PLoS ONE
Public Library of Science
Digestibility of black soldier fly larvae (Hermetia illucens) fed to leopard geckos (Eublepharis macularius)
Volume: 15, Issue: 5
DOI 10.1371/journal.pone.0232496
•
•
•
• Altmetric

### Notes

Abstract

Black soldier fly (BSF) larvae have been marketed as an excellent choice for providing calcium to reptiles without the need of dusting or gut loading. However, previous studies have indicated that they have limited calcium digestibility and are deficient in fat soluble vitamins (A, D3, and E). In this feeding and digestibility trial, 24 adult male leopard geckos were fed one of three diets for 4 months: 1) whole, vitamin A gut loaded larvae; 2) needle pierced, vitamin A gut loaded larvae; or 3) whole, non-gut loaded larvae. Fecal output from the geckos was collected daily and apparent digestibility was calculated for dry matter, protein, fat, and minerals. There were no differences in digestibility coefficients among groups. Most nutrients were well digested by the leopard geckos when compared to previous studies, with the exception of calcium (digestibility co-efficient 43%), as the calcium-rich exoskeleton usually remained intact after passage through the GI tract. Biochemistry profiles revealed possible deficits occurring over time for calcium, sodium, and total protein. In regards to vitamin A digestibility, plasma and liver vitamin A concentrations were significantly higher in the supplemented groups (plasma- gut loaded groups: 33.38 ± 7.11 ng/ml, control group: 25.8 ± 6.72 ng/ml, t = 1.906, p = 0.04; liver- gut loaded groups: 28.67 ± 18.90 μg/g, control group: 14.13 ± 7.41 μg/g, t = 1.951, p = 0.03). While leopard geckos are able to digest most of the nutrients provided by BSF larvae, including those that have been gut loaded, more research needs to be performed to assess whether or not they provide adequate calcium in their non-supplemented form.

Boykin, Carter, Butler-Perez, Buck, Peters, Rockwell, Mitchell, and Kerby: Digestibility of black soldier fly larvae (Hermetia illucens) fed to leopard geckos (Eublepharis macularius)

## Introduction

Black soldier fly (BSF) larvae (Hermetia illucens ) are a popular feeder insect because they are the only commercially produced insect that has been found to potentially have a natural positive calcium to phosphorous (Ca:P) ratio (2.5:1) based on thediet they are provided [15]. A positive calcium to phosphorous ratio (ideally 2:1 or greater) is considered essential for reducing the incidence of nutritional secondary hyperparathyroidism (NSHP) in insectivorous reptiles [6,7]. The black soldier fly larvae's high calcium level is due to an abundance of calcium carbonate impregnated within the exoskeleton [8]. However, previous studies and anecdotal reports have indicated that these larvae, in particular the calcium-rich exoskeleton, are poorly digestible and may not be capable of providing sufficient calcium to prevent disease. Dierenfeld and King (2008) looked at BSF larvae digestibility in mountain chicken frogs (Leptodactylus fallux ) by analyzing fecal matter for nutrients and found that calcium digestibility was only 44% for frogs ingesting whole BSF larvae as compared to 84% when they were fed dusted crickets. When the BSF larvae (and exoskeleton) were mashed with a mortar and pestle, calcium digestibility increased to 88%. This suggests that physical disruption of the exoskeleton, such as would occur with mastication, could improve the calcium availability to the consumer. Dierenfeld and King theorized that species that were more apt to chew their food prior to ingestion would likely see higher levels of calcium digestibility [2]. Producers of the larvae have also made the recommendation that piercing the exoskeleton with a needle prior to feeding could help to improve issues with digestibility, but neither theory has been tested.

Of course, calcium is not the only nutrient that needs to be considered when constructing a diet for insectivorous species. In addition to NSHP, hypovitaminosis A is another common condition associated with insectivorous reptiles [9,10]. Most feeder insects, including BSF larvae, are severely deficient in the fat soluble vitamins (A, D3 , and E), and thus still require multivitamin dusting or gut loading [3,1113]; however, recent research has found that some insects can produce vitamins D2 and D3 secondary to ultraviolet B radiation exposure, similar to vertebrates [14]. Vitamin A is integral to many bodily functions, including growth and development, immunity, vision, reproduction, and health and function of glands, ducts, and mucous membranes [10, 1517]. Although true requirements for vitamins are not known for any reptile species, generic recommendations are currently extrapolated from requirements of laboratory rats [12, 18]. Previous research from the authors has proved the feasibility of gut loading vitamin A into BSF larvae [1], but determination of reptile digestibility and absorption requires further study.

The goal of this research was to determine if BSF larvae, given their potential issues with digestibility and vitamin deficiencies, can provide an adequate source of nutrition for leopard geckos (Eublepharis macularius ). Leopard geckos were selected as a model because they are a common insectivorous species in the pet trade with well-documented nutritional disorders such as NSHP and hypovitaminosis A [6,9]. Additionally, their smaller size makes them more apt to at least partially chew their food as compared to larger species. Our specific objectives were to determine digestibility of whole versus needle-pierced BSF larvae in a leopard gecko model and to determine if calcium and vitamin A can be absorbed in sufficient quantities by the target species without the use of dusting powder supplementation. We hypothesized that 1) digestibility of BSF larvae would increase by piercing the exoskeleton with a needle prior to being offered to the leopard geckos, and that 2) geckos receiving vitamin A gut loaded BSF larvae would have higher liver vitamin A concentrations compared with those receiving non-gut loaded BSF larvae.

## Materials and methods

### Animals and husbandry

This research was approved and conducted in accordance with the rules and regulations set by the Louisiana State University's Animal Care and Use Committee (protocol #17–083). Twenty-four male leopard geckos of unknown age were obtained from a private breeding colony with an average initial weight of 50.7 ± 11.5 grams (range 31.6–72.1 g). Males were recruited to rule out any potential bias associated with female reproduction. Each gecko was individually housed in a clear plastic terrarium (43 cm x 21 cm x 25 cm) without substrate and maintained at 28–30°C (83–86°F) and 30–40% relative humidity. Overhead fluorescent lamps provided lighting on a 12:12 light:dark cycle. The lighting did not provide any ultraviolet B radiation. The geckos had access to hide houses and water ad libitum. The baseline diet fed during the acclimation period was comprised of fasted crickets (4-week-old nymphs from Fluker Farms, Inc., Port Allen, LA) and BSF larvae (size large from Fluker Farms) at 3% of the gecko’s body weight. Vitamin A concentrations in these insects were well under 100 μg/kg (as fed), which is considered low. Twenty of the geckos were maintained on this diet for 75 days before starting the feed trial, while four geckos underwent a 26 day acclimation period with the latter group needed to replace four original subjects that refused to eat BSF larvae.

### Physical and ophthalmic exams

Prior to the start of the feeding experiments, each gecko underwent a full physical exam. Fecal samples were collected and no parasites were seen on fecal floatations using zinc sulfate solution or direct 0.9% saline smears. Repeat physical exams were performed at the end of the study. In order to assess the geckos for ocular changes associated with potential hypovitaminosis A [19], ophthalmic exams were performed on awake geckos by a boarded veterinary ophthalmologist (RTC) at the beginning and end of the study. Exams included full visual assessment of the external structures, cornea, anterior chamber, and lens by slit-lamp biomicroscopy and evaluation of intraocular pressure (IOP) by rebound tonometry (Icare® Tonovet, Vantaa, Finland).

### Sedation and bloodwork values

Each animal was sedated to facilitate handling for physical exams and venipuncture. Sedation was achieved by using a combination of dexmedetomidine (Dexdomitor, 0.1 mg/ml; Zoetis Services LLC, Parsippany, NJ) at 0.1 mg/kg and midazolam (1 mg/ml; West-Ward Pharmaceuticals Corp., Eatontown, NJ) at 1 mg/kg, subcutaneously in the axillae [20,21]. Once the examinations were completed, the geckos were reversed with atipamezole (Antisedan, 5 mg/ml; Zoetis Services LLC, Parsippany, NJ) at 1 mg/kg and flumazenil (0.1 mg/ml, manufacturer) at 0.05 mg/kg, subcutaneously in the axillae. Due to issues with recovery in another leopard gecko study running simultaneously, the dexmedetomidine dose was decreased to 0.05 mg/kg and flumazenil was discontinued for all sedation events performed after the baseline blood draws. Sedation level remained appropriate for venipuncture with this dexmedetomidine dose.

Blood was collected via the cranial vena cava using a 3 ml syringe and 25-gauge needle. Maximum volume that could be safely drawn was 1.5% body weight (0.5–1.1 ml). Samples were placed into lithium heparin microtainers, centrifuged within 30 minutes of collection, and chilled on ice. Within three hours of collection, all plasma was separated and frozen at -80°C until further processing could be performed. Two blood samples were obtained during the acclimation/vitamin A depletion period. On Day -50, blood was collected for a baseline plasma vitamin A concentration (measured as retinol, n = 13). These samples were sent to Michigan State University (MSU Veterinary Diagnostic Laboratory, Lansing, MI) for evaluation using ultra high performance liquid chromatography (UPLC). A second sample, obtained on Day -35 (n = 20), was used for an in-house plasma biochemistry panel (VetScan VS1 Chemistry Analyzer, Abaxis, Inc., Union City, CA). Two separate sampling periods were required due to the blood volume required for each test (>0.25 ml for UPLC, >0.1 ml for biochemistries) and difficulties in drawing enough blood at one time. For the four replacement geckos, a baseline vitamin A plasma concentration from MSU was not performed and the samples for the biochemistry profile were obtained on Day -14 (n = 4). An additional blood sample was drawn during the course of the study on Day 35 for a repeat plasma biochemistry profile (n = 23). On Days 105 and 140, samples from individual geckos were pooled together for a final plasma vitamin A concentration using UPLC from MSU (n = 24 and 24, respectively).

### Feeding experiments

The 24 geckos were divided into three diet groups using a random number generator (random.org). The treatment groups received either vitamin A gut loaded BSF larvae that were intact (Group 1, n = 8) or pierced once with a 21-gauge needle (Group 2, n = 8). The control group (Group 3, n = 8) received only non-gut loaded BSF larvae that remained intact. BSF larvae were gut loaded for 24 hours using a wheat bran and corn meal based diet with an added water-soluble vitamin A supplement (Rovimix-A 500-WS [178,500 μg retinyl acetate/g, equal to 150,000 μg retinol equivalents/g]; DSM Nutritional Products, Ames, IA). The expected final concentration of vitamin A in the diet was 20,000 μg/kg (dry matter basis, DMB) and the final larval vitamin A concentration was expected to be 1,000 μg/kg (as fed basis or 3,636 μg/kg DMB) [1]. Over the course of the study, samples of the wheat bran diet (n = 4) and larvae (treatment groups, n = 8; control, n = 8) were sent to SDK Laboratories, Inc. (Hutchison, KS) for vitamin A concentration analysis performed by high performance liquid chromatography (HPLC). All geckos were offered larvae equal to 5% of their body weight three times per week. The weights of BSF larvae ingested by the geckos were recorded over the course of the study (140 days) to calculate total vitamin A ingestion.

