Effects of inclusion of fodder tree leaves mix meal in rearing media on the body size, protein and lipid content of larvae of black soldier fly (Hermetia illucens)

Marthen L Mullik, Twen O Dami Dato, Y M Mulik, Cornelia Imul and Gustaf Oematan¹

Faculty of Animal, Marine and Fishery Sciences, Universitas Nusa Cendana, Kupang, 85001 Indonesia
marthenmullik@staf.undana.ac.id
¹ Politeknik Pertanian Negeri, Kupang NTT, Indonesia

Abstract

This study aimed to evaluate the impact of a fermented substrate containing various proportions of fodder tree leaf mix meal at 0% (LM0), 10% (LM1), 20% (LM2), 30% (LM3) and 40% (LM4) on the body dimensions and nutrient content of black soldier fly (BSF) larvae (maggots). The fodder tree were Leucaena leucocephala and Moringa oleifera in a fixed ratio of 1:1. Completely randomized design was deployed to test those five levels of the legume mix meal. Each experimental unit consisted of 2 kgs of substrate, which was placed randomly in an open-air hut for oviposition by wild BSF. Maggots were harvested 10^th days after substrate placement. Variables measured were maggot’s fresh and dry weight, length, crude protein, and lipid content. Analysis of variance was used in analysis of the data. Results showed that levels of fodder tree leaf meal improved maggots protein and lipid content at a quadratic fashion. However, larval weight (0.13-0.18 g), length (15.7-18.2 mm), and DM content (41.0-45.7%) were not effected. It can be concluded that the levels of leucaena-moringa leaf meal in the growth substrate can increase the protein and lipid content of black soldier fly maggots but not the larva’s weight and length.

Key words: insects, leucaena, maggot, moringa

Introduction

Larvae (maggot) of black soldier fly (BSF; Hermetia illucens) is one of the alternative high quality protein sources for animal feed (Makkar et al 2014), due to its ability to decompose organic biomass from waste to synthesize and accumulate protein and fat in the body (Gold et al 2020; Lu et al 2022). Rate of growth, size, nutrient composition, lifespan, and substrate conversion efficiency to larval biomass is determined by substrate quantity and quality (Nguyen et al 2013; Barragan-Fonseca et al 2018; Laganaro et al 2021) and other rearing environments (Cheng et al 2017; Lalander et al 2019; Ma et al 2018). Consequently, larvae fed on different substrates exhibit varying degrees of body protein (37.0 - 62.7%) and fat (6.6–39.2%) content (Barragan-Fonseca et al 2017; Ewald et al 2020; Eriksen 2022).

However, the experiments described in the published literatures have focused heavily on understanding the relationship between substrate and larval nutrition, without taking into account secondary metabolic compounds possibly present in the rearing media that could potentially alter metabolic pathways within the larvae's body. This metabolic interference is likely to affect growth and nutrient content of the maggots. Leguminous and certain fodder trees produce abundant high quality organic biomass in tropical areas of the world and are potential rearing media for maggots. Unfortunately, they possess a plethora of secondary metabolite compounds, such as polyphenols, alkaloids, and saponins, that serve as vital defense compounds against insects, herbivores and pathogens (Gupta 1987; Wang et al 2019).

To date, there has been a lack of research on the utilization of fodder plants biomass as a nourishment substrate for BSF larvae to characterize their effects on growth dynamics and nutrient density. Hence, the present experiment was designed to evaluate the effects of including two fodder trees namely leucaena (Leucaena leucocephala) and moringa ( Moringa oleifera) leaf meal in the rearing media on body dimensions, protein content, and fat content of the resulting maggots. The fodder tree leaf mix meal serve as protein source and at the same time confer secondary metabolites that might have negative effects on the larvae.

Materials and methods

This study employed a Completely Randomized Design (CRD) with 5 treatments and 5 replications to evaluate the effects of incorporating a mixture of 50% leucaena and 50% moringa leaf mix meal into maggot rearing substrate on weight, length and nutrient content of resulting maggots. The treatments consisted of a full basal substrate (LM0) and the addition of legume mix meal at varying proportions of 10% (LM10), 20% (LM20), 30% (LM30), and 40% (LM40). The basal substrate comprised 80% rice bran and 20% yellow maize flour. The substrate's raw materials and their nutritional composition are presented in Table 1.

