A comprehensive evaluation of the potential of semiterrestrial isopods, Ligia exotica, as a new animal food

The semiterrestrial isopod, Ligia exotica represents one of the oldest documented species introductions of marine organisms and is known as an intermediate form between marine and strictly terrestrial isopods. In order to explore the potential value of Ligia as an animal food source, this study focused on the growth rate under laboratory rearing conditions and conducted a detailed analysis of the overall nutrient content of the species in comparison to two other marine food media (krill and fish meal). Evaluation of the growth rate of juveniles suggests it is a relatively fast-growing species of the Ligiidae family. The essential amino acids content Ligia meal is the lowest amongst the three studied media but the proportion of flavor amino acids, and in particular taurine, was higher. The most restricted amino acids of isopod meal are methionine and cysteine. The significantly unbalanced amino acid composition of Ligia meal may affect the absorption and utilization by consumers. In terms of fatty acids, the total polyunsaturated fatty acids in the isopod is very low. A total of 12 vitamins were examined. The VK1, VE, VB2, VB3, VB5 content of isopod meal were significantly higher than those of krill meal and fish meal. Similarly, most of the 11 mineral elements are highest in the isopod meal. Ligia therefore offers potential as an alternative natural food source in animal given the growth rate under culture and the overall nutrient content. But Ligia collected in most of the field would be deemed unfit for human consumption because of the relatively low nutritional value and heavy metal content exceeding the provided standard. Further study is warranted to elucidate the biological characteristics of isopods and how its diet is reflected in its nutritional value to consumers.


Materials and methods
Growth rate determination of juveniles Ligia exotica. Ligia exotica specimen were collected at the embankment of Tiaoshun Island in Zhanjiang City, Guangdong Province, China (N 21° 28′, E 110° 39′) in June to September 2017. The species identification was confirmed by the morphological character, which usually has 37-40 segmental number in 2nd antenna 27 . Dozens of adults were cultured in a 40 cm × 20 cm × 30 cm aquarium, with oyster shells stacked on the right side and a plastic baffle with small holes through which seawater could pass was fixed to the bottom left 10 cm of the aquarium. Sea water reached half the height of the oyster stack and a filter pump was installed. Ligia exotica were fed daily with tilapia fish pellet feed placed on dry oyster shells. When gravid females were observed, especially where fertilized eggs in the marsupium were found to change color from orange to black, they were immediately isolated into a plastic box with a layer of cotton covered with a layer of gauze on the bottom. A piece of paper was placed on the gauze and thoroughly wetted with clean seawater. Excessive tilapia pellet feed was spread on the paper as a food source. The cotton, gauze and seawater were changed every two days.
When sufficiently developed, the 50-60 juveniles crawled out from the marsupium of the brooding female. The time of birth was recorded, and the mothers removed from the plastic box to avoid cannibalism. Juveniles from each female were divided into groups of ten individuals and cultured in a plastic test tube separately and in a constant temperature incubator at 28 °C. Hence the 50-60 juveniles were divided evenly into 5-6 groups, which consist of 5-6 check points waited to be sampled. A total of 15 check points for 70 days lasted culture experiment were established in this way and cultured under similar conditions as described that of gravid females.
Each group of juveniles was weighted every 5 days. After being frozen at − 20 °C, the samples were placed at room temperature for 20 min to volatilize the water on the body surface and were weighed together with a high-precision electronic balance. The total weight of ten juveniles then transformed into the value of average individual weight that facilitate to growth analysis. The check points were set up in triplicate and the sampling procedures was performed as described above.
Analysis of nutritional components of Ligia exotica and comparative substrates. The frozen field collected Ligia exotica were subsequently dried at 75 °C for one day in an oven, ground into powder and stored at − 20 °C until analysis.
Two readily available aquatic food substrates were used for comparative purposes. Antarctic krill (Euphausia superba) meal was purchased from the China National Fisheries Corporation. It had originally been cooked at 80 ~ 95 °C for 20 ~ 25 min, dehydrated and dried on board when caught at sea and was stored in the laboratory at − 20 °C.
Fishmeal was white fish meal (degreased) imported from Russia, PJSC Nakhodka Active Marine Fishery Base, which was processed directly on board and mainly composed of the pacific cod Gadus macrocephalus. When delivered to the laboratory, the samples were divided into several bags, stored at − 20 °C and sampled at random during the experiment.
A range of nutrient components were analyzed from Ligia and the comparative substrates as described in Supplementary Table S1 online.