### Apparent digestibility of BSF larvae

In order to measure BSF larvae digestibility, fecal samples produced during the first and last month of the study were collected and pooled together by month and feeding group (n = 2 for each group). Any water dishes that contained feces were transferred to a glass dish and dried in an oven at 100°C for several hours before being combined with the rest of the fecal material. Fecal samples were frozen at -80°C until they could be further processed. Nutrient content analysis was performed by Dairy One Forage Lab (Ithaca, NY). Nutrient analysis was also performed on representative samples of BSF larvae (gut loaded and control). Average daily intake was determined for each group of geckos and apparent nutrient digestibility was calculated based on the dry matter intake and excretion of each nutrient using the following formula:

$Apparent\phantom{\rule{0.25em}{0ex}}digestibility\phantom{\rule{0.25em}{0ex}}\left(%\right)=\frac{\text{Average}\phantom{\rule{0.25em}{0ex}}\text{daily}\phantom{\rule{0.25em}{0ex}}\text{intake}-\text{Average}\phantom{\rule{0.25em}{0ex}}\text{daily}\phantom{\rule{0.25em}{0ex}}\text{output}}{\text{Average}\phantom{\rule{0.25em}{0ex}}\text{daily}\phantom{\rule{0.25em}{0ex}}\text{intake}}×100$

### Liver biopsies

At the conclusion of the study, all geckos underwent anesthesia and surgical liver biopsies for determination of liver vitamin A concentrations. Geckos were fasted for 12 hours prior to pre-medication with dexmedetomidine (0.1 mg/kg), midazolam (1 mg/kg), and hydromorphone (2 mg/ml, Hospira, Inc., Lake Forest, IL) at 0.25 mg/kg subcutaneously in the axillae. The geckos were maintained on isoflurane inhalant gas via face mask during the procedure. Anesthesia was monitored throughout the procedure via respiratory rate, Doppler heart rate, and muscle tone. The surgical site was prepared aseptically with chlorhexidine scrub 2% and sterile 0.9% saline. A #11 scalpel blade was used to make a 1–1.5 cm left paramedian incision starting 1–1.5 cm caudal to the xiphoid process. Both lobes of the liver were visually assessed for any abnormalities. The left lobe was then gently exteriorized using sterile cotton tipped applicators and digital manipulation. The caudal half of the left lobe was clamped using Hemoclips (blue or medium size, Weck Hemoclip® Traditional; Teleflex Medical, Research Triangle Park, NC) and removed with a scalpel blade, placed in a Whirl-Pak® bag (Nasco, Fort Atkinson, WI), and frozen at -20°C until processed. The surgical site was closed using a 2-layer method and 4–0 Vicryl suture (polyglactin 910, Ethicon US, LLC, Somerville, NJ); the body wall was closed in a simple continuous pattern and the skin was closed using a horizontal mattress pattern.

The geckos were reversed with atipamezole following the same protocol noted previously. Meloxicam (OstiLox TM, VetOne, Boise, ID) at 0.2 mg/kg subcutaneously was also given post-operatively. A second dose of hydromorphone (0.25 mg/kg) was given 18–24 hours after surgery, and meloxicam at 0.2 mg/kg subcutaneously was continued once per day for four days. Seven geckos (Group 1, n = 3; Group 2, n = 2; Group 3, n = 2) were euthanized intra-operatively after collection of the biopsies due to marked weight loss toward the end of the study period. All seven were submitted for full necropsy.

### Statistical analysis

Sample size for this study was determined using the following a priori information: an alpha = 0.05, a power = 0.80, an expected difference in vitamin A liver concentrations of 20 μg/g, and a standard deviation for the treatment and control groups of 12 μg/g. The Shapiro-Wilk test, skewness, kurtosis, and q-q plots were used to evaluate the distributions of the data. Data that were normally distributed are reported as mean and standard deviation (SD), while non-normally distributed data are reported as median and 25–75 percentiles (%), and min-max. Non-normal data were log transformed for parametric testing. Outliers were identified using the Dixon's Q test and removed if the calculated Q value was larger than the critical value given a 95% confidence interval. The amount of vitamin A ingested per group was dependent on voluntary gecko intake, therefore, larvae intake was analyzed for significance among groups using a one-way ANOVA. Fecal digestibility coefficients were also analyzed using a one-way ANOVA. Paired plasma biochemistry data was analyzed using a repeated measures ANOVA. None of the ANOVA tests required post-hoc analysis to differentiate significance between diet groups. For final vitamin A concentrations in the plasma and liver, Groups 1 and 2 were combined into a single vitamin A gut loaded group versus the non-gut loaded control group (Group 3). The two groups were then analyzed using a one-tailed independent t-test. A Pearson’s correlation test was used to determine if there was a relationship between the presence of a mucoid ocular discharge and the liver or plasma vitamin A concentrations. A commercial software (SPSS 25.0; IBM Statistics, Armonk, NY) was used to analyze the data; p<0.05 was used to determine statistical significance.

## Results

### Physical exams and necropsy results

All geckos were determined to be healthy at the beginning of the study. Over the course of the experiment, 10 (41.6%) out of 24 leopard geckos lost weight (mean ± SD: -13.76 ± 7.71% weight loss, range: -0.14% to -23.91%)(Group 1, n = 4; Group 2, n = 3; Group 3, n = 3). Seven (29.2%) of these geckos experienced inappetence and weight loss severe enough to require early removal from the study (Group 1, n = 3; Group 2, n = 2; Group 3, n = 2). These geckos were euthanized intra-operatively (after collection of hepatic biopsies) and then submitted for necropsy. Necropsy results revealed stomatitis (n = 4 total; mild to moderate, n = 3; moderate to marked, n = 1), hepatic lipidosis/vacuoles (n = 4), infection with Cryptosporidium spp. via histopathology and/or fecal floatation (n = 3), lymphoplasmacytic enteritis (n = 3), renal tubular necrosis (n = 2), and pulmonary xanthomatosis (n = 1). None of the necropsied specimens had evidence of epithelial squamous metaplasia that could be associated with hypovitaminosis A.

### Ophthalmic exams

No obvious ophthalmic lesions were observed at the beginning of the study. Exams were repeated at the end of the study. Seven (29.2%) of the geckos did have mild mucoid discharge within the medial canthus of one or both eyes (Group 1, n = 2; Group 2, n = 2; Group 3, n = 3). Three of these animals (Group 1, n = 1; Group 2, n = 1; Group 3, n = 1) were among those that were necropsied and showed no evidence of squamous metaplasia or any other ocular disease. Correlation analysis showed no significant correlation between the presence of the discharge and the liver (r = 0.371, p = 0.074, two-tailed) or plasma (r = -0.47, p = 0.105, two-tailed) vitamin A concentrations. The authors are unsure as to the significance of the mucoid discharge, as no other significant ocular disease process was found.

### Diet vitamin A concentrations and intake amounts

The target vitamin A concentrations for the larval diet and larvae were 20,000 μg/kg (DMB) and 1,000 μg/kg (as fed or 3,636 μg/kg DM), respectively. When analyzed samples were averaged together over the course of the experiment, the vitamin A supplemented diet contained 32,753 ± 15,350 μg/kg (DMB) and the larvae for the two treatment groups analyzed at 835 ± 232 μg/kg (as fed or 3,036 ± 844 μg/kg DMB). The average vitamin A concentration of the control group diet and larvae were 391 ± 40 μg/kg (DMB) and 23 ± 30 μg/kg (as fed or 84 ± 109 μg/kg DMB), respectively. Geckos were allowed to eat the larvae ad libitum up to 5% of their body weight. On average, the geckos ate 54.7 ± 12.2% of the food offered to them per feeding (2.74% body weight per feeding). When intake was analyzed as the average amount of grams eaten per group per feeding, there was no significant difference among diet groups (F = 0.587, p = 0.558).

### Apparent digestibility of BSF larvae

The average nutritional composition of BSF larvae used in this study is presented in the first column of Table 1. The mean digestibility coefficient ± standard deviation for the pooled feces from each group is listed in the remaining columns of Table 1. There was no significant difference in digestibility among groups for any of the nutrients analyzed. Since there were no significant differences among groups, all digestibility coefficients were averaged together and reported as a single mean ± standard deviation in the first column of Table 2. The second and third columns of Table 2 show the reported values for BSF larvae digestibility from the mountain chicken frog study [2]. With the exception of calcium and phosphorous, leopard geckos were more capable of digesting whole BSF larvae than mountain chicken frogs.

Table 1
Nutritional composition of BSF larvae and apparent digestibility coefficients for leopard geckos fed an exclusive diet of BSF larvae prepared in one of three ways.
NutrientAverage BSF larvae compositionGroup 1: Intact, vitamin A gut loaded (n = 2)Group 2: Pierced, vitamin A gut loaded (n = 2)Group 3: Intact, non-gut loaded (n = 2)
Dry Matter, %27.4 ± 0.8170 ± 0.972 ± 9.371 ± 4.5
Crude Protein, %56.1 ± 0.9181 ± 0.582 ± 3.880 ± 1.7
Crude Fat, %23.2 ± 11.7865 ± 11.774 ± 13.767 ± 5.9
Ash, %9.85 ± 0.3349 ± 9.354 ± 11.354 ± 6.1
Calcium, %2.14 ± 0.0841 ± 8.942 ± 18.144 ± 6.1
Phosphorous, %1.15 ± 0.0342 ± 4.147 ± 10.846 ± 3.2
Magnesium, %0.39 ± 0.0250 ± 5.852 ± 8.852 ± 3.0
Potassium, %1.35 ± 0.0377 ± 1.481 ± 2.980 ± 3.4
Sodium, %0.13 ± 0.0162 ± 3.965 ± 13.817 ± 90.9
Iron, mg/kg204 ± 7.7258 ± 4.960 ± 2.658 ± 2.8
Zinc, mg/kg131 ± 5.2437 ± 5.439 ± 1.043 ± 2.5
Copper, mg/kg11.2 ± 0.3826 ± 6.031 ± 4.433 ± 0.3
Manganese, mg/kg232 ± 8.9424 ± 8.120 ± 15.927 ± 5.1
Molybdenum, mg/kg1.24 ± 0.0844 ± 8.549 ± 10.952 ± 2.1
Sulfur, %27.4 ± 0.841 ± 0.747 ± 9.045 ± 4.5
No significant difference was found among groups for any nutrient.
Table 2
Apparent digestibility coefficients for leopard geckos fed a long term (140 days) exclusive diet of BSF larvae compared to published digestibility coefficients for mountain chicken frogs (Leptodactylus fallux) fed either whole or mashed BSF larvae for a period of only one day each.
NutrientLeopard Geckos, whole and pierced larvae (n = 6)Frogs, Whole Larvae* (n = 5)Frogs, Mashed Larvae* (n = 5)
Dry Matter, %71 ± 4.726 ± 9.976 ± 3.2
Crude Protein, %81 ± 2.044 ± 7.577 ± 3.1
Calcium, %43 ± 9.5a44 ± 7.5a88 ± 1.7
Phosphorus, %45 ± 5.8a51 ± 6.5a91 ± 1.3
Magnesium, %51 ± 5.06.3 ± 1340 ± 8.0
Potassium, %79 ± 2.724 ± 1060 ± 5.3
Sodium, %48 ± 48-378 ± 64-489 ± 78
Copper, mg/kg30 ± 4.6-61 ± 2150 ± 6.7
Iron, mg/kg59 ± 2.9-284 ± 5140 ± 7.9
Molybdenum, mg/kg49 ± 7.2-126 ± 5623 ± 9.6
Zinc, mg/kg40 ± 3.823 ± 1072 ± 3.7
*Data reported in study by Dierenfeld and King, 2008
a The digestibility coefficients for calcium and phosphorous were the only nutrients to not differ significantly when comparing our data to the values reported for intact larvae from Dierenfeld and King (2008) using a single value t-test

### Blood values

Baseline plasma vitamin A (measured as retinol) concentrations were all found to be <50 ng/ml (lower limit of quantification for small volume samples, <1 ml). For the final plasma vitamin A samples, only 13 out of 24 samples were of a high enough volume to obtain readings above the limit of quantification. One sample in the non-gut loaded group was returned as <20 ng/ml, and for purposes of including for statistical analysis, was read as 20 ng/ml. For the 13 samples that were able to be analyzed, vitamin A concentrations were significantly higher (t = 1.906, p = 0.0415) in the treated groups (n = 8; 33.38 ± 7.11 ng/ml) compared with the control group (n = 5; 25.8 ± 6.72 ng/ml) (Table 3).

