Final report for GNE17-159
Increased consolidation and specialization of dairy farms have created larger point-sources of cow manure and left farmers dangerously vulnerable to fluctuations in the price of milk. Thus, safe and effective management of manure and diversification of on-farm revenue streams are priorities for the Northeast region.
Our goal was to address both issues by creating a system for producing housefly (Musca domestica) larvae meal (LM) using dairy cow manure. We hypothesized that LM had potential as a feed ingredient in the diets of farmed fish, and that it might additionally confer disease resistance for these fish. Since production of LM also has desirable effects for manure management - reduction in volume and amelioration of antibiotic resistance - this system has the potential to both strengthen existing dairy farms and kickstart sustainable aquaculture in the region.
To test these hypotheses, Housefly larvae were raised on dairy cow manure. Third stage larvae were processed into LM that was included in diets for rainbow trout (Onchorhynchus mykiss). The control diet was based on a typical modern diet, and included 10% fishmeal and 11% fish oil by dry weight, with the remaining protein and fat coming from soy-, corn-, and wheat-derived ingredients (Table 2). High- and low-inclusion LM diets replaced these plant-based sources so that the final diets were 30% or 5% LM by dry weight. After 8 weeks, trout fed a diet comprised of 30% LM showed significantly increased growth compared to the control diet, while the 5% LM diet showed a non-significant increase in growth (Figure 4). High mortality (~20%) was observed in the 5% LM diet over the course of the 8 week trial, but no clear cause was identified (Figure 3).
We next addressed the question of whether housefly LM could improve resistance to disease. We suspected that if such an effect were mediated by stimulation of the fish immune system, it might be short-lived. Thus we included groups that were exposed to the 5% and 30% LM diets for 2 and 8 weeks (Figure 2). We measured serum lysozyme activity from these fish, and found that it was significantly increased in fish from 5% and 30% LM groups after 2 weeks, but reduced after 8 weeks (Figure 5). This supports the hypothesis that immune stimulation due to LM is temporary. We next conducted an infection trial by intraperitoneal injection of Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD). Although we had determined an optimal bacterial dose for 50% mortality in an earlier pilot infection study, this dose proved to be too low and we had insufficient mortality across all treatment groups to draw any conclusions about possible protective effects of LM (Figure 6). We are currently investigating possible mechanisms by which LM might confer disease resistance, and plan to repeat the challenge study once we have a clear hypothesis about the mode of action.
Overall, the results of this study are encouraging - LM produced from cow manure was a superior aquaculture feed ingredient when compared to common plant-based alternatives, and it did provide an apparent boost in immune activity after two weeks of feeding - but several questions remain unanswered. Understanding if and how LM confers resistance to disease is an open question. More systematic research that incorporates insect biology, fish immunology, and microbiology will be needed to untangle these connections. At the same time, developing scalable methods of LM production is necessary if LM is to compete with established feed ingredients. The goals of this project - 1) mitigation of biosafety concerns surrounding manure, 2) diversification of dairy farm revenue streams, 3) stimulation of Northeast regional aquaculture production - remain attainable and realistic with proper support and guidance.
1) Raise housefly larvae on organic dairy manure. Our group’s previous publication on production of housefly larvae (M. domestica) used manure from the Cornell teaching barn. To demonstrate this technique using manure from an actual dairy farm, we partnered with Jerry Dell Farm, a local organic dairy producer.
2) Produce diets for rainbow trout using insect meal (IM). Diets with both high and low inclusion of IM will be formulated to meet the nutritional requirements of juvenile rainbow trout (O. mykiss). Modern fish feeds typically contain some fishmeal (FM), with most protein coming from plant ingredients to save on cost. In this preliminary stage, we aim to demonstrate that IM is a superior replacement for fishmeal than the modern alternatives (soy protein concentrate and corn gluten meal). This design gave our experiment the best balance of relevance (using IM in the context of a realistic modern diet) and control (direct comparison of FM replacements). Future studies should investigate the feasibility of using IM to cut down FM content even further.
3) Conduct trout feeding trial using IM diets. Juvenile rainbow trout were fed diets with both high and low inclusion of IM to evaluate nutritional value of these feeds. A secondary hypothesis we are interested in evaluating is that IM diets are less pro-inflammatory than diets with high levels of plant ingredients, owing to lower levels of pro-inflammatory omega-6 fatty acids and trout’s evolutionary adaptation to eating insects. In addition to growth parameters, assays of immune health and inflammatory stress were conducted.
