworms and superworms. Calcium content and calcium-to-phosphorus ratios for larvae in the high-calcium cricket feed group were the highest among the diet treatments for all treatment intervals and for both larval species.

CONCLUSIONS AND CLINICAL RELEVANCE A 2-day interval with the high-calcium cricket feed achieved a larval nutrient composition sufficient to meet National Research Council dietary calcium recommendations for nonlactating rats. Mealworm calcium composition reached 2,420 g/1,000 kcal at 48 hours, and superworm calcium composition reached 2,070g/1,000 kcal at 48 hours. These findings may enable pet owners, veterinarians, insect breeders, and zoo curators to optimize nutritive content of larvae fed to insectivorous animals.

Abstract

OBJECTIVE To evaluate whether the nutritive quality of Tenebrio molitor larvae and Zophobas morio larvae, which are commonly cultured as live food sources, is influenced by 4 commercially available diets used as nutritional substrates; identify which diet best improved calcium content of larvae; and identify the feeding time interval that assured the highest calcium intake by larvae.

ANIMALS 2,000 Zophobas morio larvae (ie, superworms) and 7,500 Tenebrio molitor larvae (ie, mealworms).

PROCEDURES Larvae were placed in control and diet treatment groups for 2-, 7-, and 10-day intervals. Treatment diets were as follows: wheat millings, avian hand feeding formula, organic avian mash diet, and a high-calcium cricket feed. Control groups received water only. After treatment, larvae were flash-frozen live with liquid nitrogen in preparation for complete proximate and mineral analyses. Analyses for the 2-day treatment group were performed in triplicate.

RESULTS The nutrient composition of the high-calcium cricket feed groups had significant changes in calcium content, phosphorus content, and metabolizable energy at the 2-day interval, compared with other treatment groups, for both mealworms and superworms. Calcium content and calcium-to-phosphorus ratios for larvae in the high-calcium cricket feed group were the highest among the diet treatments for all treatment intervals and for both larval species.

CONCLUSIONS AND CLINICAL RELEVANCE A 2-day interval with the high-calcium cricket feed achieved a larval nutrient composition sufficient to meet National Research Council dietary calcium recommendations for nonlactating rats. Mealworm calcium composition reached 2,420 g/1,000 kcal at 48 hours, and superworm calcium composition reached 2,070g/1,000 kcal at 48 hours. These findings may enable pet owners, veterinarians, insect breeders, and zoo curators to optimize nutritive content of larvae fed to insectivorous animals.

Insects have been commercially cultured as live food sources for zoological collections and pet insectivores and omnivores for more than 40 years. The earliest invertebrate nutrient composition study1 evaluated mealworms as a dietary option for captive new world primates. As mealworms and other insects became recognized as diet options for captive insectivores, the nutritional growth and propagation requirements for many invertebrates were established.2,3 Zoo collections worldwide have since published culturing protocols for commonly fed insects.4,5 A complete review of the nutritive value of insects commonly used as live food (ie, feeder insects) is available.6

Bilby and Widdowson7 found a correlation between the nutritive quality of earthworms and insects fed to nestling birds and the digestive tract content of the earthworms and the substrate used to house the feeder insects. The practice of intestinal loading of invertebrates with a highly nutrient dense diet has become an accepted method to provide dietary supplementation with vitamins and minerals to insectivores and omnivores. This method involves feeding the invertebrate prey a nutrient dense diet for a specific time interval before the prey is offered to insectivorous animals. A number of studies7–21,a,b have led to the development of equations to ensure nutritive improvement on the basis of specified recommendations for dietary supplementation.

Several composition studies1,3,22,23 confirmed that feeder insects are poor in vitamin and mineral content, particularly mealworms and crickets. Diseases caused by nutritional deficiencies are commonly seen in captive insectivores, including avian, amphibian, and reptile species in zoological collections and in those kept as privately owned pets. In efforts to improve overall vitamin and mineral content of whole invertebrate prey, it has been found that the digestive tract content of feeder insects could be altered to improve their nutritive quality.7,8,a

Several diseases manifest as a result of hypocalcemia in animals kept in zoological collections and in those that are part of the pet trade industry, including nutritional hyperparathyroidism, dystocia, fibrous osteodystrophy, tetany, and pathological fractures.6,24,25 Improving the dietary calcium intake of captive insectivores has profound clinical importance in disease prevention. Various intestinal loading protocols for crickets and mealworms have been described; however, intestinal loading protocols have not been published for the popular feeder superworms (ie, Zophobas morio larvae).22,23,26 To our knowledge, longitudinal studies evaluating the nutritive quality of mealworms or superworms with diet exposures for > 48 hours are unavailable.

