Abstract
The present study investigated the potential use of defatted black soldier fly (Hermetia illucens) meal as a protein source in the formulated feed for juvenile lobsters, Panulirus ornatus. A total of 160 heads of juvenile lobsters with initial weight of 0.24 ± 0.01 g and initial length of 21.77 ± 0.32 mm were distributed evenly into 16 rectangular fiberglass aquaria and kept individually for 8 weeks of the feeding experiment. Four formulated feeds containing defatted BSF meal as fishmeal replacements at 0%, 25%, 35%, and 50% and total dietary protein to energy ratio of ~ 26 g CP MJ−1 were prepared and assigned as BSF0, BSF25, BSF35, and BSF50, respectively. Fishmeal is technically a suitable aquafeed ingredient in terms of crude protein, moisture, and amino acid compositions. The partially substituted defatted BSF meal has higher crude lipid and ash content than fishmeal. At the end of week 8 of feeding trial, the specific growth rate, length increment, survival rate, and moulting rate of juvenile lobster were not significantly (P > 0.05) affected by the fishmeal replacement. The fishmeal replacement with the defatted BSF meal did not significantly (P > 0.05) alter the whole-body proximate composition of the juvenile lobster. However, it significantly (P < 0.05) affected the superoxidase dismutase of juvenile lobsters. The cytokine cell expression analysis indicated that up to 35% of fishmeal replacement increased the inflammatory cytokine cells (Interleukin 8 and 17) of juvenile lobster, while an adverse impact was observed on juvenile lobster receiving 50% of fishmeal replacement by BSF meal. Histopathological analysis showed that the hepatopancreas cells of juvenile lobsters were damaged following fishmeal replacement beyond 35%. The present study indicated that up to 35% of defatted BSF meal can be used as fishmeal replacement in the formulated feed of juvenile lobster.
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Introduction
Since
the 1980s, dietary insect meal has been recognized as an alternative to fishmeal in livestock food and aquaculture industries. With the increase in awareness of sustainability related to the use of fishmeal in aquaculture, research on insect meal as an aquadiet has progressed. The potential use of insect-based ingredients in aquadiets has been reviewed by many authors and one of the most researched insect species that has emerged is black soldier fly (BSF), Hermetia illucens (Alfiko et al.
Growth and moulting data
The physiological data including survival rate and specific growth rate of the juvenile lobster were collected at the beginning and at the end of the feeding experiment. The total length increment, moult interval, and moulting rate data were based on daily records. All data parameters were calculated as follows:
-
(1)
Survival rate (%) = \(\frac{\mathrm{Final number of survived spiny lobster juvenile }}{\mathrm{Initial number of spiny lobster juvenile}} x 100\)
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(2)
Specific growth rate (g.%.day−1) = 100 x [ln final weight (g)–ln initial weight (g)]/days
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(3)
Total length increment (%) = (Wt total length – W0 total length)/ (initial total length) × 100
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(4)
Moult interval (days) = duration in days between two consecutive molts
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(5)
Moulting rate (%) = (Number of molted lobster juvenile)/ (Number of total lobsters juvenile) × 100
Statistical analysis
The biological data parameters were analyzed by using SPSS IBM for Windows (version 25.0). One-way analysis of variance was used to evaluate any significant differences among the variables of the parameters of the juvenile lobster and Turkey’s HSD post hoc test analysis were performed when a significant interaction occurred. The statistical significance was evaluated at P < 0.05.
Results
All formulated diets have similar proximate composition (Table 2). The amino acid contents in fishmeal were higher compared to the amino acid in the defatted BSF meal (Table 3). The amino acid content in feeds without defatted BSF supplementation was higher than the amino acid in the supplemented-BSF feeds. The increase of percentage defatted BSF in the diets have a negative correlation with the availability of amino acids in the feeds (Table 5).
Physiological evaluation on juvenile lobster indicated that there were no significant differences in the mean of final weights, specific growth rates, final lengths, length increments, survival rates, moulting rates, and moult interval of juvenile lobster fed dietary defatted BSF meal (P > 0.05) (Table 6).
The composition of protein and lipid in the whole-body of juvenile lobster were not significantly different (P > 0.05). The ash and SOD of juvenile lobster’s whole-body fed with the BSF50 dietary were significantly high (P < 0.05) (Table 7).