4) Immune challenge with BCWD. At the end of the feeding trial, juvenile trout were exposed to a bacterial infection. The objective was to broadly screen for possible immune-stimulatory effects of insect meal, as these have been reported in recent work (Ido et. al 2015). BCWD is a common infection in salmonids in both the hatchery and aquaculture context. Mortality can be as high as 85% in fry and fingerlings and fish that survive the infection often present with scoliosis and lordosis as a result of muscle fiber destruction. There is no commercial vaccine for this disease, therefore development of an IM diet that enhances resistance to BCWD would provide additional value for both hatcheries and commercial fish farms.
Dairy cow manure is increasingly concentrated due to consolidation of farming operations, and is increasingly treated as a potential hazard to human and environmental health. In the Northeast there is particular concern about what to do with manure during the winter months when it cannot be spread safely or effectively for fear of runoff. Building a storage facility can be costly – anywhere from $100 to $1000 per cow depending on the design – and farmers who spend the money to build safe containment structures rarely save enough money on commercial fertilizers to benefit financially from being environmentally responsible. Meanwhile, consolidation and specialization of dairy farms has increased the vulnerability of dairy farmers to fluctuations in the market for fluid milk. Diversification of dairy farm revenue streams could help to insulate farmers from future volatility.
To help address these issues, our group has developed a method for raising Housefly larvae (Musca domestica) on dairy cow manure. Insect larvae are high in protein and fat, have rapid generation times, and can be cultivated using waste streams. So-called “upcycling” of organic waste into valuable ingredients is gaining traction worldwide, and insects are a core component of the strategy. Through conversion of manure into insect protein, the value of stored manure is increased - shifting the cost-benefit equation of storage for farmers. After processing by larvae, dairy cow manure retains 75% of its wet weight (95% of dry weight), 80% of organic nitrogen (75% total nitrogen), and 94% of phosphorus. Moreover, processed manure has been shown to have multi-log fewer antibiotic resistant bacteria. Thus, the production of insect larvae from manure results in several benefits: a modest reduction in the volume of manure, an added-value product from the manure, and increased biological safety by reduction of antibiotic resistant bacteria. There is little tradeoff for farmers in terms of loss of raw nutrients (after larval processing the manure can still be used as fertilizer), and insect larvae can be processed into feed ingredients.
In this project we followed previous work from our group to its next practical step – demonstrating the economic utility of insect larvae produced in the manner in the Northeast context. The goal of this study was to demonstrate a use for insects raised on dairy cow manure that 1) has potential to be profitable, 2) reduces manure volume, providing environmental and public health benefits, and 3) paves the way for growth of new agricultural industries in the Northeast.
We investigated the utility of insect larvae meal (LM) produced from dairy cow manure as a feed ingredient for cultivated rainbow trout (Oncorhynchus mykiss). The appeal of LM to fish farmers and hatchery managers is clear: production of high value farmed fish species like trout and salmon is currently limited by reliance on fishmeal (FM) and fish oil (FO) as feed ingredients. FM and FO are finite resources being exploited at close to or above sustainable limits, meaning that prices for FM and FO have increased rapidly. There is thus a strong push from both industry and environmental groups to develop alternatives to FM and FO.
Initial results using IM as a supplementary feed ingredient are promising, and one hypothesis for this is that insects represent a more natural diet for fish than commonly used ingredients (e.g. soybeans). The combination of high protein content, favorable amino acid profile, and evolutionary adaptation by freshwater fish makes LM an ideal candidate feed ingredient. Additionally, a recent study by Ido et al. showed that a diet incorporating Housefly ingredients can protect red sea bream (P. major) against bacterial infection. This result has not been repeated, but bears investigating: pro-immune effects would greatly increase the value of LM.
Thus, our project sought to solve multiple current problems in agriculture through an integrated approach. We produced Housefly LM using dairy cow manure, made a fish feed, conducted a growth trial in juvenile Rainbow trout, and then performed an infection challenge to determine whether LM could protect against bacterial cold water disease (BCWD).
- (Educator and Researcher)
Larva meal was produced as described in our group’s recent publication (Hussein et. al 2017). Housefly pupae purchased from a distributor (Spider Pharm, Yarnell, AZ) were used to establish fly breeding colonies. Adult flies were kept in mesh cages and provided a mixture of dry milk and sugar (1:1) as a source of food. Drinking water was provided in a closed water container with a protruding cotton wick. Flies were kept in a room at 25 ± 2°C with a photoperiod of 12 hours per day.