Table 3
Plasma and liver vitamin A concentrations from geckos receiving vitamin A gut loaded BSF larvae versus those receiving non-gut loaded BSF larvae.
Vitamin A supplemented (Groups 1 & 2)Non-gut loaded (Group 3)
Final Plasma Vitamin A concentrations (retinol, ng/ml)33.38 ± 7.11 (n = 8)25.80 ± 6.72* (n = 5)
Final Liver Vitamin A concentrations (total vitamin A, μg/g)28.67 ± 18.90 (n = 16)14.13 ± 7.41** (n = 7)
All samples were run by Michigan State University's Veterinary Diagnostic Laboratory using UPLC. Eleven plasma samples were not included due to insufficient plasma quantities reading below the limit of quantification for this assay (LOQ: <50 ng/ml).
*One sample was reported as <20 ng/ml. This value was included as a reading equal to 20 ng/ml.
**A single outlier of 61.35 μg/g was removed from this group prior to statistical analysis.

Plasma biochemistries were sampled on Day -35 (baseline) and Day 35. A final sample was not performed due to the plasma volume restrictions of the vitamin A test. No significant differences were seen among groups during either of the two time periods; however, when baseline values (averaged across all three groups) were compared to Day 35 values using a repeated measures ANOVA, there were significant decreases in calcium, total protein, albumin, globulin, and sodium (Table 4). One value for calcium during the baseline time period was >16 mg/dL (upper limit of quantification for the rotors used) and one value for albumin at the Day 35 time period was <1 g/dL (lower limit of quantification for the rotors used). For statistical purposes these values were removed, however, their removal did not change the significance found for these analytes. One value from potassium at Day 35 was also removed for obvious hemolysis interference. Additionally, bile acids were under 35 umol/L for all individuals tested with the exception of one individual at the Day 35 time period that was reported as 44 umol/L. Bile acids are not reported in Table 4.

Table 4
Average biochemistry results from all geckos over time (baseline vs. day 35).
Biochemistry AnalytesBaseline ValuesDay 35 ValuesSignificance
AST (U/L)54.6 ± 20.367.9 ± 37.2F = 2.314, p = 0.144
Creatinine kinase (U/L)458 (25% = 275, 75% = 1006)698 (25% = 286, 75% = 1256)F = 0.877, p = 0.361
Uric acid (mg/dL)3.4 ± 1.53.8 ± 1.4F = 1.359, p = 0.257
Glucose (mg/dL)166.5 ± 14.4161.6 ± 20.1F = 1.207, p = 0.285
Calcium (mg/dL)14.2 ± 1.113.0 ± 1.1F = 25.299, p<0.001*
Phosphorous (mg/dL)3.8 ± 0.63.6 ± 0.8F = 1.38, p = 0.254
Total Protein (g/dL)5.9 ± 0.95.1 ± 0.7F = 19.061, p<0.001*
Albumin (g/dL)1.8 ± 0.31.5 ± 0.3F = 30.076, p<0.001*
Globulin (g/dL)4.1 ± 0.73.7 ± 0.5F = 10.717, p = 0.004*
Potassium (mmol/L)5.2 ± 0.65.0 ± 0.8F = 1.993, p = 0.174
Sodium (mmol/L)147.8 ± 5.8138.8 ± 3.1F = 94.955, p<0.001*
No significance was found between groups at either time period. All data was normally distributed with the exception of creatinine kinase which was able to be log transformed for analysis. Mean ± standard deviation is reported for normally distributed data. Median and 25–75 percentiles (%) are reported for creatinine kinase. F statistics and p values are reported for all analytes; those with an asterisk (*) indicate significance, p<0.05.

### Liver biopsies and surgical outcomes

Liver biopsies collected at the end of the study revealed a significantly higher hepatic vitamin A concentration (measured as total vitamin A) for the gut loaded group versus the control group (Vitamin A supplemented: 28.67 ± 18.90 μg/g, Non-gut loaded: 14.13 ± 7.41 μg/g; t = 1.951, p = 0.0325)(Table 3). A single outlier was removed from the control group (liver vitamin A concentration of 61.35 μg/g). Age, genetic factors, or diet prior to the study could have played a role in the unusually high liver concentration of this gecko compared to the rest of the geckos in this group. There were no anesthetic complications with any of the surgeries. All non-survival surgeries (n = 7) were determined prior to the start of anesthesia. All survival surgeries (n = 17) were performed successfully with only one animal requiring the placement of Gelfoam® Sterile Sponge (Pfizer Inc., New York, NY) for more controlled hemostasis. Post-operatively, there were no issues with hemorrhage, dehiscence, or any other wound complications. As of one-year post-surgery, no additional issues or concerns were noted.

## Discussion

The results of this study confirm that leopard geckos are capable of digesting BSF larvae. Overall, BSF digestibility was higher in this reptile species compared to a single study with an amphibian [2]. Average dry matter digestibility of intact BSF larvae was 71 ± 2.8% when fed to leopard geckos and 26 ± 9.9% for mountain chicken frogs. Protein, magnesium, potassium, sodium, iron, zinc, copper, and molybdenum also saw significant gains in digestibility (Table 2). Some of the differences in digestibility could be related to differences in larval composition between the studies. However, given that most of our digestibility coefficients were more similar to the values reported for mashed larvae from their study (Table 2, column 3) rather than the values for intact larvae (Table 2, column 2), we believe that most of the differences were due to a higher degree of mastication by the leopard geckos which would allow for digestive enzymes to breach the tough exoskeleton and breakdown the inner portions of the larvae. This would also explain why piercing the BSF larvae with a needle did not result in improved digestibility among the three groups (Table 1), as the geckos were already accomplishing this through mastication. It is possible that needle-piercing would still be of benefit to species that swallow their prey whole. Transecting the BSF larvae could also be considered, but this can lead to decreased movement by the larvae and reduced acceptance rate by the geckos (personal observation).

Calcium digestibility, on the other hand, did not improve and was similar between the two species (43% for geckos, 44% for frogs) when fed intact BSF larvae [2]. It would appear that gecko mastication or needle piercing does not provide enough disruption to the exoskeletal matrix to allow for calcium carbonate digestion. Visual assessment of the geckos' feces supports this hypothesis, as there would frequently be whole exoskeletons passed through the digestive tract (personal observation). This poor level of calcium digestibility should raise concerns over whether non-supplemented BSF larvae can support the calcium needs of captive reptiles. The estimated minimum dietary calcium requirement for growing leopard geckos is between 6.1 and 8.5 g Ca/kg diet (DMB) [22]. When adjusted for digestibility, BSF larvae in our study provided 9.2 g Ca/kg diet (DMB), which should be adequate to support calcium needs. It is possible that the calcium digestibility was low in these leopard geckos because they were adults and calcium absorption was impacted by normal feedback mechanisms. A study in fast-growing juvenile geckos would be helpful in further discerning whether low digestibility is a function of the BSF exoskeleton, as it would be expected to be higher in growing animals with a higher calcium requirement.

However, the authors would still recommend caution due to the fact that physiological needs may vary based on species, age, reproductive status, and/or vitamin D status of the animal. Additionally, calcium content of BSF larvae can vary greatly based on their rearing diet’s composition. Recent studies have shown calcium:phosphorous ratios can range anywhere between 0.3:1 to 14.9:1 [4,5]. Thus, more research would be needed to prove that non-supplemented BSF larvae can, in fact, provide enough calcium to insectivorous reptiles based on how the insects are reared.

When comparing the paired blood samples, there was a significant decrease in calcium over time for all treatment groups. Poor calcium digestibility or low levels of vitamin D could be possible causes for this decline. Another explanation could be that the geckos received high levels of calcium supplementation prior to entering the study and that the decline represented a return to more physiologic levels. Unfortunately, due to plasma volume restrictions at the conclusion of the study, a final biochemistry panel to determine whether or not values continued to trend down could not be performed. As all of the geckos were males, reproductive status did not play a role in this decline. At this time, we do not have enough data to recommend whether calcium supplementation is needed when offering BSF larvae to reptiles. Additional research needs to be conducted to establish true calcium requirements for reptiles and varied diets should always be offered to insectivores to limit the incidence of nutritional deficiencies.

The biochemistry panels also revealed significant decreases in total protein, albumin, globulins, and sodium in the geckos over time for all treatment groups. Compared to other feeder insects, BSF larvae have lower concentrations of protein and sodium [3,11]. The decrease in protein could indicate a change from a primarily protein-rich cricket diet to one of a higher fat, lower protein larval diet. Hyporexia could also play a role in the decreased values, but with an average intake of 2.7% body weight and no correlation between blood values and the amount of food ingested, this is less likely. In regards to sodium, there is added concern about the possibility of poor or negative digestibility. Negative digestibility indicates that more sodium is being lost in the feces than was initially present in the food. This was seen in both mountain chicken frogs and corn snakes (Pantherophis guttatus ) fed diets containing BSF larvae [2,23]. It was also seen in a single replicate from Group 3 of this study. Dierenfeld and King originally theorized that the BSF larvae may be irritating the gut and causing a "diarrhea"-type syndrome that could lead to hyponatremia if fed exclusively over time [2]. Unfortunately, no other studies have addressed digestibility of sodium or looked at sodium biochemistry data from animals ingesting BSF larvae. To the authors' knowledge, the present study is the first to report serum or plasma sodium concentrations for any animal consuming BSF larvae as the main ingredient of the diet. When compared to reference intervals for this species, the sodium levels obtained from Day 35 of this study do indicate a possible hyponatremia, however, these reference intervals may not be representative of the true physiologic range for sodium in leopard geckos [24]. Feeding a variety of insects or protein sources, should alleviate any concerns that could be associated with the nutritional deficits of an exclusive BSF larval diet.

The second objective regarding digestion and absorption of vitamin A from gut loaded larvae was more challenging to confirm. Previous research in multiple animal species suggested that plasma vitamin A concentrations are not usually representative of whole body vitamin A status, nor do they correlate well with liver vitamin A concentrations [2527]. Retinol-binding proteins within the blood tend to maintain homeostatic concentrations of circulating vitamin A, except in cases of extreme hypo- or hypervitaminosis A [16, 28]. Liver concentrations are considered the more accurate representation of body status, but clinically can be more challenging to obtain in sick or small patients. Ideally, liver biopsies from each gecko would have been obtained at the beginning and end of the study period. Without baseline hepatic concentrations of vitamin A for individual geckos, it is impossible to truly evaluate a change in vitamin A status, but random assignment of the geckos to treatment groups should have minimized any bias. Unfortunately, due to the geckos' small size and the minimum requirements for biopsy weight (0.25 grams), only one hepatic biopsy would be able to be performed unless the study became non-survival. The other issue was the possibility of fatal complications early on in the study period leading to reduction in sample population sizes. A recent study reported a high complication rate following liver biopsies in this species, so the authors had concerns about multiple surgeries [29]. Due to these limitations and the fact that there are no published references for plasma vitamin A concentrations in leopard geckos, the authors decided to proceed with collecting plasma concentrations as well.