The proportional ingredient mixture for each treatment was uniformly mixed in a plastic container and added with 50% (v/w) inoculum stock solution before being fermented for 7 days in an air-tight plastic bag. The stock solution consisted of 98.6% distilled water, 1.0% granulated sugar, 0.4% nonfat probiotic drink (Yakult®), and a sufficient quantity of seasoning powder (Royco®) to enhance aroma and attract wild black soldier flies (BSF). After fermentation, 2 kgs of the substrate for each experimental unit was weighed into a medium-sized plastic container. Fresh banana leaves were placed over the substrate to create a dark environment for the medium and serve as a site for BSF oviposition.

The filled containers were randomly placed on a mat-covered floor in a semi-walled hut to allow the medium to be easily accessed by wild BSF. Following a 10-day exposure, the containers were removed, and maggot harvesting was initiated by manually separating the maggots from the medium.

Two thousand fully grown maggots from each experimental unit were taken as samples. The maggots were weighed using an analytic scale, body dimension was measured using millimeter block paper, and then the maggots were oven-dried at 60°C for 48 hours for dry matter and other nutrient determination. The substrates and maggot samples were subjected to proximate analysis following procedures of AOAC (2005).

The collected data were subjected to analysis of variance according to the CRD principle. Treatments effect was detected at an alpha value of 0.05, and between-treatment differences were tested using Duncan multiple range test. The data analysis process was aided by SPSS version 25 software (IBM 2017).Bottom of Form

Results and discussion

Weight and body dimension

Average fresh weight, dry weight, and length of the individual maggots are presented in Tabel 2. The values ranging from 147 to 180 mg, 60 to 74 mg, and 14 to 18 mm for fresh weight, dry weight and length respectively. It appears that the increased in the level of fodder tree leaf meal mixture in the rearing media improved its nutrient density (Table 1) resulting in a better larval growth (Table 2). These results confirm other findings (Tendonkeng et al 2017; Veldkamp et al 2021; Myers et al 2008) that maggot’s weight correlate positively with substrates nutritional content, especially CP and EE.

However, the statistical analysis did not reveal any significant treatment effects on the fresh weight (p=0.38), dry weight (p =0.55), or length (p=0.401) of the maggots. Lack of response in body dimension to substrate enrichment using fodder tree leaf meal could potentially be attributed to the following two factors. Firstly, there may have been a large variation in age of the maggots. Since the substrates were not restricted to wild BSF access, oviposition most likely took place continuously during the study period resulting in large variability in the sampled maggots as shown by the relatively large standard error values (SEM) for fresh weight, dry weight and length (Table 2). In a 30-day experiment, Widyaswara et al (2021) used substrates of chicken manure and household waste to investigate the growth of BSF larvae. They found that the larval body dimension increased linearly with age, with regression coefficients of 0.0035x and 0.07882x for weight (g) and length (mm), respectively, until they reached the prepupal stage. Liu at al (2017) has also constructed a sigmoidal growth-age dependent model for maggots, where different age exhibit different growth rates. A recent dynamic modelling of feed assimilation and growth by BSF larvae by Erikson (2022) have also confirm this growth pattern.

Secondly, the potential inhibitory effects of secondary metabolites in the mixed the fodder tree leaf meal on nutrient assimilation within the body of maggots could have been elucidated through substrate fermentation prior to use. Although the metabolic compounds were not characterized in the present study, literatures (Mulik et al 2016a; Wang et al 2019), suggests that anaerobic fermentation significantly reduces secondary metabolic compounds in legume plant tissues which can improve their nutritional quality as a growth substrate for BSF larvae.

Nutrient contents

Table 2 presents the selected nutritional composition of the maggots. The dry matter (DM) content ranging from 41.0% to 45.6%, which did not differ significantly among the five treatments. This is predictable, since all treatments were formulated using raw materials with similar moisture content (79.1% to 84.4%; see Table 1). The DM content obtained in this study was classified as intermediate compared to the results reported by various researchers, which ranged from 27.3% to 40.5% (Nguyen et al 2015; Spranghers et al 2017; Ewald et al 2020). However, the values recorded in our study were still lower than those reported by Barraga’n-Fonseca et al (2018).