The purposes of the study presented here were to evaluate whether the nutritive quality of Tenebrio molitor larvae and Zophobas morio larvae were influenced by 4 commercially available diets, identify which diets significantly improve calcium content of larvae, and identify which time interval assured the highest calcium intake by larvae. Our null hypotheses were that the fed avian diets and cricket feed would influence the nutritive quality of both larval groups, the cricket feed would have significantly improve the calcium content in each larval group, and the highest calcium content in larvae would be identified at 48 hours of diet exposure.

Materials and Methods

Study design and measurement outcomes

A multiarm controlled diet trial was planned to evaluate the proximate and mineral compositions of mealworms (Tenebrio molitor larvae) and superworms (Zophobas morio larvae) fed 4 commercially available diets at various time intervals. The study protocol was approved by the University Of Pennsylvania Institutional Animal Care and Use Committee.

Primary outcomes for each treatment group included calcium expressed as percentage DM and calcium energy density expressed as grams per kilocalorie (g/kcal). Phosphorus, fat, and protein expressed as percentage DM, Ca:P expressed as a ratio, and ME and net energy expressed as kilocalorie per kilogram (kcal/kg) of DM were examined as secondary outcomes.

Animals

Tenebrio molitor larvaec (mealworms) were purchased from a commercial insect breeder. Insects were selected, weighed in grams and placed in 150-g replicates into 8.75 × 14.5 × 9.75-inch clear plastic containers with ventilated topsd (1 container/treatment for all time intervals). Each treatment group was given 48 hours to acclimate and allow for complete digestive passage of previous diets, which was newspaper bedding provided by the supplier. Mealworms were supplied a diet of wheat bran prior to transport from the vendor. Water was supplied as moistened cotton balls during the acclimation period and was changed daily.

Zophobas morio larvaee (superworms) were purchased from the commercial insect supplier. Insects were selected, weighed in grams, and placed in 40-g replicates into 8.75 × 14.5 × 9.75-inch clear plastic containers with ventilated topsd (1 container/treatment for each time interval separately). Treatment groups were given 48 hours to acclimate and allow for complete digestive passage of previous diets, which was newspaper bedding provided by the supplier. Superworms were supplied a diet of wheat bran prior to transport from the vendor. Water was supplied as moistened cotton balls during the acclimation period and was changed daily.

Diets

At the end of the acclimation period, the mealworm treatment groups received 450 g of the following trial diets as substrates: wheat millings,f avian hand feeding formula,g organic avian mash diet,h and high-calcium cricket feed.i The control group received water only. The substrate volume was determined to assure a 3-fold surface area allotment for the 150-g insect density in each treatment group. Treatment durations for diet exposure were designated at 2-, 7-, and 10-day intervals (Figure 1).

Figure 1—
Figure 1—

CONSORT flow diagram for the Tenebrio molitor (mealworms) diet trial.

Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.178

At the end of the acclimation period, the superworm treatment groups received 120 g of the following trial diets as substrates: wheat millings,f avian hand feeding formula,g organic avian mash diet,h and high-calcium cricket feed.i The control group received water only. The substrate volume was determined to assure a 3-fold surface area allotment for the 40-g insect density in each treatment group. Treatment durations for diet exposure were designated at 2-, 7-, and 10-day intervals (Figure 2).

Figure 2—
Figure 2—

CONSORT flow diagram for the Zophobas morio (superworms) diet trial.