The minimum plasma volume required for the vitamin A assay used in this study was 0.15 ml; however, the limit of quantification for sample volumes below 1 ml is 50 ng/ml. For samples that are larger than 1ml, the limit of quantification can go as low as 10 ng/ml. Unfortunately, due to size restrictions, our baseline sample volumes all fell below 1 ml of plasma and all results were returned as <50 ng/ml. Previous literature has reported plasma vitamin A (measured as retinol) concentrations for various squamates and amphibian species, including green iguanas (Iguana iguana, 52–75 ng/ml), eastern indigo snakes (Drymarchon couperi, 9 ng/ml), anacondas (Eunectes murinus, 80 ng/ml), Mississippi gopher frogs (Rana capito servosa, 36–43 ng/ml), marine toads (Bufo marinus, 60 ng/ml), Cuban tree frogs (Osteopilus septentrionalis 83 ng/ml), and Puerto Rican crested toads (Bufo lemur , 130 ng/ml) [2527, 3033]. To the authors' knowledge, there is no other literature reporting plasma vitamin A concentrations in leopard geckos, or any other insectivorous lizard, so these baseline values either indicate that leopard geckos have lower than average circulating plasma vitamin A concentrations compared to other squamates and insectivorous amphibians or that these geckos were already deficient prior to being included in this study. Given that all individuals were deemed healthy at the beginning of the study, and that no geckos developed lesions that would be considered classical for diagnosis of hypovitaminosis A (epithelial squamous metaplasia), the former is probably more likely.

Only 13 of the 24 final plasma samples were reported back with values reading below 50 ng/ml (Group 1, n = 7; Group 2, n = 1; Group 3, n = 5). Thus, for comparative analysis between treatments, data from Groups 1 and 2 were combined, providing a single vitamin A gut loaded treatment group versus the control (Group 3). Final plasma concentrations did show a significant difference between groups (t = 1.906, p = 0.0415), with the vitamin A supplemented geckos having higher plasma vitamin A concentrations. Liver vitamin A concentrations also differed significantly between the vitamin A gut loaded groups and the control group (t = 1.951, p = 0.0325). Across both treatment groups, liver vitamin A concentrations ranged from 2.9–77.98 μg/g, with only a few individuals near the upper end of this range. These geckos likely had higher liver concentrations at the start of the study compared to the others. Age, diet, and husbandry conditions prior to the study (all of which are unknown), are likely contributors to the wide range seen. Paired liver samples would have helped limit this variance, but due to the limitations already discussed, were not performed.

One other study has measured liver vitamin A concentrations in leopard geckos. Cojean et al . (2018) used female leopard geckos to determine if differences existed between liver vitamin A uptake and storage when geckos were given pre-formed vitamin A supplementation versus beta-carotene [29]. Similar to the present study, Cojean's group also only performed biopsies at the end of the study and did not collect paired samples. Those authors found that the beta-carotene fed group displayed higher overall liver concentrations of vitamin A compared with the pre-formed vitamin A group, despite the theory that many carnivorous/omnivorous reptiles are not capable of converting beta-carotene to the active form (mean 13.43 μg/g, min-max 2.31–24.05 μg/g vs. mean 9.49 μg/g, min-max 6.76–13.33 μg/g, respectively). The values from the present study are much higher, with differences in sex (all female vs. all male study designs) and age being major influencing variables. Females would potentially have lower body stores of vitamin A due to large quantities being stored in the developing eggs. Cojean’s geckos were also 6–9 months old compared to ours that were mostly thought to be adults (> 10 months old). Vitamin A tends to accumulate in the liver as an animal ages leading to potential differences in the two populations.” [34].

In contrast to Cojean’s study, we experienced no major surgical or anesthetic complications. Seventeen geckos underwent a successful liver biopsy of the left lobe (median weight = 0.22g, min = 0.08g, max = 0.59g). Lower overall vitamin A stores or some other aspect of folliculogenesis may have contributed to poor wound healing and dehiscence. Additionally, different anesthetic protocols and biopsy techniques (guillotine vs. hemoclip) may have led to better success in the present study. The authors would strongly encourage further studies involving liver biopsies on leopard geckos in the future, if they are indicated. One such study would be to determine plasma and liver vitamin A concentrations in wild-caught leopard geckos. This much needed study would help researchers to determine if our various methods of supplementation in captivity are meeting the physiological needs of this species. As far as which supplementation (beta-carotene or pre-formed vitamin A) is better for leopard geckos, there is still some debate, but both supplements appear to have some degree of absorbance and assimilation into the gecko and should probably be used in combination along with other carotenoid sources.

Without supplementation with either pre-formed vitamin A or appropriate precursors, insectivorous reptiles are at a high risk for developing hypovitaminosis A. The lesions most often associated with this disease are hyperkeratosis and epithelial squamous metaplasia, with the most classical presentations manifesting as ocular lesions (periocular edema, conjunctivitis, blepharitis, ocular discharge and debris) [9,10,15]. Wiggans et al . (2018) reported that 46% of all leopard geckos that visited a veterinary teaching hospital between 1985 and 2013 had some form of ocular lesion. Lack of vitamin A in the diet was one of the major risk factors associated with development of ocular disease [19]. In the present study, mild mucoid discharge was seen in seven geckos across all treatments and was not correlated with plasma or liver vitamin A concentrations.

Other symptoms may vary by species and have included nasal discharge, gular edema, swollen or thickened lips, stomatitis, vertebral kinking, hemipenal impaction, and renal tubular hyperkeratosis leading to visceral gout [10,15,17,35]. The timeline for developing these lesions is still currently unknown. Kroenlein et al. (2008) found no difference in liver vitamin A concentrations nor histological evidence of squamous metaplasia in red-eared sliders (Trachemys scripta elegans ) after six months of vitamin A depletion. Research in humans and other adult vertebrates confirm that >6 months of depletion is usually needed before clinical signs of hypovitaminosis A are detectable, but will obviously depend on the vitamin A status of the individual prior to the start of depletion [3638]. None of the seven geckos (Group 3, n = 2) that underwent necropsy had histopathological lesions consistent with hypovitaminosis A. A case could be made for stomatitis lesions being associated with hypovitaminosis A, but lesions were seen in treatment and control groups and were not correlated to vitamin A concentrations. Even though there was a significant difference seen between groups in regards to liver and plasma vitamin A concentrations, without histopathological evidence of disease, the authors are unable to conclude that the control group was or was not trending towards vitamin A depletion status. The difference in concentrations only provides evidence that gut loaded BSF larvae are capable of being digested enough to provide vitamin A to the consumer and to produce levels higher than geckos not receiving any supplementation.

Although BSF larvae appear to be well digested and able to offer gut loaded nutrients to leopard geckos, 10 out of the 24 individuals developed issues with inappetence and weight loss during the course of the study. The reason for this was likely multi-factorial, with a main issue being palatability. Even before the study started, four geckos had to be replaced due to strict refusal to eat BSF larvae. Most of the geckos that experienced weight loss during the study period would consistently eat less than the other geckos. Whether food preferences were due to palatability or a lack of movement of BSF larvae compared to crickets was outside the scope of this study. Another contributor to inappetence and weight loss was infection with Cryptosporidium sp. At least three geckos were confirmed to be infected at necropsy, but none showed obvious signs of infection until after the acclimation period had been completed. The stress of being transitioned to an exclusive diet of BSF larvae, instead of mixed feedings with crickets, may have increased the severity and progression of the disease. Knowing that the prevalence of cryptosporidiosis is relatively high in the commercially bred population of leopard geckos, the authors were well aware that this parasitic disease could disrupt results for a digestibility study, but these animals ultimately represent what is available in the current pet trade. Fortunately, the geckos that were infected rarely ate well and, therefore, did not contribute much to the pooled feces. The other possible contributor that was found on necropsy was stomatitis. These lesions were not seen grossly, but inflammation was seen on histopathology. The cause of the stomatitis is unknown, but nutrient deficiencies or irritation from ingestion of the BSF larvae are possible. Once again, the authors strongly encourage feeding insectivores a wide variety of insects in order to provide the diverse nutrients they require.

## Conclusions

Overall, leopard geckos are able to digest intact BSF larvae better than previously reported for the mountain chicken frog. Despite high nutrient digestibility for proximate constituents, calcium digestibility remained low. While calcium levels were likely adequate for leopard geckos based on estimated calcium requirements for the species, further research is needed to verify this assumption and to determine calcium requirements for other insectivorous species. This study demonstrated that leopard geckos are able to utilize gut loaded nutrients such as vitamin A incorporated into feeder BSF larvae. Additional research is necessary to determine gut loading amounts for other nutrients (e.g., vitamins D3 and E, etc.). Plasma and liver vitamin A concentrations for leopard geckos can be challenging to obtain in a clinical setting, but can be used in research to better study true requirements for this species.

## Acknowledgements

The authors would like to thank the Division of Laboratory Animal Medicine at LSU for assisting us with the care of these animals.

## References

1

KL Boykin, K Butler-Perez, MA Mitchell. . Evaluation of vitamin A gut loading in black soldier fly larvae. Proc ARAV. 2018: , pp.981.

2

ES Dierenfeld, J King. . Digestibility and mineral availability of Phoenix worms, Hermetia illucens, when fed to mountain chicken frogs, Leptodactylus fallux. J Herp Med Surg. 2008;18(3/4): , pp.100–105.

3

M. Finke. Complete nutrient content of four species of feeder insects. Zoo Biol. 2013; 32(1): , pp.27–36. , doi: 10.1002/zoo.21012

4

T Spranghers, M Ottoboni, C Klootwijk, A Ovyn, S Deboosere, B De Meulenaer, et al. Nutritional composition of black soldier fly (Hermetia illucens) prepupae reared on different organic waste substrates. J Sci Food Agric. 2017; 97: , pp.2594–2600. , doi: 10.1002/jsfa.8081

5

M Tschirner, A Simon. . Influence of different growing substrates and processing on the nutrient composition of black soldier fly larvae destined for animal feed. J Insects as Food Feed. 2015;1: , pp.249–259.

6

TH Boyer, PW Scott. Nutrition In: SJ Divers, SJ Stahl editors. Mader's reptile and amphibian medicine and surgery. St Louis: Elsevier; 2019 pp. , pp.201–223.

7

E Klaphake. . A fresh look at metabolic bone disease in reptiles and amphibians. Vet Clin Exot Anim. 2010;13(3): , pp.375–392.

8

OA Johannsen. . Stratiomyiid larvae and puparia of the north eastern states. J NY Entomol Soc. 1922;30(4): , pp.141–153.

9

TH Boyer. Hypovitaminosis and hypervitaminosis A In: SJ Divers, SJ Stahl editors. Mader's reptile and amphibian medicine and surgery. St. Louis: Elsevier, 2019 pp. , pp.1316–1317.