The relative expression level of cytokine genes in juvenile lobster is shown in Fig. 2. In this study, the gene expression of IL-1β and IL-17 in juvenile lobster fed with BSF25 and BSF35 was higher than those in juvenile lobster fed with BSF0 in terms of fold changes. The IL-17 cells in juvenile lobster given with BSF25 and BSF35 showed a five-fold of gene expression from the control (BSF0). In contrast, those cytokine cells decreased as the juvenile lobster fed the dietary BSF50. The BSF dietary replacement at any level in the formulated diets downregulated the fold of IL-10 cells gene expression in juvenile lobsters. The juvenile lobster given with BSF50 dietary had higher fold of ProPO, GPO, and ALFHa-1 gene expression compared to other dietaries. On the contrary, the ALFHa-2 gene of juvenile lobsters decreases as the inclusion of BSF levels in the formulated diets increases (Fig. 1).
Relative expression level of cytokines gene (IL-1β, IL-10 and IL-17) and immunological genes (ProPO, GPO, ALFHa-1, and ALFHa-2) of juvenile lobster with respect to defatted BSF meal replacement
Histology analysis on the hepatopancreas of juvenile lobster shows that necrosis occurred on the lobster fed 50% of fishmeal replacement (Fig. 2). The presence of hemocyte infiltration and sloughing on the hepatopancreas tissue were found in all treatments. The atrophy conditions of hepatopancreas cells were found only in juvenile lobster fed with BSF50 feeds (Table 8).
Histology of hepatopancreas of juvenile lobster fed with dietary of defatted BSF meal inclusion (0%, 25%, 35% and 50%) at 100 × magnification. Hepatopancreas tissue infiltrations and sloughing occur in all of the treatments. Hepatopancreas of juvenile lobster fed with BSF50 experienced necrosis and atrophy tissue. In, infiltration; Sl, sloughing; Ne, necrosis; Ar, atrophy; bar: 300 µm
Discussion
The present study is the first study attempting to use defatted BSF meal as the protein source in a formulated feed for juvenile lobster. The result of the present study indicated that the fishmeal replacement up to 50% with defatted BSF meal did not affect the physiological response and immunological response on juvenile lobster. Measured water salinity and temperature in this study were at optimum level for culturing spiny lobster (Jones 2009) and dissolved oxygen was acceptable for captive environment (Vijayakumaran et al. 2014). Water quality has significant effect on the growth and survival of juvenile lobster and the changes on that parameters can lead to the suboptimal growth (Ratunil 2017) and decrease on the immune response (Verghese et al. 2007). The readings of pH were higher than recommended level (Jones 2009; Vijayakumaran et al. 2014) and it might be caused by the error of the equipment since the physiological response of the lobster is within the normal ranges. The average survival rate in this present study was similar to other reports (Kurnia et al. 2017; Marchese et al. 2018; Sang and Fotedar 2010; Smith et al. 2003; Smith et al. 2005) and was higher to studies conducted in clean water system (Haryanti et al. 2017; Kropielnicka-Kruk et al. 2019; Ratunil 2017). In this study, the replacement of fishmeal with defatted BSF meal up to 50% of protein content did not affect the growth of juvenile lobsters, showing a specific growth rate between 1.77 and 1.82 g % day−1. It is not possible to compare the growth rate of the juvenile lobster in this study with any other study as this is the first study testing defatted BSF meal as protein source for juvenile lobster. However, that growth rate is in agreement with the said nutritional studies on juvenile lobster (Saputra and Fotedar 2022; Smith et al. 2003; Smith et al. 2005) and somewhat lower than other reports (Haryanti et al. 2017; Kropielnicka-Kruk et al. 2019). In addition, any level of defatted BSF diets that resulted to the same growth rate as to the BSF0 or control diet indicated that those diets are suitable for juvenile lobster where fishmeal is used as protein source. The data suggested that fishmeal replacement by defatted BSF meal up to 50% will increase the protein and lipid content of juvenile lobster’s whole body.