Although the effect of moisture content was not the focus of the present experiment, compiled data from various literatures (Spranghers et al 2017; Ewald et al 2020; Veldkamp et al 2021) suggests that there was no relationship between substrate moisture content and BSF larval growth. However, the relationship between substrate moisture content and maggots DM has not been conclusively established in the existing literatures, as the types of substrates used have varying physical and chemical characteristics, including differences in nutrient content, and were not specifically designed to isolate the effects of factors other than moisture content.

Contrary to DM, both the protein and lipid content in the maggots increased significantly (p<0.01) with the incremental levels of fodder tree mix meal in the basal substrate (Table 2). The protein increased from 27.3% in maggots on the control substrate (LM0) to 36.6% in those on the 40% fodder tree leaf mix substrate (LM40). Similarly, the maggots lipid content also increased from 12.4% in control treatment to 16.4% in the LM40 treatment. However, within the limit of the current substrates levels, the maggots’ protein and lipid improvement rates did not exhibit a linear trend, rather a quadratic fashion with a diminishing rates of -0.0045, and -0.0008 for protein and lipid, respectively. Figure 1a and 1 b showed that both protein and lipid improvement rate nearly plateau when the level fodder tree leaf meal comprised 40% of the growth substrate. At this level, crude protein and lipid content of the substrate were 12.6% and 6.2% for protein and lipid, respectively. It is almost certain that addition of extra protein and lipid into this type of media will not stimulate a better protein and lipid assimilation by the maggots.

The results of the present study could be used to explain why Perez-Pacheco et al (2022) did not observe difference in protein and lipid of maggots rearing on a variety of substrates (restaurant waste, fresh fruit, fish waste, and commercial feed) with crude protein ranging from 14.5% to 40.1%. They reported that the maggots have protein merely around 28% to 33%. Similarly, other studies (Shumo et al 2019; Ewald et al 2020; Lu et al 2022) reported that the CP content of maggots from various substrates, with CP content ranging from 12.2% to 52.6%, was only around 27% to 41%.

Figure 1. Relationship between the levels of fodder tree leaf mix meal in the growth substrates and the protein (a) and lipid (b) contents of the maggots

The crude lipid content of maggots reported in the existing literatures is highly variable. Lipid assimilation in maggots is a dynamic process mainly dictated by their age (Eriksen 2022) and less affected by lipid content in the growth substrates (Barraga’n-Fonseca et al 2019; Chia et al 2020; Velkman et al 2021; Mahmoud et al 2022). Liu et al (2017) showed that the lipid content of 1-day-old BSF larvae was 4.8% and increased sharply to 28.4% when it reached 14 days old, but then declined to 8% at the early pupal stage. This has also been confirmed in Erikson's dynamic model (Eriksen 2022).

The quadratic regression trend of the maggots' lipid content against the incremental levels of lipid in the substrates used in the present study (Figure 1b) suggests that a linear increase in the substrate lipid density does not stimulate lipid assimilation in the maggots at the same rate. However, since the age of the sampled maggots in the present study is likely heterogeneous, it is not possible to draw a firm conclusion about the relationship between lipid in substrate and maggot. Nonetheless, it could be proposed that the range of the maggots' lipid requirement is narrow, as found in the present study (4.7% to 6.5%)

It appears that, pretreatment (bio-fermentation) of the substrates resulted in a positive response in maggots EE content along with incremental levels of legume leaf mix meal in the growth substrates, suggesting that the suppressive nature of secondary metabolite compounds in leucanea and moringa on body fat biosynthesis (Mulik et al 2016b) has been elucidated.

Conclusion

It can be concluded that the weight and length of the maggots were not significantly affected by fermented basal substrate containing varying levels of leucaena-moringa leaf mix meal. Nevertheless, increasing the levels of the fodder tree leaf mix meal in the substrate resulted in a quadratic trend in the crude protein and crude lipid content of the maggots, which almost plateau at 40% leaf meal inclusion.

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