10

C Mans, J Braun. . Update on common nutritional disorders of captive reptiles. Vet Clin Exot Anim. 2014;17(3): , pp.369–395.

11

MD Finke. . Complete nutrient composition of commercially raised invertebrates used as food for insectivores. Zoo Biol. 2002;21(3): , pp.269–285.

12

MD Finke. . Gut loading to enhance the nutrient content of insects as food for reptiles: a mathematical approach. Zoo Biol. 2003;22(2): , pp.147–162.

13

M Pennino, ES Dierenfeld, JL Behler. . Retinol, α-tocopheral and proximate nutrient composition of invertebrates used as feed. Int Zoo Yearb. 1991;30: , pp.143–149.

14

DGAB Oonincx, P van Keulen, MD Finke, FM Baines, M Vermeulen, G Bosch. . Evidence of vitamin D synthesis in insects exposed to UVb light. Nature—Scientific Reports. 20188(10807): , pp.1–10.

15

AL Abate, R Coke, G Ferguson, D Reavill. . Chameleons and vitamin A. J Herp Med Surg. 2003;13(2): , pp.23–31.

16

DN D'Ambrosio, RD Clugston, WS Blaner. . Vitamin A metabolism: an update. Nutrients. 2011; 3(1): , pp.63–103. , doi: 10.3390/nu3010063

17

GW Ferguson, JR Jones, WH Gehrmann, SH Hammack, LG Talent, RD Hudson, et al. Indoor husbandry of the panther chameleon Chamaeleo [Furcifer] pardalis: effects of dietary vitamins A and D and ultraviolet irradiation on pathology and life-history traits. Zoo Biol. 1996;15(3): , pp.279–99.

18

National Resource Council (NRC). Nutrient requirements of laboratory animals. Washington, DC: National Academy Press; 1995.

19

KT Wiggans, D Sanchez-Migallon Guzman, CM Reily, C Vergneau-Grosset, PH Kass, et al. Diagnosis, treatment, and outcome of and risk factors for ophthalmic disease in leopard geckos (Eublepharis macularius) at a veterinary teaching hospital: 52 cases (1985–2013). JAVMA. 2018;252(3): , pp.316–323. , doi: 10.2460/javma.252.3.316

20

GA Doss, DM Fink, KK Sladky, C Mans. . Comparison of subcutaneous dexmedetomidine–midazolam versus alfaxalone–midazolam sedation in leopard geckos (Eublepharis macularius). Vet Anaesth Analg. 2017;44(5): , pp.1175–83. , doi: 10.1016/j.vaa.2017.03.007

21

DM Fink, GA Doss, KK Sladky, C Mans. . Effect of injection site on dexmedetomidine-ketamine induced sedation in leopard geckos (Eublepharis macularius). J Am Vet Med Assoc. 2018;253(9): , pp.1146–50. , doi: 10.2460/javma.253.9.1146

22

Allen ME. Nutritional Aspects of Insectivory. PhD Dissertation, Michigan State University. 1989. Available from: https://d.lib.msu.edu/etd/16494

23

KL Boykin, K Butler-Perez, MA Mitchell. . Feeding trial and health assessment of corn snakes (Pantherophis guttatus) fed an insect-based sausage diet. Proc ARAV. 2018: , pp.975.

24

S Alberton, O Cojean, R Froment, E Maccolini, C Vergneau-Grosset. . Determination of leopard gecko (Eublepharis macularius) packed cell volume and plasma biochemistry reference intervals and reference values. Proc ARAV. 2018: , pp.933–934.

25

CN Berkvens, A Lentini, CJ Dutton, DL Pearl, IK Barker, GJ Crawshaw. . Serum and hepatic vitamin A levels in captive and wild marine toads (Bufo marinus). Zoo Biol. 2014;33(6): , pp.536–543. , doi: 10.1002/zoo.21169

26

C Dutton, A Lentini, C Berkvens, G Crawshaw. . The effect of supplementation with vitamin A on serum and liver concentrations in Puerto Rican crested toads (Peltophryne lemur) and its lack of impact on brown skin disease. Zoo Biol. 2014;33(6): , pp.553–557. , doi: 10.1002/zoo.21167

27

KE Sullivan, G Fleming, S Terell, D Smith, F Ridgley, EV Valdes. . Vitamin A values of wild-caught Cuban tree frogs (Osteopilus septentrionalis) and marine toads (Rhinella marina) in whole body, liver, and serum. J Zoo Wild Med. 2014;45(4): , pp.892–896.

28

FJ Schweigert, S Uehlein-Harrell, GV Hegel, H Wiesner. . Vitamin A (retinol and retinyl esters), α-tocopherol and lipid levels in plasma of captive wild mammals and birds. J Vet Med A. 1991;38: , pp.35–42.

29

O Cojean, S Lair, C Vergneau-Grosset. . Evaluation of β-carotene assimilation in leopard geckos (Eublepharis macularius). J Anim Physiol Anim Nutr (Berl). 2018;102(5): , pp.1411–1418.

30

J Raila, A Schumacher, J Gropp, FJ Schweigert. . Selective absorption of carotenoids in the common green iguana (Iguana iguana). Comp Biochem Physiol A. 2002;132(2): , pp.513–518.

31

SE Knafo, TM Norton, MA Mitchell, DJ Stevenson, N Hyslop, R Poppenga, et al. Health and nutritional assessment of free-ranging eastern indigo snakes (Drymarchon couperi) in Georgia, United States. J Zoo Wild Med. 2016;47(4): , pp.1000–1012.

32

PP Calle, J Rivas, M Munoz, J Thorbjarnarson, ES Dierenfeld, W Holmstrom, et al. Health assessment of free-ranging anaconda (Eunectes murinus) in Venezuela. J Zoo Wild Med. 1994;25(1): , pp.53–62.

33

Ploog C, Clunston R, Morris C, Iske C, Blanner W, Pessier A. Hypovitaminosis A: influence of three diets or topical treatment on hepatic, adipose, and plasma retinoid concentrations and presence of squamous metaplasia in Mississippi gopher frogs (Rana capito servosa). In Bissell H, Brooks M Eds. Proceedings of the Eleventh Conference on Zoo and Wildlife Nutrition, AZA Nutrition Advisory Group, Portland,AZA NAG. 2015.

34

B van der Loo, R Labugger, CP Aebischer, M Bachschmid, V Spitzer, J Kilo, et al. Age-related changes in Vitamin A status. J Cardiovasc Pharmacol Ther. 2004;43(1): , pp.26–30.

35

E Ariel, PW Ladds, GN Buenviaje. . Concurrent gout and suspected hypovitaminosis A in crocodile hatchlings. Aust Vet J. 1997;75(4): , pp.247–249. , doi: 10.1111/j.1751-0813.1997.tb10089.x

36

KR Kroenlein, JM Sleeman, SD Holladay, PH Joyner, JD Brown, M Griffin, et al. Inability to induce tympanic squamous metaplasia using organochlorine compounds in vitamin A-deficient red-eared sliders (Trachemys scripta elegans). J Wildl Dis. 2008;44(3): , pp.664–9. , doi: 10.7589/0090-3558-44.3.664

37

EM Hume, HA Krebs. . Vitamin A requirement of human adults: an experimental study of vitamin A deprivation in man. Medical Research Council Special Report Series. 1949; , pp.264.

38

PT McCarthy, LR Cereoedo. . Vitamin A deficiency in the mouse. J Nutr. 1952;46(3): , pp.361–376. , doi: 10.1093/jn/46.3.361

6 Feb 2020

PONE-D-19-33399

Can reptiles digest black soldier fly larvae (Hermetia illucens)? Evaluating their digestibility using leopard geckos (Eublepharis macularius) as a model

PLOS ONE

Dear Dr. Boykin,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please address the comment by the reviewers to clarify the concerns brought up.

We would appreciate receiving your revised manuscript by Mar 22 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Jake Kerby, Ph.D.

PLOS ONE

I was able to obtain three reviews for this manuscript and the reviewers had similar comments on many points. Please address these comments in a reply. Some are merely grammatical, but others are perhaps important to the overall conclusion of the paper.

Journal Requirements:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. As part of your revision, please complete and submit a copy of the ARRIVE Guidelines checklist, a document that aims to improve experimental reporting and reproducibility of animal studies for purposes of post-publication data analysis and reproducibility: https://www.nc3rs.org.uk/arrive-guidelines. Please include your completed checklist as a Supporting Information file. Note that if your paper is accepted for publication, this checklist will be published as part of your article.

3. In your Methods section, please provide additional details regarding the BSF larvae used in your study and ensure you have described the source. For more information regarding PLOS' policy on materials sharing and reporting, see https://journals.plos.org/plosone/s/materials-and-software-sharing#loc-sharing-materials.

4. Thank you for providing the following Funding Statement:

"Primary funding for this study was provided to KB and MM by the Louisiana State University VCS Corp grant (#8262). Additional support was provided by Fluker's Cricket Farm, Inc. (flukerfarms.com) and Abaxis, Inc. (abaxis.com). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

We note that one or more of the authors is affiliated with the funding organization, indicating the funder may have had some role in the design, data collection, analysis or preparation of your manuscript for publication; in other words, the funder played an indirect role through the participation of the co-authors.

If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study in the Author Contributions section of the online submission form. Please make any necessary amendments directly within this section of the online submission form.  Please also update your Funding Statement to include the following statement: “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If the funding organization did have an additional role, please state and explain that role within your Funding Statement.

Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.

Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to  PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and  there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests

[Note: HTML markup is below. Please do not edit.]

Reviewer's Responses to Questions

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

Reviewer #1: Interesting study; specific comments appended in the MS.

In particular you need to pay detailed attention to the actual compound measured regarding vitamin A status - likely retinol - and associated units. Vitamin A, per se, is typically reported as IU or UI - based on the underlying compound measured. Especially with exotic species and the limited knowledge of nutrient requirements, physiology and metabolism - and particularly how they are evaluated and utilized, one must be explicit with defining actual compound(s) measured.

Reviewer #2: As detailed in my comments tot he authors my main concern is the conclusion that BSFL should be supplemented with calcium before being fed to leopard geckos. The data as presented does not seem to support that conclusion. Hence why I replied partly to question #1.

Reviewer #3: this is a well written manuscript and a well designed and important study. Some minor comments listed below.

Line 33-34: report p values as 0.04 and 0.03

Line 77: Please provide a reference for this statement (leopard geckos prone to vit a deficiency, etc).

Line 105: provide reference that leopard geckos develop ocular changes due to vitamin A deficiency

Line 191, 220, 230: the 7 emaciated geckos were from all 3 treatments groups? Would be worth reporting here.

Line 254-256: the statement regarding the frogs is inappropriate for the result section and should be moved to the discussion section.

Line 291: possible that this animal was a female, and not a male?

Line 323-324: were the larvae fed in your study and in the frog study of same size/age? Possible that older/larger larvae are harder to digest or have different nurtritional values than smaller/younger ones?

Line 350-359: Or it could be due to a vitamin D deficiency. What was the vit D3 content of the larvae. Did they receive enough vit D3? Since no UVB source was provided, dietary vit D3 content of the offered diet should be discussed here as a potential cause for lower Ca levels on day 35….