Studies evaluating fishmeal replacement using defatted BSF meal in spiny lobster are scant. In the present study, the replacement of fishmeal with defatted BSF meal at any level had no effect on the final total length, final weight, length increment, moulting rate, and moult interval of juvenile lobster. Results of the present study in juvenile lobster are similar to a crustacean study (Richardson et al. 2021). For L vannamei, shrimp fed with a diet of 70% defatted BSF inclusion had similar final bodyweight and survival as shrimp fed with a control diet without defatted BSF meal. In addition, the increase of defatted BSF meal amount to fishmeal replacement in the practical diet improved growth rate and survival rate (Richardson et al. 2021). On the contrary, on a study involving the same species with the said study, the shrimp fed with dietary of BSF meal > 7% on dry weight basis exhibited the decrease in their specific growth rate, final weight and feed conversion ratio (Cummins et al. 2017). The results of the present study were contradicted to study on Cummins et al. (2017) but in line with Richardson findings. The different nutritional compositions of BSF meal used in those two studies might be the ones which are responsible for the different growth and survival responses of the shrimp. The defatted BSF meal has approximately 60% crude protein and 7% of lipid which is used in the experiment conducted by Richardson et al. (2021), while BSF meal used by Cummins et al. (2017) has approximately 52% crude protein and 15.1% lipid. The different growth and survival response of treated animal not only occurred in crustaceans, but also in finfish (Fasakin et al. 2003; Karapanagiotidis et al. 2023). A set of feeding experiments conducted on gilthead seabream (Sparus aurata) indicated that the defatted BSF is more suitable in terms of promoting growth for the fish. While full-fat BSF replacement at any level would experience a decrease in the growth of the fish, only fish that received feeds containing more than 30% of fishmeal replacement with defatted BSF exhibited a suboptimal growth (Karapanagiotidis et al. 2023). The said findings indicate that with similar replacements, the defatted BSF meal is more suitable as fishmeal replacement than the full-fat BSF meal.
In the present study, the survival rate of juvenile lobster ranges from 50.00 to 62.50% without any statistical significance differences. That survival rate was higher than the survival rate of the same juvenile lobster species reared using the similar rearing system reported by Kropielnicka-Kruk et al. (2019) and somewhat beyond the survival rate of juvenile lobster in the research conducted by Marchese et al. (2018). In a study on crustaceans, the mortality rate was reported to be influenced by the moulting rate (Taugbøl and Skurdal 2008) and moulting rate itself has strong association with the availability of supporting moulting hormone (Hosamani et al. 2017). In the present study, the mortality rate was mainly caused by insufficient energy to complete the moulting process which could be observed from an incomplete moult of the juvenile lobster, and it is in agreement with the findings from Irvin and Williams (2009). The survival rate of spiny lobster P. ornatus is influenced by the rearing system both on juvenile (Kropielnicka-Kruk et al. 2019; Marchese et al. 2018) and adult lobster (Vijayakumaran and Kumar 2010). Indeed, the availability of the feed in closed compartment will provide an ease for juvenile lobster to access food. Also, the rearing system affects the feed intake. In addition, the growth rate, final carapace length, and moult rate of the juvenile lobster were also higher than juvenile lobster kept in individual compartment (Marchese et al. 2018).
The amino acids pose an important role in supporting growth and some of them are known as limiting factors in L. vannamei (Cummins et al. 2017; Richardson et al. 2021). In this study, the availability of amino acids in defatted BSF meal was lower than the amino acids in the fishmeal. Among others, amino acids which were analyzed, serine, cysteine, and phenylalanine in the defatted BSF meal, were holding the closest value of amino acids in the fishmeal. In addition, the sum squared calculation indicated that there were variations in the amino acids between formulated diets. The amino acid values in this study were decreasing as the increase of defatted BSF meal inclusion, and similar results were also reported from other crustacean feeding trials (Cummins et al. 2017; Richardson et al. 2021). For instance, the formulated diets with 36% of fishmeal replacement had a higher value of total amino acids sum squared of 140.66 than formulated diets containing 7% of fishmeal replacement with value of total amino acids sum squared as much as 6.87 (Cummins et al. 2017). It is impossible to get same nutrition from particular BSF with others because the nutritional composition of each BSF is highly dependent on the substrate of its culture (Shumo et al. 2019), life stage (Liu et al. 2017), and the form of BSF (Irawan et al. 2020). In the present study, the amino acid values might not have any impact to physiological response of juvenile lobster. However, it can be a foundation for future research in spiny lobster nutritional studies.