Evaluation of nutritional quality of amino acids. Based on the amino acid scoring standard model recommended by FAO & WHO 28 and the amino acid model using egg protein as an ideal protein reference, the Amino Acid Score (AAS), Chemical Score (CS) and Essential Amino Acid Index (EAAI) from eight essential amino acids for humans were calculated from the following formulae 29 . The higher the scores and indices that the substrates received, the more similarity they are with the ideal protein model, and the better the protein quality for human consumption.   Table 3); AA egg is the content of the same amino acid in whole egg protein (%); n is the number of essential amino acids compared (n = 9). A, B, C, ⋯; I is the content of essential amino acid of sample protein (mg/g N), AE, BE, CE, ⋯; IE is the content of essential amino acid of whole egg protein (mg/g N).
Statistical analysis. As far as the juveniles development/growth analysis is concerned, by using the function of data regression analysis in Microsoft Excel software, a power function regression model with individual body weight on ages was established, in order to obtain the growth curve (trendline) and the regression determination coefficient (r 2 ). Meanwhile the residual statistics was conducted in SPSS 26.0 to test whether the weight gain values follow the normal distribution. Following the Chinese national determination standard method, the analysis of each samples was repeated three times by the same tester to obtain data for statistical analysis. When conducting the fatty acid and vitamin content analysis, the concentration of some parameters that were too low to be detected (ND) and were considered zero with no statistical analysis undertaken. The normality of the original raw data was confirmed by the Shapiro-Wilk method in SPSS 26.0 prior to statistical analysis. It indicated that all the original data follow the normal distribution.
Levene's test was adopted to test the homogeneity of data of the nutritional parameters of the three food materials (isopod meal, antarctic krill meal and fishmeal), in advance for Analysis of Variance (ANOVA, two tailed). In the case of homogeneity of variances, Duncan's multiple range test (multiple F test) was used to identify any difference in mean values. Meanwhile Fisher's least significant difference (LSD) was employed (assuming that isopod meal is controlled and then compare it respectively with krill and fish meal) as references to verify the statistical differences. If the data violated the assumption of homogeneity of variances, Welch's Anova was used and post-hoc methods of Dunnett's T3 test employed to identify the significance or otherwise of the differences. P < 0.05 was considered significant. Mean ± 95% confidence interval (constructed with t-distribution) was used to describe the statistical data.
By employing the analytic hierarchy process (AHP) technique, a structural analysis model was established for evaluating the nutritional value of fish meal, isopod meal and krill powder in relation to amino acids, fatty acids, vitamins and minerals (Fig. 2).
Nutritional value was assessed based on the considered views of 3 nutritional experts in a small advisory committee, including expertise in human nutrition education (Lingnan Normal University. China), in swine nutrition (Jiangsu AnYou Biotechnology Group Co., Ltd. China), and in aquaculture nutrition (Ocean University of China, China). According to the scoring criteria in Table 1, each expert rated the nutritional components of the substrates and assessed the accuracy of the nutritional value judged by the four nutritional indicators, i.e., amino acids, fatty acids, vitamins and minerals.
The judgment matrices of each expert were imported into the group decision system and tested for consistency by YAAHP (Yet Another AHP) V.10.0 software. Upon testing, all the matrices derived from the scores of three experts met the consistency requirement (consistency ratio = 0.0981, 0.0000, 0.0398, respectively). The total sequencing weight value was calculated through arithmetical average, which was generated from the matrices provided by experts.
Animal welfare statement. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes. Consent for publication. The authors give our permission for the following manuscript to appear in the print, online, and licensed versions of Scientific Reports and for the Journal to grant permission to third parties to reproduce this manuscript.

Results
Growth performance of juvenile Ligia exotica. The  General nutritional components of Ligia. As shown in Fig. 4, the crude protein of isopod meal as a percentage of wet weight is less than both the krill meal and fishmeal (F = 225.18, df = 2). The crude fat (F = 224.02) and cholesterol content (F = 1430.29) of the isopod meal is also lower whereas the crude ash content (F = 237.64) is higher than both krill and fishmeal. Table 2 shows the composition and content of 18 amino acids and taurine in isopod meal, antarctic krill meal and fish meal. The total amino acid content (ΣAA) of isopod meal is higher than that of krill meal but significantly lower than that of fish meal (F = 989.81, all df = 2). The contents of the nine    Table 2. Amino acids composition of isopod meal, antarctic krill meal and fish meal (%, dry weight). ΣAA is total amino acids, ΣEAA is total essential amino acids, Σ NEAA is total nonessential amino acids, and ΣFAA is total flavor amino acids. Amino acids marked e means essential amino acids, while f means flavor amino acid. Numerical values marked with the same letter a,b or c are not statistically significantly different. www.nature.com/scientificreports/ well than that of krill powder (F = 91.28), and its proportion to the total amino acid (ΣFAA/ΣAA) is also higher than both of krill meal and fish meal (F = 51.10).

Amino acids composition.