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Reviewer #1: No

Reviewer #2: Yes: Mark D Finke

Reviewer #3: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

1 Apr 2020

We would like to submit a revised version of the manuscript entitled “Digestibility of black soldier fly larvae (Hermetia illucens) fed to leopard geckos (Eublepharis macularius)" by KL Boykin, RT Carter, K Butler-Perez, CQ Buck, JW Peters, KE Rockwell, and MA Mitchell. Please see the following outline to review changes and comments that were made regarding the requested revisions. Line numbers correspond with the manuscript version (not the tracked changes version).

Sincerely,

Kim Boykin

Overall: In particular you need to pay detailed attention to the actual compound measured regarding vitamin A status - likely retinol - and associated units. Vitamin A, per se, is typically reported as IU or UI - based on the underlying compound measured. Especially with exotic species and the limited knowledge of nutrient requirements, physiology and metabolism - and particularly how they are evaluated and utilized, one must be explicit with defining actual compound(s) measured.

Comment to reviewer: I believe we have made the changes requested. Plasma vitamin A was measured as retinol and liver vitamin A was measured as total vitamin A. As for food, we wrote the units out in mcg/kg to follow convention with other papers that had been published this way, as well as due to the fact that IU seems to be on the way out. The new nutrition labels for the FDA require vitamins be listed in mcg or mg instead of IU.

Line 1: Changed title to be more succinct

Line 26: “between” changed to among

Live 39: “nutrition” and “reptiles” have been added as keywords

Line 67: changed “Vitamin A is important…” to “Vitamin A is integral to many different…”

Line 70: added the word generic

Line 70: changed “based on the known requirements” to “extrapolated from requirements…”

Line 72-73: changed “but determination of whether it can be digested and absorbed by a reptile” to “but determination of reptile digestibility and absorption requires further study.”

Line 77: changed “that are well-known to be susceptible to nutritional disorders…” to “with well-documented nutritional disorders…”

Line 79: Is this true? Do they have teeth or palatine ridges? Suggest rephrase or omit.

Response to reviewer: Leopard geckos have pleurodont teeth that are replaced every couple of months (this has been well studied). I also have videos of them partially chewing the larvae prior to swallowing that I used to show at a conference where this research was originally presented. I’d be happy to email the video to you if you would like.

Line 82: changed “dusting powders” to “dusting powder supplementation”

Line 85: Changed “than” to “compared with”

Line 98-101: “Each gecko was started on a vitamin A depleted diet (fasted crickets and BSF larvae) during the acclimation period.” Did you analyze diet to confirm deficiency in vitamin A? Size and amounts of insects fed.

Changed to “The baseline diet fed during the acclimation period was comprised of fasted crickets (4 week old nymphs) and large BSF larvae at 3% of the gecko’s body weight. Vitamin A concentrations in these insects were well under 100 µg/kg (as fed), which is considered very low.”

Response to reviewer: More specific feeding amounts cannot be easily provided as we would only feed crickets periodically. They were usually only fed to those that were losing weight or not showing interest in eating BSF larvae for a week or more.

Line 102-103: “while four geckos underwent a 26 day acclimation period. The latter group was needed to replace four original subjects that would not eat BSF larvae.” Changed to “while four geckos underwent a 26 day acclimation period with the latter group needed to replace four original subjects that refused to eat BSF larvae.”

Line 103: Comment- Palatability probably deserves a comment in discussion, almost 20% refusal is substantial.

Comment to reviewer: Palatability is mentioned in discussion, line 508-513.

Line 109: Added the word potential “ocular changes associated with potential hypovitaminosis A”

Line 132: Added n=13. We were not able to get blood out of everyone this round hence the decreased value.

Line 131-132: Comment- Was this a pooled sample or from each individual?

Comment to reviewer: The first vitamin A sample was not pooled as we were unaware of the need to make it a pooled sample at this time. The second and final sample was pooled as was mentioned in the manuscript (line 142).

Line 132: Comment- As just retionol? Or other compounds and retionoids too? Need to be specific

Comment to reviewer: Added “measured as retinol” to the sentence.

Line 132-133: We did not make the sentence reformatting change you suggested here because according to journal submission guidelines, Michigan State University has to be written out in its full form before it can be abbreviated and your suggested way would have needed parentheses inside of parentheses, making it look jumbled.

Line 134: Changed to “A second sample, obtained on Day -35 (n=20), was used…”

Line 136-138: Two separate sampling periods were required “due to” the blood volume required for “each test (>0.25 ml for UPLC and >0.1 for biochemistries)” and difficulties in drawing “adequate quantities in a single sampling.”

Line 143: Added (n=24 and 24, respectively).

Line 145: Removed the word “randomly”

Line 153: Comment- Provide this on a DM basis as well. This has been added (3,636 µg/kg DMB).

Line 157: Comment- Suggest you add a similar statement for the acclimation period. Did you not feed crickets during the trial period?

Comment to the reviewer: Geckos were fed 3% BW during the acclimation period but was bumped to 5% BW for the study because several were losing weight despite a good appetite. We don’t think it is necessary to the paper to add all of the details of acclimation and trial period as it would just continue to make the manuscript bulky.

Crickets were occasionally fed during the study period (approximately 2-3 times per month) to ensure that some geckos would not lose too much weight. Feces were not collected near these days to ensure only BSF larvae were in the feces.

Line 157-159: Changed to “The weights of BSF larvae ingested by the geckos were recorded over the course of the study (140 days) to calculate total vitamin A ingestion.”

Line 163: Comment- If you actually did this, and not by individuals, then you cannot run statistics as you’d have n=1 only for each treatment group

Comment to reviewer: Although each group was pooled, we still had two batches from each pool that were able to be analyzed. We were supposed to have more, but we had issues with our lab animal workers cleaning cages and throwing away fecal samples when they weren’t supposed to.

Line 163: Changed to “Any water “dishes” that contained feces “were transferred to” a glass…”

Line 168: Comment: This needs better definition. Did you collect over different phases (i.e. 0-50 days, 51-100 days, 101-140 days, etc.) to get your subsamples? Unclear as currently written.

Comment to reviewer: Although we did collect feces over the entire time period, we had issues with fecal samples being thrown away and leaving us with unusable data. We have clarified which days were actually used in our final analysis which ended up being the first and last month of the study. See line 161-162.

Line 203 and 204: changed “by the” to “as”

Line 208: changed between to “among”

Line 207-209: Comment- You had 3 groups—did you also measure control?

Comment to reviewer: Wording seemed a little confusing. We calculated larvae intake (in grams) per group not the estimated vitamin A intake per group, sorry for any confusion. We changed it to say, “larvae intake was analyzed for significance among groups.” So yes, the control group was also calculated here.

Line 213: did not make suggested change (adding a hyphen between words “gut loaded”) as it does not match the format already established

Line 217: changed “data; p<0.05”

Line 224-228: Comment- Confusing- so only 4 of the 7 were replaced?

Comment to reviewer: Four geckos were removed before the study even started. These seven were removed approximately 1 month prior to the end of the study (day 140) due to inappetence and weight loss.

Line 239: Changed “Correlation analysis showed no significant…”

Line 244: Comment- Are you sure? This looks more like a target level in the diet of 20,000 IU/kg DM. If this is actually the target, conversion of µg retinol would equate to 66,667 IU/kg DM for the diet. Similar for the larvae- 1000 µg/kg would equate to 3333 IU/kg AFB in the larvae or close to 10,000 IU/kg DM.

Comment to reviewer: The values are correct as written. The larvae diet has to have an extremely high vitamin A content to be able to reach appropriate levels in the larvae. One of our cited papers (Finke 2003) can be used to confirm the high amounts needed for vitamin gut loading in insects.

Line 245: Comment- Convert this to DM as well. Probably something like 3000-3500.

Comment to reviewer: Added “1000 µg/kg (as fed or 3,636 µg/kg DM)”

Line 246: Changed “was” to “contained”

Line 247: changed “were” to “analyzed at”

Line 247- 248: Did not accept change to combine the sentences because it makes the resulting sentence too long and it reads incorrectly.

Line 250: Comment- Ug is not a proper unit for reporting vitamin A per se- it is the unit for retinol and other retinoids. Convert to vitamin A in IU if you want to talk about that nutrient or keep using ug units referring to retinol and provide justification for your values. Conversions can be tricky but can be justified.

Comment to reviewer: As stated above, we reported food vitamin A in mcg/kg to follow another paper that was published regarding vitamin A gut loading of insects (Finke 2003) and because the FDA is starting to change nutrition labels to show mcg or mg instead of IU. The measurements we received back from the lab were in IU, but we converted them to this format because of the reasons outlined.

Line 253: Changed “between” to “among”

Line 256: Changed “no significant difference in digestibility among groups for any of the nutrients analyzed.”

Line 261: Remove the word “much”

Line 266: Changed “between” to “among”

Line 276: Changed “bloodwork values” to “blood values”

Line 277: Changed “Baseline plasma vitamin A (measured as retinol) concentrations were all <50 ng/ml...”

Line 279: Changed “give” to “obtain”

Line 281: Comment about using 20 ng/ml in place of <20 ng/ml: Cannot really do this

Comment to reviewer: We agree that this is not the best way to perform statistics. Unfortunately, if we do not keep this number in, our sample size becomes too small and we lose significance. With that in mind, we assigned it the highest value we possibly could knowing that it is likely lower than that value (which would better support our data). Even with it as the highest number that it could be, we were still able to show significance between the two groups.

Line 282: changed “read” to “analyzed”

Line 282: did not add “(retinol)” here as it seems repetitive given the change that was made to Line 277

Line 282: Change “vitamin A was” to “vitamin A concentrations were”

Table 3: Comment- Perhaps need to call your “groups” “treatments” throughout the MS

Comment to reviewer: The authors believe that the word “group” is less confusing to people overall and would prefer to keep it as is.

Line 295: Changed “between” to “among”

Line 297: Did not change “group” to “treatment” as was mentioned above.

Line 298: Changed “at” to “during”

Line 301: Comment- This is not valid. Really better to exclude.

Comments to Reviewer: Omitted the two values for calcium and albumin as their removal did not change our p values and did not significantly change the mean, SD, or F statistics. However, in this particular instance, we still believe that they can be used as adequate place holders. For calcium- we were seeing that values decreased over time. By changing the >16 mg/dL baseline value to 16, we are actually shortchanging ourselves. For albumin- we were seeing decreases by day 35. By changing a less than 1 value to 1, we are again shortchanging ourselves of potentially an even greater change in values, but again still able to see a significant difference. If the rounding had changed values in the opposite directions from how things were trending, then we would definitely had removed them from the first submission.

Line 307: Comment- Title for Table 4 needs to be more comprehensive for table to stand alone.

Comment to reviewer: Title has been changed to “Average biochemistry results from all geckos over time (baseline vs. Day 35)”

Line 316: Comment- What is the baseline vitamin A content of your BSFL?

Comment to Reviewer: See line 249: 23 ± 30 µg/kg (as fed or 84 ± 109 µg/kg DMB)

Line 331: Changed “whole” to “intact”

Line 332-334: Comment- This is not a sentence- rewrite.

Comment to reviewer: “Protein, magnesium, potassium, sodium, iron, zinc, copper, and molybdenum also saw significant gains in digestibility (Table 2).”

Line 340: Changed “between” to “among”

Line 342-343: Changed “but this led to decreased…” to “but this can lead to decreased…”

Line 346: Added “when fed intact BSF larvae.”