The body composition of crustaceans has been reported to be affected by the different dietary ingredients in the formulated diets. While some studies reported that the ash content in the body of crustaceans was lower compared to the one in the experimental diets (Cummins et al. 2017; Goda 2008; Hu et al. 2008; Huo et al. 2014), another study with contradicted results reported that the ash content in the body was higher than the one in the experimental diet (Xu et al. 2013). The body composition analysis of juvenile lobster in this study indicated that the ash content in the whole-body juvenile lobster was higher than the one in the formulated diet, and it is in agreement with the said latter study (Xu et al. 2013). These results argued to a consideration that different ash analysis is the highest possible consequence on the said mixed results. In this study, analyzed juvenile lobster whole body with shell included resulted in high ash content. In crustaceans, any non-edible and edible body part have different ash value with non-edible body part with shell included having a higher ash content than the edible body part (Ali et al. 2018). In terms of the BSF meal replacement within this study, the ash content increased as the defatted BSF meal inclusion in the formulated diet increased, whereas protein, lipid, and moisture content were the same with formulated diet without BSF included. Those results on ash content in the present study is similar to Richardson et al. (2021) which reported that the ash content in the diet with 70% fishmeal replacement is higher than the ash content of the diets with 30% and 50% replacement of BSF meal. In the present study, high ash content was found in the juvenile lobster fed with defatted BSF meal inclusion at 35% and 50%. This result was considerably affected by the nutritional composition of defatted BSF meal, where those inclusions have slightly higher ash content compared to fishmeal. In addition, the inclusion would directly influence the ash content in the whole body of juvenile lobster (Table 2), although physiologically it did not cause a significant difference in growth rate and survival rate. It might come into consideration that ash content on the juvenile lobster whole body relates to a high level of hepatopancreas cell damage. The damage occurred to juvenile lobsters fed with defatted BSF meal replacement at 50% only. The growth rate and survival rate of P. ornatus juvenile reared in sea cage fed with high-ash formulated diet are lower than those of lobster fed with dietary low-ash diet (Kurnia et al. 2017). Ash content is an important source of minerals for fish. Nevertheless, an excessive ash amount in feed may reduce the digestible ability of other ingredients which resulted to a poor growth of the fish (Shearer et al. 1992). There is no exact information of optimum ash content amount required by spiny lobster to achieve their maximum growth. However, it is found on the wild-caught lobster Panulirus japonicus that the ash content in their body is 8–12%, and those values can be used as an acceptable reference (Takeuchi and Murakami 2007). In this study, the ash content of the formulated diets ranged from 8.21 to 8.80 g/100 g, while in juvenile lobster whole body, it ranged from 11.26 to 13.59 g/100 g. In addition, it was found that ash content in the juvenile lobster whole body fed with 50% defatted BSF meal inclusion was significantly different. The differences of ash content amount affected the immunology of juvenile lobster by the occurrence of severe hepatopancreas cell damage evidence but did not affect to physiological parameters of juvenile lobsters.
Histopathology has been used in analyzing the effect of feeding treatments in crustaceans (Ambasankar et al. 2022; Chen et al. 2021; Duan et al. 2018; Jatobá et al. 2018; Li et al. 2022) and also to determine the tissue of certain organs changes due to disease infection (Shields et al. 2012; Zha et al. 2018). The effect of BSF meal inclusion on gut health has been demonstrated in crustaceans (Chen et al. 2021; He et al. 2022), while the effect of defatted BSF meal inclusion on hepatopancreas of spiny lobster is not yet available. However, the histopathology of hepatopancreas has been used as the reference of nutritional status in some crustaceans (Chen et al. 2017; Fernández Gimenez et al. 2004; Sukor et al. 2016). In the present study, the histopathology analysis of intestine failed due to the intestinal tissue sample damage. Therefore, only the histopathology result of hepatopancreas was used for further discussion. In the histopathology analysis, juvenile lobster fed with defatted BSF meal fishmeal replacement at 50% experienced a more severe hepatopancreas cell damage than juvenile lobster fed with control diets (BSF0) and other treatments (BSF25 & BSF35). The histopathology of hepatopancreas juvenile lobster fed with fishmeal replacement at 50% exhibited severe sloughing of cells tissue, hemocyte infiltration, necrotizing, and atrophy cells. Meanwhile, there was only decent sloughing of cells tissue and hemocyte infiltration found on hepatopancreas cells of juvenile lobster fed with other dietary treatments. Those detrimental effects on hepatopancreas cells in this study might be accounted by the overuse of defatted BSF meal inclusion at 50%. This result, however, contradicted with the physiological response of shrimp receiving similar defatted BSF meal dietary which has no differences in shrimp growth. The optimum growth performance is achieved on L. vannamei juvenile receiving 20% of BSF meal inclusion, and beyond that level, the shrimp juvenile experienced a reducing growth rate (Chen et al. 2021). A similar result was also reported by Richardson et al. (2021), with limiting BSF meal inclusion to 25%, which is in converse with a recent study in 2022 on the same species which reported that higher than 25% inclusion of BSF level can be used to promote growth and immunology status of the L. vannamei juvenile (He et al. 2022). The different responses of animals used in the said research which uses BSF meals as fishmeal replacement were considerably caused by the diverse nutritional value particularly the amino acids of used BSF (Chen et al. 2021; Cummins et al. 2017; Richardson et al. 2021) which also occurred as result in the present study.
