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Both the degradation and mass reduction of the manure with increasing larval density resulted in a corresponding proportional increase in the mass of dry manure per egg mass produced (r2 = 0.75, p<0.05). According to the initial hypothesis that there will be optimal conditions to support larval growth and survival, we compared the consequences of varying the moisture content on the mass of the manure produced per egg mass. Increasing the moisture content from 50% to 75% of manure total weight had no significant effect on mass reduction (Fig 3C). However, manure moisture content greater than 75% of total weight (relative to total weight of egg mass) led to significantly decreased mass of manure per egg mass as compared to controls (Fig 3D). Moisture content of manure greater than 70% thus does not appear to be conducive to feeding larvae. It is unclear why the relationship between relative moisture content and growth-related fitness of larvae shifted at moisture contents greater than 70% in our system. However, these experimental results indicate that there is an upper limit to moisture content that allows for effective degradation of manure, and therefore larval growth and survival. Our results suggest that manure degradation has been optimized in housefly larvae production systems.
Manure samples from the 28 day experiment were further processed to identify nutrients that were limiting larval growth. These samples were analyzed for phosphorus, calcium, and magnesium. Samples were loaded into a lyophilized form and analyzed using an elemental flame photometer (Cobas Integra; Roche Diagnostics, Indianapolis, IN, USA) for phosphorus, magnesium, and using a nitrate/nitrite colorimetric kit (Cobas Mira; Roche Diagnostics) to analyze the calcium levels. Total nitrogen content of the manure samples was analyzed using the Kjeldahl method (Kjeltec 2300 Analyzer; Tecator, Horest, Sweden). The efficiency of nitrogen digestion was based on changes in nitrogen content of the manure (Fig 4A). In addition, the percentage of nitrogen that was available for the larvae was calculated by subtracting the total amount of nitrogen present in the manure samples (mg/kg manure) from the total amount of nitrogen in the eggs (mg/g of eggs) (Fig 4B). Manure samples produced at all larval densities (1, 2, 4, 8, 16 eggs/g of manure) had significantly increased amounts of nitrogen (Fig 4C), compared to controls (0/g of eggs of manure; p<0.0150). Larvae produced at all densities, except the density of 16 eggs/g of manure (Fig 4D), reached similar levels of N.N availability (p<0.05)Fig 4C).
In terms of possible non-nutritive effects of manure degradation, there was no significant change in moisture content of manure over the 5-day experimental period (p>0.05). While there was a small decrease in ammonia content of manure with increased larvae density (p0.05). A potential concern is that increased manure degradation as larvae density was increased would lead to increased contamination with fecal microbial metabolites. However, the ammonia levels in this study were significantly lower than those found in other studies using similar treatments (2332).
For each sample, a single dose of HPLC-grade water (1.0 ml) was added to 250 mg of manure and vortexed for 30 seconds. The liquid phase was then separated from the manure matrix by centrifugation (15,000 × g, 5 minutes). The upper phase was filtered through a 1.2-µm Teflon filter and the filtrate was concentrated in a CentriVap concentrator at −40C for at least 5 hours and freeze-dried using a Micropack Freeze-Dryer (model 54100-10, Robert Hill Scientific, Mega-process, Manchester, UK). The freeze-dried sample was then weighed and milled to a homogeneous powder using a mortar and pestle. The resultant pellet was stored at −20C in a desiccator and ground again to a homogeneous powder before analysis. Samples were analyzed for 9 amino acids. The analyses of amino acids were performed using previously described methods [35] with the following modifications. In brief, 5 g of the ground mixture was pre-digested in 500 ml 0.5 M hydrochloric acid in a shaker bath at 55C for 3 hours with agitation (500 rpm). After centrifugation, the supernatant was adjusted to pH 7 using 1 M sodium carbonate (NaCO3) and centrifuged again. The filtrate was then mixed with a buffer solution containing 5% ninhydrin (1,4-dimethyl-5-phenyl-1,3,5-triazinylamine hydrate) and 3% sodium phosphate (sodium trihydrogenphosphate). After standing for 15 minutes, the mixture was extracted with diethyl ether, and the organic phase was washed with water. The organic phase was then dried over anhydrous sodium sulphate and evaporated to dryness under a stream of nitrogen. The residue was dissolved in 0.2 M sodium hydroxide. After filtration with a 0.45-µm syringe filter, amino acid analysis was performed using a HPLC system Gold with 32 Karat software (Beckman-Coulter, Inc., Fullerton, CA, USA). Detection was at 340 nm following the ninhydrin reaction post column derivation. Separations of amino acids were carried out on a cation exchange column (4 x 150 mm, P/N 0354100, Pickering Laboratories, Mountain View, CA, USA) using a three-buffer step gradient (Li292, Li365 and Li375, Pickering Laboratories, Mountain View, CA) and a column temperature gradient (33, 42, 60 and 70C); detection was at 340 nm following the ninhydrin reaction post column derivation. Results were standardized using cysteine as the internal standard. Analyses of amino acids were performed in duplicate on the same day.
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