Line 346-348: Comment- Probably needs rephrased. The calcium is not “released” per se, but that likely that stomach acid has more surface area to act upon it if in smaller particles (i.e. ground).

Comment to reviewer: We agree, just difficult to say succinctly! How is this? “It would appear that gecko mastication or needle piercing does not provide enough disruption to the exoskeletal matrix to allow for calcium carbonate digestion.”

Line 368: Comment- In all treatment groups? Or which?

Comment to reviewer: Yes, all treatment groups saw a decline over time. Added “… for all treatment groups.”

Line 376: Changed “done” to “conducted”

Line 379: Comment- With all diet treatments?

Comment to reviewer: Yes, all treatment groups saw a decline over. Added “… for all treatment groups.”

Line 380: Changed “drop” to “decrease”

Line 391: Removed the word “on”

Line 393: Removed “…consuming either whole BSF larvae or as the main ingredient…”

Line 399: Changed “Proving the second hypothesis regarding…” to “The second objective regarding…”

Line 400: Removed “a little”

Line 407-410: Changed to “Without baseline hepatic concentrations of vitamin A for individual geckos, it is impossible to truly evaluate a change in vitamin A status, but random assignment of the geckos to treatment groups should have minimized any bias.”

Line 411: Removed “per MSU”

Line 412: Did not accept change from “unless the study became non-survival” to “unless the study design were altered” because we really want to highlight the fact that we were able to perform survival surgeries. Keeping the wording as is, is important to us here.

Line 413: Changed “sample size population” to “sample population sizes”

Line 421-422: Comment- With 8 animals per treatment, could you have pooled at least some each sampling time to get longitudinal samples?

Comment to Reviewer: We could have, had we known at the beginning that their values were going to be low enough to require the higher sampling volumes. But we had already turned in our full samples to MSU and ended up not getting useable results back. We tried to use an ELISA plate assay for retinol during the middle part of the study (required less volume), but our results were very inconsistent with that method and we opted to leave those results out of the manuscript (hence why there is a big gap between blood draws in the middle of the study).

Line 423: Added “(measured as retinol)”

Line 438-439: Changed to “Group 1 and 2 were combined, providing a single vitamin A gut loaded treatment group versus the control (Group 3).” We did not use the phrase “value” as suggested because it made sound like there was only one value (n=1) for all of the treatment group (instead of n=8).

Line 441-442: Change to “Liver vitamin A concentrations also differed significantly between the vitamin A gut loaded and control dietary treatment groups.”

Line 443: Comment- redundant; This line has been omitted.

Line 443-448: Comment- Interesting but see if you can condense.

Comment to reviewer: “Across both treatment groups, liver vitamin A concentrations ranged from 2.9-77.98 µg/g, with only a few individuals near the upper end of this range. These geckos likely had higher liver concentrations at the start of the study compared to the others. Age, diet, and husbandry conditions prior to the study (all of which are unknown), are likely contributors to the wide range seen. Paired liver samples would have helped limit this variance, but due to the limitations already discussed, were not performed.”

Line 457-463: Comments- Already reported. Do not repeat but rather incorporate the comparison more concisely. Tighten up (whole section).

Comment to Reviewer: “The values from the present study are much higher, with differences in sex (all female vs. all male study designs) and age being major influencing variables. Females would potentially have lower body stores of vitamin A due to large quantities being stored in the developing eggs. Cojean’s geckos were also 6-9 months old compared to ours that were mostly thought to be adults (> 10 months old). Vitamin A tends to accumulate in the liver as an animal ages leading to potential differences in the two populations.”

Line 476-477: Comment- Or other carotenoids—mixes have been shown more effective for some insectivorous species… Don’t discount that… and its probably more realistic.

Comment to reviewer: We added “should probably be used in combination along with other carotenoid sources” to the end of the paragraph.

Line 478-479: Added “pre-formed” and changed “beta-carotene” to “appropriate precursors”. Added “insectivorous” and changed “will eventually become at risk” to “are at a high risk”.

Line 486: Added “across all treatments and was not correlated with measured plasma or liver vitamin A concentrations.”

Line 488-505: Left this paragraph in as the authors wanted to highlight what lesions would be consistent with hypovitaminosis A.

Line 508-509: Changed “multi-factorial, with a main issue being palatability.”

Line 511-513: Changed “Whether food preferences were due to palatability or a lack of movement of BSF larvae compared to crickets was outside the scope of this study.”

Line 513-514: Changed “Another contributor to inappetence and weight loss was infection with Cryptosporidium sp.”

Line 514-515: We did not accept addition of colon and merger of sentences. Makes the resulting sentence too long. We did shorten the second sentence with your other suggested changes though. Changed “At least three geckos were confirmed to be infected at necropsy, but none showed obvious signs of infection until after…”

Line 516: Changed “having intermittent” to “mixed”.

Line 520: Removed two sentences.

Line 522: Removed one sentence.

Line 522: Changed to “The other possible health limitation found on necropsy was stomatitis; lesions were not seen grossly…”

Line 528: Added “intact”. Removed “much” and “was”.

Line 529-530: Added “Despite high nutrient digestibility for proximate constituents, calcium digestibility, remained low.”

Line 533-534: Added “such as vitamin A incorporated into feeder BSF larvae.”

Overall: As detailed in my comments to the authors my main concern is the conclusion that BSFL should be supplemented with calcium before being fed to leopard geckos. The data as presented does not seem to support that conclusion. Hence why I replied partly to question #1.

Comment to reviewer: Thank you for pointing out your concerns. We do agree with you that theoretically it should be enough calcium to support physiological needs based on the estimated minimum requirements that we have reported for leopard geckos. But we do think that people should still be cautious, especially given the comment you made about not all BSF larvae having a positive calcium to phosphorous ratio. We have changed our comments around accordingly (see below for specifics).

Line 28-29 & 34-36: I’m not sure the data supports these statements. See comment #9 for more details why this might be.

Comment to reviewer: For the abstract we changed the lines to read “… with the exception of calcium (digestibility co-efficient 43%), as the calcium-rich exoskeleton usually remained intact after passage through the GI tract” and “While leopard geckos are able to digest most of the nutrients provided by BSF larvae, including those that have been gut loaded, more research needs to be performed to assess whether or not they provide adequate calcium in their non-supplemented form.”

Line 42-44: While 3 years ago I would have agreed with this statement there is now data showing the calcium content of BSFL is highly variable (more so than I ever would have guessed) and diet dependent. A review of 3 articles (shown below) shows calcium levels from 0.12 (similar to that for other insects) to 6.66% (dry matter basis) and Ca:P ranging from 0.30:1 to 14.9:1. It would be good for the authors to mention this somewhere so veterinarians and zoo nutritionists understand that the diet plays a critical role in BSFL calcium content and that live soldier fly larvae sold commercially can varying widely in calcium content.

Spranghers T., Ottoboni M., Klootwijk C., Ovyn A., Deboosere S., De Meulenaer B., Michiels J., Eeckhout M., De Clercq P De Smet S., 2017. Nutritional composition of black soldier fly (Hermetia illucens) prepupae reared on different organic waste substrates. Journal of the Science of Food and Agriculture 97:2594-2600.

Tschirner M., and Simon A., 2015. Influence of different growing substrates and processing on the nutrient composition of black soldier fly larvae destined for animal feed. Journal of Insects as Food and Feed 1:249-259.

Wang S.Y., Wu L., Li B., Zhang, D. 2019. Reproductive potential and nutritional composition of Hermetia illucens (Diptera: Stratiomyidae) prepupae reared on different organic wastes. Journal of Economic Entomology doi: 10.1093/jee/toz296

Comment to reviewer: Thank you for this information! We have added these sources and changed the line slightly. We also addressed this subject more fully in the discussion.

“…they are the only commercially produced insect that has been found to potentially have a natural positive calcium to phosphorous (Ca:P) ratio (2.5:1) based on their diet.”

In discussion, line 362-364: “Additionally, calcium content of BSF larvae can vary greatly based on their rearing diet’s composition. Recent studies have shown calcium:phosphorous ratios can range anywhere between 0.3:1 to 14.9:1 [4-5].”

Line 65: There is now data available to show that insects can be an excellent source of vitamin D if exposed to UV light which might help explain how nocturnal insectivores get their vitamin D. That article is Oonincx DGAB, van Keulen P, Finke MD, Baines FM, Vermeulen M, Bosch G. 2018. Evidence of vitamin D synthesis in insects exposed to UVb light. Nature - Scientific Reports DOI:10.1038/s41598-018-29232-w

Comment to reviewer: This information has been added.

“…however, recent research has found that some insects can produce vitamins D2 and D3 secondary to ultraviolet B radiation exposure, similar to vertebrates [14].”

Line 222-226: It might be nice to tell the reader the breakout of the 10 geckos that lost weight and the 7 that were removed (i.e. how many came from each of the three groups)

Comment to reviewer: Each breakout has been provided. The breakout for the seven necropsied ones are listed in two separate places now though. If this is a concern, please let us know which one to remove.

Line 195: “Seven geckos (Group 1, n=3; Group 2, n=2; Group 3, n=2) were euthanized intra-operatively after collection of the biopsies…”. This was the original placement.

Line 222-226: “Over the course of the experiment, 10 (41.6%) out of 24 leopard geckos lost weight (mean ± SD: -13.76 ± 7.71% weight loss, range: -0.14% to -23.91%)(Group 1, n=4; Group 2, n=3; Group 3, n=3). Seven (29.2%) of these geckos experienced inappetence and weight loss severe enough to require early removal from the study (Group 1, n=3; Group 2, n=2; Group 3, n=2).” These were the ones that were added per reviewer suggestions.

Line 243-247: I wonder if the authors might graph wt change by food intake (either % of body wt or perhaps ME) and they might be able to generate a nice regression line showing the maintenance energy requirements of these geckos. Might be a nice figure to add if there is a good correlation between intake and weight change.

Comment to reviewer: We looked at this information per your request but there was not a good correlation. Example: I had two geckos that ate approximately 80% of the food I offered them (food amounts were based on 5%BW) and one lost over 8% of his weight over time and the other gained 20% of his starting weight over time. Almost all of them did weird things like this.

Table 2: Perhaps here or somewhere else it might be a good idea to show the readers the average nutrient composition of the BSFL used in this experiment. I realize the raw data is in the supplemental appendix but perhaps to help the reader the average data (at least moisture, protein, fat, ash, Ca and P) might be shown in the article.

Comment to reviewer: Added the requested nutrient composition to Table 1 (column 1).

Table 3: Does the statistical significance change for the plasma vit A data if the sample that was reported as <20 is assigned a value of 0 rather than 20? Seems unusual to assign it a 20.

Comment to reviewer: The value that was reported as <20 ng/ml was in the non-gut loaded group. Assigning it as a zero would have looked as if we were trying to skew the numbers to show that plasma vitamin A was lower in that group, so we assigned it the highest value we could which was a 20. Even with it as the highest number that it could be we were still able to show significance between the two groups, which was enough for us. If we take the value out completely, our results lose significance due to a smaller sample size which is why we decided to keep it in (p=0.04 vs 0.09).

Table 3: Does the statistical significance change for the liver vit A data if the outlier sample of 61.35 ug/g is included in the non-gut loaded group?

Comment to reviewer: Yes it does, p=0.03 (with the outlier removed) vs. p=0.15 (with it left in). Since we were able to follow the statistical rules regarding outlier removal, we felt comfortable removing it.