Nutritional evaluation of amino acids. The amino acid score (AAS), chemical score (CS, the limiting amino acid index) and essential amino acid index (EAAI) were calculated by converting the data in Table 2 into milligrams of amino acid per gram of nitrogen (× 62.5). The results were compared with the amino acid scoring standard pattern suggested by FAO/WHO and the standard amino acid pattern of whole egg protein.
Amino Acid Score (AAS) and Chemical Score (CS) reflect the relationship of protein composition and utilization ratio from different perspectives. As can be seen from Table 3, in most of the case, the lowest scores are from the isopod meal, with Ile and Met + Cys in particular less than half of the value of krill meal and fishmeal. Isoleucine, Methionine and cystine are therefore the main limiting amino acids of isopod as suggested by their content. EAAI index reflects how close the essential amino acid content of material is to the standard protein (egg protein). Comparing the values of EAAI indicates that the protein quality of the isopod meal is worse than that of both krill meal and fishmeal. The high AAS and CS scores of fishmeal demonstrate that fishmeal is rich in essential amino acids and it is well-balanced in composition. Table 4 shows the fatty acid composition of the three substrates.

Nutritional composition of fatty acid.
There are 12 fatty acids including 3 saturated fatty acids (SFA), 3 monounsaturated fatty acids (MUFA) and 6 polyunsaturated fatty acids (PUFA) in isopod meal. 13 fatty acids were detected in krill meal, including 4 SFA, 3 MUFA,6 PUFA, while all 17 fatty acids (4 SFA,6 MUFA and 7 PUFA) were found in fish meal. The actual content of saturated fatty acid (SFA) in isopod meal is similar to that of krill but higher than that in fish meal (F = 18.00, df = 2). The content of monounsaturated fatty acids (MUFA, F = 71.11), and EPA and DHA are the lowest in the isopod (F = 367.63 and F = 311.70 respectively) and although the content of n-6 PUFA is slightly higher in the isopod than that of krill meal (F = 55.69), the total content of PUFA is far lower than either krill meal or fishmeal (F = 117.81).
Comparison of vitamin composition. The vitamin composition of isopod meal is relatively comprehensive (Table 5). Among the four fat-soluble vitamins, the content of VA is lower in the isopod than in fish meal, while the contents of VK 1 and VE (F = 1.81 × 10 5 , df = 2) are much higher than those in krill and fish meal. In addition, the content of water-soluble vitamin VB 2 (F = 1.30 × 10 5 ),VB 3 (F = 19.13) and VB 5 is the highest in isopod meal.
Comparison of mineral composition. The mineral composition of Ligia exotica is shown in Table 6.
Ubiquitous mineral elements such as calcium (F = 3995.94, all df = 2), potassium (F = 590.06) and magnesium (F = 658.60) are most abundant in the isopod. The trace mineral element ferrum (F = 13185.74), chromium (F = 33.59) and selenium (F = 406.02) are the richest in the isopod, while copper content (F = 160.04) is higher in both isopod and krill meal than that in fishmeal. Table 3. Comparative analysis of amino acid score (AAS), chemical score (CS) and essential amino acid index (EAAI) of antarctic krill meal and isopod meal.  Fig. 5. For human or animal consumption, the importance of amino acids, fatty acids, vitamins and minerals are different. On this basis, fishmeal is the most nutritionally rich substrate (weight = 0.5407), and isopod meal is the least (weight = 0.2015), due largely to the imbalance in nutritional elements.