Citation: American Journal of Veterinary Research 78, 2; 10.2460/ajvr.78.2.178

Outcome assessments

Container weight, DM substrate (diet) weight, and insect weight gains were measured daily in grams for all treatment groups and controls. Cotton balls moistened with water were weighed (grams), offered, and changed daily for both the control groups and treatment groups. The insects were housed in a temperature-controlled environment (24° to 30°C) in ventilated containers with 12-hour light to 12-hour dark photoperiods. At the end of the specified treatment durations, mealworms and superworms were harvested in 40-g aliquots from individual treatment containers, including controls that received no diet, with tongs and placed into cryogenic vials. Each cryogenic vial was labeled and weighed in grams. The sampled larvae were flash-frozen live with liquid nitrogen, stored at −80°C in a cryogenic freezer, and shipped within 48 hours to a commercial laboratoryj for nutritional analysis. Proximate analysis (to determine ash, DM, acid detergent fiber, total digestible nutrients, digestive energy, ME, moisture, crude fat, and crude protein content) and minerals analysis (to determine calcium, phosphorus, sulfur, manganese, magnesium, sodium, potassium, zinc, iron, and copper content) were performed on all control groups and treatment groups. Proximate and mineral analyses were performed in triplicate for each group. Official methods of analysis were performed for nutritional analysis as put forth by the Association of Official Analytical Chemists.27

Statistical analysis

Statistical analysis was performed by a reviewer, who was blind to treatment groups, using a commercially available software program.k Skewness and Kurtosis tests were performed to assess mineral groups by diet for normality for both the mealworm and superworm treatment groups. An ANOVA was performed on the specific proximate and mineral analytes for each treatment group (diet) and time interval (day).28 When the ANOVA revealed a value of P < 0.05, a Tukey pairwise comparison test was performed to determine the significance of the differences among temporal comparisons.

Results

For the Tenebrio molitor larvae (mealworms) treatment groups, there was no significant difference by group or day for percentage of phosphorus, fat, protein, net energy, or ME (DM basis). The highest calcium gains were found on day 2 across all treatment groups except for the control group. All of the significant differences in percentage calcium (DM basis), calcium (energy basis), and Ca:P ratio were in the high-calcium cricket feed group, compared with other treatment groups. There were no significant differences for the mean calcium content between day 7 (0.70) and day 10 (0.78) or for the Ca:P ratio between day 7 (0.97) and day 10 (1) within the high-calcium cricket feed group.

On days 2, 7, and 10, respectively, the mean percentage DM calcium of mealworms was as follows by treatments: 0.05%, 0.04%, and 0.06% for control (water only); 0.23%, 0.16%, and 0.15% for the avian hand feeding formula; 0.05%, 0.05%, and 0.05% for the wheat millings diet; 0.22%, 0.12%, and 0.22% for the avian mash diet; and 1.02%, 0.70%, and 0.78% for the high-calcium cricket feed. There was a significant (P < 0.001) difference in calcium percentage (DM basis) by treatment group. The mean calcium percentage for the high-calcium cricket feed was 1.02% (95% CI, 0.86% to 1.18%). The mean calcium percentage differences between the high-calcium cricket feed group and the other diet trials were as follows: 0.83% (95% CI, 0.64% to 1.02%) for the control group; 0.69% (95% CI, 0.48% to 0.89%) for the wheat millings group, 0.83% (95% CI, 0.62% to 1.03%) for the avian formula group, and 0.83% (95% CI, 0.48% to 0.89%) for the avian mash diet trial.

For mealworms on days 2, 7, and 10, respectively, the mean calcium (energy basis) values were 0.12, 0.11, and 0.15 g/kcal for the control group; 0.53, 0.37, and 0.36 g/kcal for the wheat millings group; 0.13, 0.11, and 0.12 g/kcal for the avian hand feeding formula group; 0.50, 0.28, and 0.50 g/kcal for the avian mash diet group; and 2.42, 1.79, and 2.06 g/kcal for the high-calcium cricket feed group. A significant (P < 0.001) difference in mean calcium (energy basis) values was found between treatment groups. For day 2, the mean calcium energy density for the high-calcium cricket feed was 2.42 g/kcal (95% CI, 2.01 to 2.82 g/kcal) in comparison with the same day means for the control group (0.12 g/kcal; 95% CI, 0.09 to 0.59 g/kcal), wheat millings (0.53 g/kcal; 95% CI, 0.13 to 0.94 g/kcal), avian hand feeding formula (0.13 g/kcal; 95% CI, 0.07 to 0.54 g/kcal), and avian mash (0.50 g/kcal; 95% CI, 0.10 to 0.91 g/kcal) diet trials.