Line 352-366: I think this section needs to be reworded since the data does not appear to be consistent with previous statements (see comment #1). The authors estimate that “when adjusted for digestibility the larvae supplied 9.2 g Ca/kg diet” which is 8-51% HIGHER than the estimated calcium requirement for growing leopard geckos that they cite (6.1-8.5 g Ca/kg diet). Add to this the fact that these geckos were adults likely means the calcium intake was likely 200-300% of the adult requirement. It is well known that calcium regulation in most species occurs in the intestine. To quote the NRC Mineral Tolerance of Domestic Animals (page 99) “During positive calcium balance intestinal mechanism for absorption are shut down in most species”. That would appear to be the case here as even with relatively low absorption the geckos were likely consuming 2-3 times their requirement. So while low digestibility of calcium in the exoskeleton is certainly a possible explanation realistically feedback mechanisms regulating calcium absorption likely play as much if not a bigger role in the low calcium digestibility observed here.

Comment to reviewer: Thank you for the comment and we have adjusted the manuscript and our final conclusions to hopefully be a little less matter of fact regarding the larvae needing further supplementation given the points you make about adult geckos likely needing less calcium than growing juveniles. That is a very good point and we are glad you brought it up. However, I am not completely sold on the fact that the low digestibility is due to feedback mechanisms regulating the calcium absorption. Our reasoning for this goes back to Dierenfeld’s paper where she measured digestibility of dusted crickets as well. The frogs were able to digest >80% of that calcium despite a higher starting level compared to the BSF. While not a great comparison given the differences between our studies (different species, durations, etc) it is still enough to make me question the digestibility of the exoskeleton. Also, we had the personal observation of large exoskeleton pieces passing directly through the GI tract. Feedback mechanisms could definitely play a role in regulation of unbound calcium being digested, but I don’t think they would prevent the exoskeleton itself form breaking down. Here is our updated paragraph for this section:

“The estimated minimum dietary calcium requirement for growing leopard geckos is between 6.1 and 8.5 g Ca/kg diet (DMB) [18]. When adjusted for digestibility, BSF larvae in our study provided 9.2 g Ca/kg diet (DMB) which should be adequate to support calcium needs. It is possible that the calcium digestibility was low in these leopard geckos because they were adults and calcium absorption was impacted by normal feedback mechanisms. A study in fast-growing juvenile geckos would be helpful in further discerning whether low digestibility is a function of the BSF exoskeleton, as it would be expected to be higher in growing animals with a higher calcium requirement.

However, the authors would still recommend caution due to the fact that physiological needs may vary based on species, age, reproductive status, and/or vitamin D status of the animal. Additionally, calcium content of BSF larvae can vary greatly based on their rearing diet’s composition. Recent studies have shown calcium:phosphorous ratios can range anywhere between 0.3:1 to 14.9:1 [4-5]. Thus, more research would be needed to prove that non-supplemented BSF larvae can, in fact, provide enough calcium to insectivorous reptiles based on how the insects are reared.”

Line 344-345(?): The statement “The increased phosphorus content would require a subsequent increase in calcium level to maintain the proper calcium to phosphorus level” seems overstated given how little data there is on optimum calcium-phosphorus ratios in reptiles and even for most vertebrates studied the ranges are fairly broad, typically 1:1 to 2:1. Suggest changing this to “The increased phosphorus content would require a subsequent increase in calcium level to maintain the SAME (emphasis mine) calcium to phosphorus level”

Comment to reviewer: We took this line out based on the changes to the paragraph we made for the previous comment.

Line 374-377: See the previous comment about calcium regulation (comment 9). As such a blanket recommendation regarding adding additional calcium to soldier fly larvae would seem to put animals at risk for excessive calcium intake which has been shown to inhibit the absorption of trace minerals putting the animal at risk for secondary trace mineral deficiencies. Add to that is the fact that the calcium content of black soldier fly larvae is highly variable and there is no standardized data for black soldier fly larvae sold commercially. As such I think this statement needs to either be removed or extensively modified.

Comment to reviewer: We changed this statement to be much less matter of fact. It now reads “At this time, we do not have enough data to recommend whether calcium supplementation is needed when offering BSF larvae to reptiles. Additional research needs to be conducted to establish true calcium requirements for reptiles and varied diets should always be offered to insectivores to limit the incidence of nutritional deficiencies.”

Line 422-428: Ploog reports plasma vitamin A values for Mississippi gopher frogs and while not a reptile it is an insectivore. Ploog C, Clunston R, Morris C, Iske C, Blanner W, Pessier A. 2015. Hypovitaminosis A: influence of three diets or topical treatment on hepatic, adipose, and plasma retinoid concentrations and presence of squamous metaplasia in Mississippi gopher frogs (Rana capito servosa). In Bissell H, Brooks M Eds. Proceedings of the Eleventh Conference on Zoo and Wildlife Nutrition, AZA Nutrition Advisory Group, Portland

Comment to reviewer: Thank you for the comment. We originally had values for reptiles and amphibians, but removed the amphibians for concision. Since none of the reptiles mentioned are insectivorous, we have added a few of them back and removed the chelonians. Now we can focus just on comparisons between squamates and insectivorous amphibians. Their plasma values still fall lower than most.

“Previous literature has reported plasma vitamin A (measured as retinol) concentrations for various squamates and amphibian species, including green iguanas (Iguana iguana, 52-75 ng/ml), eastern indigo snakes (Drymarchon couperi, 9 ng/ml), anacondas (Eunectes murinus, 80 ng/ml), Mississippi gopher frogs (Rana capito servosa, 36-43 ng/ml), marine toads (Bufo marinus, 60 ng/ml), Cuban tree frogs (Osteopilus septentrionalis 83 ng/ml), and Puerto Rican crested toads (Bufo lemur, 130 ng/ml)[25-27, 30-33].

Line 455-456: There is now data showing carnivores can convert beta-carotene to retinol although whether they can make sufficient retinol from beta-carotene to meet their needs is unclear (see Green, Tang, Lango, Klasing, & Fascetti. Domestic cats convert [2H8]-β-carotene to [2H4]-retinol following a single oral dose. J Anim Physiol Anim Nutr 96:681-92.) Since in some studies that show no conversion the animals were fed or were previously fed diets containing high levels of retinol (often from liver) it may be that feedback mechanisms designed to prevent vitamin A toxicity were in play. Note Mississippi gopher frogs appear to be able to convert carotenoids to retinol. See Ploog at al cited in the previous comment.

Comment to reviewer: Thank you for the comment. We did not end up changing this sentence since there still there is still much more to study regarding this aspect in reptiles.

Line 493-496: Agree however the amount of time required for depletion likely depends on vitamin A stores in the liver so depending on how the animals are fed it may take less than or much more than 6 months to show signs of deficiency.

Comment to reviewer: Changed sentence to “Research in humans and other adult vertebrates confirm that >6 months of depletion is usually needed before clinical signs of hypovitaminosis A are detectable, but will obviously depend on the vitamin A status of the individual prior to the start of depletion [36-38].”

Line 530-533: I think these two sentences need to be modified based on the data shown and my comments (#9).

Comment to reviewer: “While calcium levels were likely adequate for leopard geckos based on estimated calcium requirements for the species, further research is needed to verify this assumption and to determine calcium requirements for other insectivorous species.”

Overall: this is a well written manuscript and a well designed and important study. Some minor comments listed below.

Line 33-34: report p values as 0.04 and 0.03. This change has been made.

Line 78: Please provide a reference for this statement (leopard geckos prone to vit a deficiency, etc).

References have been added to this sentence.

Line 109: provide reference that leopard geckos develop ocular changes due to vitamin A deficiency

Line 195, 224, 226, 236: the 7 emaciated geckos were from all 3 treatments groups? Would be worth reporting here.

These lines have all been updated to show distribution across groups.

Line 260-262: the statement regarding the frogs is inappropriate for the result section and should be moved to the discussion section.

Comment to reviewer: Usually we would agree with you. However, we really wanted the two digestibility tables to be placed next to each in the manuscript (and not interrupted by having the biochemistry table and vitamin A table in between). We found it easier for the purposes of flow to place it here as we were already describing how the first column represented the three treatment groups being averaged together. We also performed statistics between our data and the frog data to determine if there were any significant differences between what we reported and what was already published. If you would still like it moved, we absolutely can. These were just the reasons why we preferred it here.

Line 298: possible that this animal was a female, and not a male?

This gecko was definitely male based on external characteristics. This does not rule out the possibility of an ovotestis, which has been reported in various lizard species. Since we have now removed the value from statistical analysis, we do not believe that it needs to be discussed as a possibility in the manuscript.

Line 333-338: were the larvae fed in your study and in the frog study of same size/age? Possible that older/larger larvae are harder to digest or have different nurtritional values than smaller/younger ones?

Comment to reviewer: Thank you for the comment. This is definitely possible. In looking back at the previous research, the frog study used medium sized larvae, whereas we used large sized larvae (weighed twice as much on average). I also looked at the frog study’s nutrient composition versus our own. DM and crude protein were very similar between the two studies. However, the frogs’ mineral levels were all moderately higher meaning that it is possible that we saw higher digestibilities for those nutrients because there was less to begin with. We have added a comment to bring up this point.

“Some of the differences in digestibility could be related to differences in larval composition between the studies. However, given that most of our digestibility coefficients were more similar to the values reported for mashed larvae from their study rather than the values for intact larvae, we believe that most of the differences were due to a higher degree of mastication by the leopard geckos which would allow for digestive enzymes to breach the tough exoskeleton and breakdown the inner portions of the larvae.”

Line 350-359: Or it could be due to a vitamin D deficiency. What was the vit D3 content of the larvae. Did they receive enough vit D3? Since no UVB source was provided, dietary vit D3 content of the offered diet should be discussed here as a potential cause for lower Ca levels on day 35….

Comment to reviewer: Very astute point. We purposefully did not supplement with vitamin D3 or UVB because of its role in calcium absorption and it would have added another variable that we weren’t able to control very well. However, we did neglect to mention that vitamin D deficiency could be a cause of lower calcium levels. We have added a quick mention to two different spots.

Line 360-361: “However, the authors would still recommend caution due to the fact that physiological needs may vary based on species, age, reproductive status, and/or vitamin D status of the animal. “

Line 368-369: “Poor calcium digestibility or low levels of vitamin D could be possible causes for this decline.”

16 Apr 2020

Digestibility of black soldier fly larvae (Hermetia illucens) fed to leopard geckos (Eublepharis macularius)

PONE-D-19-33399R1

Dear Dr. Boykin,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Jake Kerby, Ph.D.

PLOS ONE

Reviewer's Responses to Questions

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: Good job of addressing reviewer concerns and comments; statistics on small sample sizes remain problematic (and likely will with most exotics), and the assignment of values below detection limits is still not statistically appropriate, but your justification is acceptable.

Footnote for Table 1 should read "No significant differences....." (rather than no significance)

Reviewer #2: See attached for some minor comment but I liked the original manuscript and this revision makes it even better. Kudos to the authors for a job well done and contributing valuable information to the field.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Reviewer #1: No

Reviewer #2: No

29 Apr 2020

PONE-D-19-33399R1

Digestibility of black soldier fly larvae (Hermetia illucens) fed to leopard geckos (Eublepharis macularius)

Dear Dr. Boykin:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Jake Kerby