Study on the search for alternative ingredients in aquafeeds does not only aim to find suitable ingredients supporting fish growth, but also the ones which are able to improve the immunity of the animals. In this study, the fold of gene expression level of juvenile lobster exhibited different responses with respect to the defatted BSF meal replacement in the experimental formulated diets. The replacement of defatted BSF meal at any level did not affect the fold of gene expression of inflammatory cytokines of juvenile lobster. However, juvenile lobster fed with the formulated diets containing defatted BSF meal replacement at 25% and 35% had higher inflammatory cytokine IL-1β and inflammatory cytokine IL-17 expression than juvenile lobster fed with the control diet. In contrast, the fold of gene expression of inflammatory cytokines on the juvenile lobster fed with the formulated diets containing defatted BSF meal replacement at 50% was lower than juvenile lobster fed with the control diet. The result of this study is similar with a study on freshwater lobster C. cainii conducted by Foysal et al. (2019). In that lobster study, the supplementation of 30% BSF in the diet improved the expression level of cytokine cells primarily IL-7, IL-10, and IL-17, which coincide with the high availability of lactic acid bacteria from the Firmicutes phylum. In another study on northern snakehead (Chana argus), the abundance of certain Firmicutes is responsible for the increase of the availability of some inflammatory cytokines (Miao et al. 2018). The inclusion of defatted BSF meal as substitutes on fishmeal in this study might have role to provide Firmicutes phylum affecting the increase of cytokine cells of juvenile lobster. However, it requires further investigation.
The changes of several immune-related gene expression have been used as parameters in assessing the immune status of crustaceans (Duan et al. 2018; Haryanti et al. 2017; Li et al. 2018; Miandare et al. 2017; Wongsasak et al. 2015). In the present study, juvenile lobster receiving high inclusion level of defatted BSF meal in the diet had higher gene expression of prophenoloxidase, glutathione, and anti-lipopolysaccharide factor-1 than others. That finding indicated that a high inclusion level of defatted BSF meal would improve those immune-related genes of juvenile lobster by providing required nutrition. The increase of gene expression fold on prophenoloxidase was also reported for juvenile lobster P. homarus fed with probiotic dietary. The essentials nutrition in the yeast combined with the function of the probiotics have responsibilities in promoting immunological status of juvenile lobster (Haryanti et al. 2017). The prophenoloxidase is a part of innate immunity system in crustacean and plays an important role in the bacterial infection process (Wang et al. 2010). A rigorous study conducted by Vogel et al. (2018) showed that BSF is an insect with 53 of potential antimicrobial peptides and ranked them as the highest insect with potential antimicrobial peptides amongst common insects used in feed. In addition, BSF also have higher potential immunomodulatory factors lauric acid and chitin (Koutsos et al. 2022). These superior attributes of BSF are considered contributions to the results of the present study.
Conclusions
The present study shows that the defatted BSF meal replacement of fishmeal at between 0 and 50% in formulated feeds for juvenile lobster has no effect on growth, survival rate, moulting rate, moult interval, and body composition compared to the standard feed. However, it considerably changes the cytokine cells and some immune-related gene expression juvenile lobster. The hepatopancreas of the juvenile lobster fed with defatted BSF meal dietary at 50% shows a severe cell damage. An increasing amount of defatted BSF meal will increase the availability of amino acid contents in the formulated diets and the superoxidase dismutase availability in juvenile lobster whole body. Further study is required to evaluate the findings as well as other potential ingredients as supplement in defatted BSF meal fishmeal replacement on the formulated diets of juvenile lobster.