For mealworms on days 2, 7, and 10, respectively, the mean Ca:P ratios were 0.07, 0.05, and 0.08 for the control group; 0.30, 0.22, and 0.22 for the avian hand feeding formula group; 0.06, 0.06, and 0.06 for the wheat millings group; 0.30, 0.18, and 0.27 for the avian mash diet group; and 1.31, 0.97, and 1.0 for the high-calcium cricket feed group. Additionally, the Ca:P ratio significantly (P < 0.01) differed by group. For day 2, mean Ca:P ratio for the high-calcium cricket feed group was 1.31 (95% CI, 1.12 to 1.49), whereas other groups had Ca:P ratios that ranged from 0.06 to 0.30. The mean Ca:P ratio differences between the high-calcium cricket feed group and other groups were 1.09 (95% CI, 0.87 to 1.30) for the control group, 0.88 (95% CI, 0.65 to 1.12) for the wheat millings group, 1.08 (95% CI, 0.85 to 1.32) for the avian formula group, and 0.89 (95% CI, 0.65 to 1.12) for the avian mash diet trial.

For the Zophobas morio larvae (superworms) treatment groups, there were no significant differences by group or day for percentage values of fat or net energy (DM basis). Significant differences were seen for percentage values of phosphorus, protein, and ME (DM basis).

On days 2, 7, and 10, respectively, the mean percentage DM phosphorus content of superworms was as follows by treatments: 0.64%, 0.71%, and 0.53% for the control (water only); 0.66%, 0.63%, and 0.53% for the avian hand feeding formula; 0.67%, 0.69%, and 0.56% for the wheat millings diet; 0.61%, 0.61%, and 0.21% for the avian mash diet; and 0.64%, 0.64%, and 0.58% for the high-calcium cricket feed. Percentage values for phosphorus (DM basis) varied by treatment group (P < 0.03). A significant (P < 0.022) decrease in the percentage of phosphorus was seen between day 2 and day 10 for the wheat millings fed superworms, which decreased from 0.66% to 0.53% (95% CI, 0.47% to 0.59%), and for the organic avian mash diet (P < 0.001), which decreased from 0.61% to 0.21% (95% CI, 0.15% to 0.27%).

Percentage values for protein (DM basis) significantly (P < 0.001) declined in the avian mash group between day 2 and day 10 from 44.6% to 16.3% (95% CI, 10.98 to 21.62). Metabolizable energy (DM basis) significantly (P < 0.032) decreased in the control group between day 2 and day 7 from 2.14 to 1.07 kcal/kg (95% CI, 1.48 to 1.90).

Significant differences in superworms for calcium content and Ca:P ratios between the treatment days were noted within the high-calcium cricket feed group. Within this group, mean percentage calcium (DM basis) significantly decreased between day 2 (0.95%) and day 7 (0.43%; 95% CI, 0.29% to 0.56%; P < 0.002) and between day 2 (0.95%) and day 10 (0.48%; 95% CI, 0.25% to 0.71%; P < 0.021). Within the high-calcium cricket feed group, significant differences were found in mean calcium (energy basis) between day 2 (2.07 g/kcal) and day 7 (0.93 g/kcal; 95% CI, 0.60 to 1.25 g/kcal; P < 0.003) and between day 2 (2.07 g/kcal) and day 10 (1.07 g/kcal; 95% CI, 0.52 g to 1.63 g/kcal; P < 0.038). Likewise, for mean Ca:P ratios in the high-calcium cricket feed group, significant differences were noted between day 2 (1.46) and day 7 (0.67; 95% CI, 1.27 to 1.66; P < 0.001) and between day 2 (1.46) and day 10 (0.83; 95% CI, 0.50 to 1.16; P < 0.024).

Larvae fed the high-calcium cricket feed met the NRC dietary calcium recommendations for rats on the basis of the percentage calcium (DM basis). The calcium recommendation for maintenance in nonlactating rats consuming a diet containing 3.8 to 4.1 kcal ME/g with 10% moisture is 0.5% calcium as fed or 0.55% calcium (DM basis). For the cricket feed–treated mealworm larvae, calcium percentages (DM basis) of 1.02% at day 2, 0.70% at day 7, and 0.78% at day 10 were achieved. In superworms, NRC calcium percentage (DM basis) recommendations were achieved at 0.95% on day 2. The calcium content significantly (P = 0.002) declined to 0.43% at day 7, but the slight increase to 0.48% at day 10 was not significant.