Fresh manure from a local organic dairy farm was used as a source of substrate for oviposition. Every three to four days, a cup of fresh manure was placed in a fly cage and eggs collected 18-24 hours later. Eggs were diluted to a density of approximately 4-8 eggs per gram of manure and maintained in a high humidity chamber. After a few days, larvae migrated out of the manure container and fell into an underlying collection tray. Larvae were collected daily and stored at -20°C. Shortly before production of diets, larvae were dried at 50°C and ground into LM using an adjustable burr grinder.
b) Production of diets
Three fish diets were prepared: a standard diet, a 5% larva meal diet, and a 30% larva meal diet. The diets were formulated as described in Table 2. Diets were prepared by mixing the different ingredients with sufficient warm water to form a dough, spreading the dough into a thin layer on baking sheets and and drying to completion (no further reduction in weight after 4 hours of drying) in an oven at 50°C. The dried feed was then crushed and ground using an adjustable burr grinder and the resulting crumble was size-sorted using stacked sieves. Final feed composition was assessed by Brookside Laboratories (New Bremen, Ohio) (Table 3). Dried feed was aliquoted and stored in the dark at -20°C for long-term storage.
c. Fish care and handling
Rainbow trout (O. mykiss) was selected for these experiments as it is an important hatchery and aquaculture species for the United States. Approximately 450 fingerlings of 1-2 g were purchased from Beaverkill Fish Hatchery (Livingston Manor, NY). Upon arrival, fish were acclimated for 4 weeks, then randomly sorted into groups of 14 fish, batch weighed, and distributed to 10 gallon recirculating tanks at a stable temperature of 10-12°C. During acclimation, fish were maintained on a commercial diet for fingerlings (Ziegler Bros, Gardners, PA) and were fed 2 times a day to a maximum of 3% body weight per day. All experimental procedures were approved by the Cornell Institutional Animal Care and Use Committee (IACUC).
d. Feeding Trial
After the acclimation period, tanks were randomly assigned to one of 5 experimental groups comprised of the 3 experimental diets. Figure 2 summarizes the 5 experimental groups. Group 1 was the control group and was fed the control diet for 8 weeks prior to receiving the immune challenge. Groups 2 and 3 were fed, respectively, a 5% and 30% larva meal diet for 8 weeks prior to challenge, whereas groups 4 and 5 were fed the control diet for 6 weeks before being switched to the 5% and 30% larva meal diets for 2 weeks prior to challenge. Groups 4 and 5 were dropped from the growth trial after week 6, when their diet changed. Each group featured 84 fish spread across 6 technical replicates (6 tanks with 14 fish per treatment).
During the feeding trial, fish were fed 2 times a day to a maximum of 3% body weight per day. Fish were batch-weighed by tank every two weeks, and feeding ration was recalculated for all fish based on the weight of the control group after each weighing. At the conclusion of the feeding trial, serum samples were collected from 10 fish per diet group and evaluated for lysozyme activity (Ellis, 1990) to assess innate immune status. 10ul serum samples were added to 190ul of a .15mg/ml suspension of Micrococcus luteus in triplicate on a 96-well plate before being read at 450nm every 15 seconds for 12.5 minutes on a microplate reader (BioTek). Over this time period, all samples were strongly linear, thus activity was calculated as the rate of change in absorbance over the full 12.5 minutes.
e. Immune Challenge
An isolate of Flavobacterium psychrophilum was obtained from Thomas Loch, Michigan State University. The strain was isolated from the kidney of a feral Steelhead trout (Oncorhynchus mykiss) and was of sequence type 31 (1). Bacteria were grown at 15°C 150rpm in Tryptone Yeast Extract Salts broth (TYES): 0.4% tryptone, 0.4% yeast extract, 0.05% magnesium sulfate, 0.05% calcium chloride, pH 7.2. Bacteria were harvested during the exponential growth phase (OD600 ≈ 0.3-0.4), washed in PBS, and suspended in PBS to an estimated concentration of 1 x 108 CFU/ml. To determine the appropriate challenge dose, a subset of fish from the experimental cohort were moved to a separate room for a preliminary study. Fish were kept under identical conditions to those in the rest of the study, and were fed the experimental control diet at 3% BW/day. Fish were anesthetized by immersion in Tricaine Methanesulfonate (MS-222) and injected in the coelomic cavity with 50µl of bacterial suspension at across a range of dilution. Dilutions of the inoculum were spread on agar plates and incubated to determine the bacterial concentration. The actual infectious dose to achieve ~50% mortality over 4 weeks was determined to be 7.5 x 106 CFU/fish. This dose was used for the main infection challenge with the same regimen of anesthesia and injection. For the main infection challenge, two rooms with the same experimental setup were used; in the infection room, fish were injected with live F. psychrophilum in PBS, while in the control room fish were injected with sterile PBS.
a) Raising housefly larvae on dairy manure
Larvae production using the method established in Hussein et. al (2017) was successful, though it should be noted that laboratory-scale production remains a significant bottleneck for research. The current study required 4 months of daily larvae collection to produce an adequate amount of LM. Future studies that use larger fish or run for a longer duration currently face significant production constraints. Parallel research into larger-scale production of larvae would be valuable for research.
b) Production of diets
The protein requirement for Rainbow trout fingerlings is 38% of dry weight, and our control diet represents a modern diet containing protein from several common sources. The design of the diets in this study sought to balance multiple considerations in assessing a novel feed ingredient; analysis of the diets confirmed that they were consistent with the desired nutritional values (Table 3). Selection of ingredients in feed production is driven by the balance between cost and performance - while fishmeal is generally regarded as the gold standard of protein ingredients, modern diet design has sought to replace fishmeal with cheaper and more sustainable alternatives. Thus, the ultimate utility of LM will hinge on both its nutritional performance and its cost of production. Cost of production for LM is hard to forecast, but it is expected to be lower than fishmeal and higher than many plant ingredients. The goal of our study was therefore to provide rigorous empirical data on the performance qualities of LM in relation to existing non-fishmeal ingredients, in anticipation of more reliable cost projections. Our control diet is based on a modern commercial-type trout diet (Fehringer et. al, 2014), featuring a typical blend of marine and plant ingredients: fishmeal (10%), soy protein concentrate (20%), corn gluten meal (18%), and wheat gluten (5%).
In both of the experimental diets, the levels of marine ingredients was held constant, while plant ingredients were replaced by LM. LM used in this study is a crude ingredient, without the quality controls and processing to ensure optimal purity and performance. Thus, there is reason to be optimistic that the full potential of Housefly LM is greater than what the current study has been able to demonstrate. However, processing can add significantly to the cost of an ingredient and may not always be optimal. Soy protein is an instructive example in this regard: soybean meal, soy protein concentrate, and soy protein isolate show increasing performance, but the optimal balance of cost and performance in salmonid diets is found with soy protein concentrate. While the current study is designed in such a way that it cannot make claims about the ability of Housefly LM to replace marine ingredients, we believe the design does allow for a rigorous comparison of LM to existing alternatives. Because a diet featuring crude LM performed better than the control diet featuring high-quality processed plant-derived ingredients, we feel confident in concluding that LM has potential to surpass these ingredients in terms of performance. It remains to be seen whether that potential manifests in further reduction of marine ingredients, and whether the cost of LM production can be reduced to a point where this is economically relevant.
c. Growth Trial
Over the course of this study, inclusion of 30% LM significantly increased growth and decreased feed conversion rate (FCR) relative to the control group (Figure 4). The 5% LM group is harder to evaluate, as not only is the growth not significantly different from the control group, the FCR is higher. This is likely an effect of the high mortality observed in group 2 (Figure 3). Mortality peaked over the first three weeks of the study before slowing down, and despite the help of the Aquatic Animal Health diagnostic team at Cornell, a satisfactory explanation for the mortality was not identified. Anecdotally, there did seem to be higher incidence of caudal fin erosion in tanks with the most mortality, suggesting that perhaps aggression between fish played some role, though this is likely not the whole story. Likewise, proximate analysis of the diets did not reveal obvious deficiencies in the formulation. A final consideration on the feeding trial is that the physical properties of the feeds appeared to be impacted by their varying levels of saturated fat; the higher levels of saturated fat in the 30% LM diet could explain the formation of clumps that stayed intact during feeding, while the control and 5% LM diets, which featured higher levels of soybean oil, tended to disperse into smaller fragments during feeding.
d. Immune Challenge
The idea behind using 5 treatment groups (Figure 2) was to control for long-term vs. short-term immune-stimulatory properties of the insect diets in addition to the growth trial. The reasons for selecting these categories were as follows. First, we wished to conduct an eight-week feeding trial comparing LM to plant ingredients at both high and low replacement levels (groups 1-3). Second, we wished to test the protective effects of these different diets against bacterial infection. Third, we wished to determine if the putative protective effects of larva meal are acquired rapidly (2 weeks), and whether they are sustained over longer feeding (8 weeks). This design allowed us to conduct a well-controlled feeding trial that also served as a broad screen for immune-stimulatory effects of IM diets.