The NRC maintenance calcium energy density (DM basis) recommendations for rats ranges from 1.34 to 1.45 g/1,000 kcal, on the basis of consuming a diet containing 4,100 or 3,800 g ME/kcal, respectively.29 This was achieved in both invertebrate groups at 48 hours. Mealworm calcium compositions reached 2.42 g/kcal or 2,420 g/1,000 kcal in 48 hours, and superworm calcium composition reached 2.07 g/kcal or 2,070 g/1,000 kcal in 48 hours.

Discussion

Several intestinal loading protocols have been analyzed in various feeder insects. Most studies have evaluated dietary supplementation with calcium in domestic crickets (Acheta domesticus),10,14,18,30,a mealworms (T molitor),9,11,12,14 and, to a lesser extent, domestic flies (Drosophila melanogaster),18 waxworms (Galleria mellonella),15 and silkworms (Bombyx mori).8 Insects that have a naturally high calcium content include larvae of the black soldier fly (Hermetia illucens)31,32 and adult wood lice (Porcellio scaber).26 Since dietary supplementation with high mineral content can be toxic to insects, calcium-enriched substrates have been evaluated for optimal intake by insects. Dietary avoidance has been shown in crickets fed calcium carbonate additives of 40.7% within the substrate medium.9

Subsequent studies investigating the bioavailability of calcium in feeder insects used are scarce. Klasing et al12 evaluated varying calcium carbonate concentrations added to chicken starter rations. These diets were then fed to crickets and mealworms. A linear correlation between the calcium content in the substrate and digestive tract calcium content in the insects was found. Results of that study12 not only showed increased bone mineralization and density in chickens fed the treated crickets and mealworms, but it also revealed that leopard geckos (Eublepharis macularius) fed the treated crickets had radiographic evidence of increased growth and bone density. Various substrate options to improve calcium content of feeder mealworms for a collection of avian species were investigated in another study.11 A mealworm diet that contained 35% calcium carbonate was developed; in this study, mealworm larvae achieved a Ca:P ratio of 1.5 within 72 hours.

In the present study, the wheat millings, avian hand feeding formula, organic avian mash diet, and high-calcium cricket feed were selected as nutritional substrates for larvae. Wheat millings were evaluated for nutritive quality because it is the traditional transport media used by most commercial insect vendors and pet stores for mealworms and superworms. Because poultry diets have been a common choice to use in the evaluation of intestinal loading in mealworms, commercially available psittacine diets were evaluated in the present study for comparative nutritive contributions in larvae composition. There are no marketed high-calcium mealworm diets; however, the high-calcium cricket feed used in the present study was chosen because it had not been previously evaluated. All diets were chosen on the basis of their commercial accessibility and availability in pet stores and veterinary clinics.

The NRC dietary calcium recommendations for poultry range from 0.9% to 1.3% of the total diet ration (DM basis).33 As age and rearing conditions vary immensely for poultry production, established ranges are recommended on the basis of the purpose of production. Many recommendations were developed for optimal laying and meat production; this may not reflect recommendations designed for optimal health and longevity. Comparatively, the well-studied maintenance recommendations for rats were chosen as a basis of comparison with our data.

The NRC dietary calcium recommendations for rats on an energy basis range from 1.34 to 1.45 g/1,000 kcal DM (on the basis of a 4,100 g ME/kcal diet and 3,800 g ME/kcal diet, respectively).29 This would result in a minimum calcium percentage of 0.55% (DM basis) and minimum phosphorus percentage of 0.33% (DM basis) in a total diet. Given the dynamic metabolic relationship between calcium and phosphorus, the Ca:P ratio, targets of 1.5 for growth in nonlactating rats is recommended.

Since the high-calcium cricket feed contained 8.2% total calcium (DM basis), it was hypothesized that the insects in this treatment group would have increased total calcium content. Also, because the toxicity of calcium and dietary avoidance of high-mineral diets have not been evaluated in superworms, the expected degree of calcium ingestion was unknown. Results of the present study revealed that mealworm and superworm larva can survive on 8% calcium (DM basis) in diet rations without risk of acute death from calcium toxicosis. The performance of the avian hand feeding formula and organic avian mash, although not significant, did reveal improvements in total calcium value for mealworms and superworms, compared with that of wheat millings. The control groups revealed expected declines in calcium percentages and overall nutritive quality of larvae on the basis of treatment duration in the face of starvation and cannibalism.

Mealworm and superworm phosphorus contents were compared with the NRC phosphorus recommendations for nonlactating rats consuming a diet containing 3.8 to 4.1 kcal ME/g with 10% moisture and 0.3% phosphorus as fed or 0.33% phosphorus on a DM basis. All of the treatment groups and controls had higher phosphorus values than NRC feed recommendations for rats for all treatment durations. The phosphorus content found in these insect species compare closely with values of previous studies22,23,26 that documented the mineral content of insects. The phosphorus content for the high-calcium cricket feed group did not vary from the phosphorus content of control insects for both mealworms (0.76% phosphorus on a DM basis) and superworms (0.63% phosphorus on a DM basis).

The Ca:P ratios in all diet trials did not attain the NRC dietary Ca:P ratio recommendations for rats (1.66) on the basis of grams per kilogram of feed. In the present study Ca:P ratios that were most comparable were found in the high-calcium cricket feed group at 48 hours exposure, achieving a 1.31 Ca:P ratio in mealworms, and a 1.47 Ca:P ratio in superworms. The high phosphorus content of the insects is the limiting factor in obtaining a Ca:P ratio of 1.66.

The study reported here was designed to be a reproducible protocol that could be used by insect breeders, curators, and pet owners in efforts to improve the nutritional quality of the insects offered to captive insectivores and omnivores. There are several variables that could have altered the results of the present study, including vendor source, the use of historical controls, environmental factors influencing diet intake, substrate, humidity, diet particle size, and treatment duration. The mealworms and superworms came from different vendor sources, which may have inherently influenced the findings as a result of insect health differences among various vendor stock. Insects were not submitted for baseline nutritional composition on the day of arrival. As whole invertebrate composition studies22,23,26 have been well documented for mealworms and superworms, these published values were used as historical controls for untreated specimens for the arrival day.

In the present study, larvae were fasted for 48 hours14 to ensure that the previous digestive tract content (ie, wheat bran and newspaper) was expelled from larvae prior to diet treatments. It is possible that fasting for 48 hours is not required to ensure maximal trial dietary intake. The findings of the present study complimented the findings of studies8,11,12 that examined dietary intake of unfasted mealworms and crickets exposed to various high-calcium food substrates. It is important to note that previous studies8,11,12 on intestinal loading have confirmed that 48 hours is a sufficient period for mealworms to maximize their calcium content when fed a high-calcium diet.

Overall diet intake, particle size, food competition, and palatability were not monitored for individual insects and reflected limitations in the present study. It has been shown that particular substrate calcium content in cricket feed is associated with dietary avoidance behavior in crickets.9 Particle size plays a substantial role in overall diet intake as well. Mashes and ground substrates were provided with the intent to ensure ingestion of the diets. Substrate amounts were chosen on the basis of recommendations of the diet manufacturer: approximately 1 part insect mass to 3 parts substrate. This is meant to ensure adequate surface area for the insects and dietary ingestion and may be associated with reduced food competition among larvae. All diet trial groups gained weight for both mealworms and superworms (Figures 1 and 2; allocation and analysis). Larval crowding and molting behavior have been well studied in Zophobas morio34; in the study reported here, fewer total insects were housed in containers in efforts to avoid crowding and food competition. Palatability was not specifically monitored for each insect; however, changes in substrate weights from the start of treatment were measured to confirm food consumption across all groups.

In the present study, environmental humidity was not measured among the treatment groups and served as a study limitation because environmental conditions may have influenced dietary intake. Fresh water was supplied daily and uniformly to all diet treatment groups. Previous studies35,36 have investigated the water-focus behaviors of mealworm beetles, and this may be an important aspect in invertebrate hydration and may influence diet intake. It is also important to note that the absence of preservatives in the avian mash diet may have affected dietary intake as a result of palatability and freshness after a 10-day interval at room temperature.