To test for stimulation of the fish immune system by LM we measured serum lysozyme activity and found that it was significantly increased in fish from 5% and 30% LM groups after 2 weeks, but reduced after 8 weeks (Figure 5). This supports the hypothesis that immune stimulation due to LM is temporary.
The results of our immune challenge were not informative. We observed less than 10% mortality in all groups (Figure 6). Without significant mortality in the control diet group, it is impossible to say whether the LM diets had a protective effect or not. One of the challenges we encountered in this study was that our pilot infection study to determine an appropriate dose was necessarily conducted on younger fish than the final infection. Since younger fish are more susceptible to BCWD, this is important to take into account in similar trials. The hypothesis that LM confers immunoprotection to fish remains to be tested, as our infectious challenge failed. We plan on repeating this phase of the experiment in the future with a more a more tractable infection model. The fact that lysozyme activity was upregulated in the 2-week but not the 8-week groups suggests that a short-term protective effect is perhaps more likely.
This study showed that Housefly larvae meal (LM) has desirable nutritional characteristics for aquaculture feed, and that feed incorporating LM performed well. We performed a rigorous comparison of LM to relevant plant-based alternative ingredients to fishmeal and fish oil, and found that high levels of LM (30%) resulted in increased growth of juvenile Rainbow trout over 8 weeks of feeding.
We also observed that after 2 weeks on diets including LM, trout displayed increased serum lysozyme activity - perhaps suggesting an immune stimulatory effect. To test this, we performed an infection challenge study using Flavobacterium psychrophilum. This part of the study was inconclusive, as we observed minimal mortality across all groups. Future studies using more robust challenge models and more precise metrics of health are likely needed to untangle the possibility of disease protection from LM-based diets.
The future of LM in aquaculture feed remains promising, with excellent performance characteristics and low environmental impact. Cost will always be a consideration, but our data suggests that even at a higher price point than existing plant protein sources, the performance of LM may be sufficient to allow for reduction of fishmeal below current best practices. Reduction and replacement of fishmeal and fish oil in aquaculture diets are huge priorities for the industry - LM may yet prove to be an important part of achieving that goal, and dairy farmers may be able to capitalize on the need for organic inputs.
Education & Outreach Activities and Participation Summary
Work from this project has been presented at the annual meeting of the New York State chapter of the American Fisheries Society in Cooperstown, NY, the 2018 National SARE Conference in St. Louis, MO, and at the International Symposium of Fish Nutrition and Feed in Las Palmas, Spain. Numerous conversations with farmers, nutritionists, and feed producers have come from these presentations, and some have continued beyond the conferences.
After the results of this study are published in a peer-reviewed journal, an article intended for the general public will be submitted to New York State Conservationist magazine, a bi-monthly periodical put out by the Department of Environmental Conservation. In consultation with the editorial staff of the magazine and if allowed, this article will be submitted to similar state and national publications.
This study is part of a larger effort to understand the nutritional benefits of insect-derived feed ingredients. Farmed insects hold tremendous potential for the future of sustainable agriculture. By allowing local upcycling of organic waste into valuable feed commodities, insect farmers are likely to produce a number of benefits for existing farms: 1) By utilizing existing organic waste streams (e.g. cow manure), these operations will add value to a number of farm and farm-adjacent industries, 2) by producing high quality sustainable aquaculture feeds, these operations can improve the profitability and consumer acceptance of farm-raised fish, and 3) by operating locally these operations can serve as a link between farmers and help to promote local circular economies.
This project demonstrated the importance of fundamental science for improving sustainable agriculture. It also helped me to understand the importance of cultivating both rigorous scientific communication and the ability to communicate science to a general audience. Conferences like the International Symposium of Fish Nutrition and Feed are explicitly about sustainable agriculture, and yet almost no effort is made to make the results accessible to farmers. However, I found this to be the most useful and inspirational conference for planning my future research. Simplifying research projects for a general audience is important, but so is thinking those projects through in full technical detail beforehand. I would say that my goal as an applied scientist has shifted from occupying a space in between farmers and basic scientists to being able to occupy both spaces as necessary.
Research into scaleable production of insects, or even a centralized production facility, would be incredibly useful for research in this area. Producing enough larvae to conduct this study was a big commitment of time and labor, and we remained limited to relatively small numbers of juvenile fish.