Sample size calculations were not determined for the present study because of limited information in the literature on the commercially available diets and species-specific intestinal loading. This reflects a limitation of the present study. The sample sizes chosen were estimated from a previous study11 that evaluated dietary intake and calcium modification in mealworms, which included minimums of 5-g aliquots for mineral processing. Proper randomization (randomized group assignment after insects were divided into aliquots) and blinding of the personnel to diet exposure prior to harvest would have limited additional selection bias in this multiarm controlled diet trial.

Time intervals that were evaluated in the present study were chosen to mimic the harvesting scenarios most commonly encountered by zoological collections, curators, and pet owners. The 2-day treatment interval was chosen to allow for complete passage of the previous diet, ensure ingestion of the new diet, and allow for adequate time for rehydration. Larvae in transport and those housed in pet stores are often housed in media that do not contain food or water and are kept in refrigerators prior to being sold to prevent molting. This time interval reflects anticipated high-food intake by the insects, as most are starved and dehydrated from prior transport. Although not evaluated in the present study, other studies8,11,14 have revealed that increases in nutritive quality of mealworms occur as early as 24 hours after a diet is offered.

The 7-day treatment interval was chosen to assess whether specimens could thrive on the diets and to ascertain whether larval calcium content declines under prolonged treatment. The 10-day treatment interval was chosen to evaluate mealworms and superworms prior to their undergoing morphological change into beetles. Mealworm larvae molt into beetles after 14 days of ideal environmental conditions.5 This would substantially alter their nutritional composition. Superworms can continue to molt into new larval forms every 14 days for up to 5 months if population densities are high; however, continued larval molting may also alter nutritional composition of larvae.34,37

Future aims based on findings of the present study include evaluating other diets and protocols for other popular feeder insects. Evaluation of amino acid and vitamin composition in intestinal loaded larvae and assessment of nitrogen availability through chitin estimates38 in select invertebrate feeders are also warranted. The authors of the present study make no claims about the digestibility of the insects or the calcium bioavailability in treated larvae, as few studies have evaluated the complexity of the species specific digestive energetics of insectivorous amphibians,31 lizards,39,40 birds, primates, and mammals. Despite the reported presence of chitinases in some herptiles,41,42 cuticle density and invertebrate composition can substantially alter digestion of larvae by insectivores. The results of the present study can used by zoos, curators, insect breeders, veterinarians, and pet owners to increase the digestive tract calcium content of T molitor and Zophobas morio larvae and optimize nutrition for insectivorous animals.

Acknowledgments

Presented in oral form at the joint meeting of the Annual Conference of the Association of Zoos and Aquariums and the AZA Nutrition Advisory Group, Tulsa, Okla, October 2009.

ABBREVIATIONS

Ca:P

Calcium to phosphorus

CI

Confidence interval

DM

Dry matter

ME

Metabolizable energy

NRC

National Research Council

Footnotes

a.

Attard L. The development and evaluation of a gut-loading diet for feeder crickets formulated to provide a balanced nutrient source for insectivorous amphibians and reptiles. MS thesis, Department of Animal and Poultry Science. University of Guelph, ON, Canada, 2013.

b.

McClements RD LB, Slifka KA. Calcium and insect gut-loading: the development of a protocol for achieving the best Ca:P ratio for insectivorous animals (abstr), in Proceedings. 5th NAG Conf Zoo Wildl Nutr 2003;80.

c.

Bassett's Cricket Ranch, Visalia, Calif.

d.

Lee's Kritter Keepers, San Marcos, Calif.

e.

Komodo Reptiles, Verplanck, NY.

f.

Fluker's Mealworm Bedding, Port Allen, La.

g.

Kaytee Exact Hand Feeding Formula for Birds, Chilton, Wis.

h.

Harrison's High Potency Mash for Birds, Brentwood, Tenn.

i.

Mazuri Hi Ca 5M38 Diet, PMI Nutrition International, St Louis, Mo.

j.

MidWest Laboratories, Omaha, Neb.

k.

STATA, StataCorp LP, College Station, Tex.

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Contributor Notes

Address correspondence to Dr. Latney (llatney@vet.upenn.edu).