In order to better visualize the differences of nutrient composition of the three food materials, a radar chart (Fig. 6) was constructed including essential amino acids, flavor amino acids, essential amino acid index, Σ PUFA, vitamins (eight parameters) and minerals (nine parameters). As can be seen there are clear differences between the assessed nutritional value of isopod, krill and fish meal. Isopod substrate scores better in minerals and vitamin content, and has a certain flavor stimulating effect (based on ΣFAA/ΣAA (%)). However, fatty acid content, especiallyΣPUFA, is far lower than that of krill meal and fish meal. Fish meal scores best in EAAI andΣPUFA. Unsaturated fatty acids (including PUFA) are known to have beneficial physiological functions such as improving blood microcirculation and increasing the activity of brain cells. While the closer the protein composition is to

Discussion
Ligia species are distributed globally and Ligia exotica is probably the most widely distributed among about 30 species of the genus. Based on the data presented in Hourado et al., 2018 30 , the sites sampled in the current study and after reviewing the literature on Ligia exotica, we have compiled a comprehensive list of locations where Ligia are known or generally available (see Supplementary Table S2 online) and constructed a global distribution map (Fig. 7) generated by ArcGIS software. This is useful to illustrate the widespread nature of the species and therefore its broad availability as a potential medium for food. Ligia exotica represents one of the oldest documented introductions of marine organisms, originally described by Roux 31 at the docks in Marseille, France (ID = 61, Fig. 7). This is the northernmost location, while the southernmost location is Sunday Island, Australia 32 (ID = 58, Fig. 7). They are widely distributed in tropical and temperate regions, including the Seychelles archipelago and Hawaiian Islands suggesting significant colonization ability, but the species have not been found in the Antarctic or Arctic. The southern coast of the United States and the coast of East Asia are two major hot spots. East Asia is traditionally considered to be the origin of L. exotica. In China, L. exotica is distributed all over the rocky coast 1 , including Taiwan (ID = 35, 36. Figure 7). Areas in eastern China account for 70% of Chinese human population, whose activities not only bring a large amount of nutrients to the coastal waters by way of waste discharge and disposal, but also provide habitats to L. exotica in Table 6. Minerals composition of isopod meal, Antarctic krill meal and fishmeal. Numerical values marked with the same letter a,b or c are not statistically significant different at the p < 0.05 level.    Tables 2, 3 and 4. For other two factors, three food materials are ranked based on the top rank having the highest value in the number of parameters, followed by the second and third respectively. For example, isopod substrate has six highest parameters in the mineral category, followed by krill with two and fishmeal one (Table 6), so they are ranked first, second and third respectively. To facilitate comparison with other factors in the radar chart, numbers "50", "40", "30" were assigned to the first, second and third ranked materials respectively.  www.nature.com/scientificreports/ able to grow from 56.5 mg to 111.6 mg over 40 weeks of culture 33 . The resulting specific growth rate (SGR) is only 0.24%. In contrast, for the Pacific white shrimp Litopenaeus vannamei, the SGR of genetically selected high growth lines could reach 29.25% 34 . In present study, juvenile Ligia exotica has an SGR of 6.97% after 70 days of culture. Juvenile are expected to keep growing for a long time until they mature. Usually isopods live for 1.5 to 2 years. The breeding occurs in the spring and early summer, with some females carrying winter broods of eggs (Carefoot, field observation 3 ). Hence it can be deduced the isopods mature sexually around 6 months to 1 year. In China, crustacean farming for food (represented by species such as Pacific white shrimp Litopenaeus vannamei and American crayfish) is a significant industry. The annual output of white shrimp is more than 1.5 million tons, and its gross value is more than 8.7 billion dollars. Litopenaeus vannamei also has a mineralised cuticle that sheds regularly to allow for growth. From the aspect of academic research, Ligia extocia, given its abundance and large geographic range, has the potential to become a model animal for crustacean studies related to aquaculture, and to better understand some of the physiological properties of crustacea such as the shrimps that are economically important. For example, the calcium translocations and transepithelial movement during the moulting cycle of L.vannamei, and dietary calcium requirement in low salinity environments 35 . This undoubtedly has significant theoretical and applied value.
The primary aim of present study was to examine the nutritional value of Ligia as a potential new natural food source in aquaculture based on our previous study that confirmed that L. exotica provides a good diet for juvenile cuttlefish 21 . In comparison to both krill and fish meal, the nutritional value of protein and amino acids of Ligia isopod is lower in almost all evaluation indexes, such as crude protein content, ΣEAA/ΣAA (%), ΣEAA/ ΣNEAA (%), and EAAI. In particular the two amino acids with the lowest values for L. exotica, methionine and cystine, are present at less than half of that of krill meal and fishmeal. The imbalance of these amino acids may affect the digestion and absorption of predators from isopod food. However, isopods have a relative high value of ΣFAA/ΣAA (%) and the contents of taurine are 4 to 5 times those of fish meal and krill. As a sulfonic acid, taurine is a found in high concentrations in animal tissues and has been attributed a wide diversity of roles for food additives. It is added to cat food, chicken feed, energy drinks, infant formula, dietary supplements, cosmetics, inert ingredients in pesticides and pharmaceuticals 36 . For instance, a number of studies (Salze et al. for review 37 ) have demonstrated the essentiality of dietary taurine for many commercially relevant species, especially marine teleosts. In the European Union and China, taurine is authorized for fish feed in all species. Consequently, combining with the flavor amino acid, they may possibly be the most useful ingredients that L. exotica provided to develop into food additive, especially in feeding stimulation, for animal culture.
Ligia contains more saturated fatty acid (SFA) than fishmeal and krill, but carries fewer polyunsaturated fatty acid (PUFA), which is important food element especially in the cardiovascular health of consumers. This also reduces its potential nutritional value. Interestingly, however, the isopod has superior vitamin content as concentrations of VK 1 , VE, VB 2 , VB 3 and VB 5 are all far higher than that in krill and fishmeal. It should be noted that the vitamin content in substrate is highly variable, influenced by several factors, such as origin and composition of the animal, meal processing method, and product freshness 38 . Under the processing methods of this study, the three substrates went through a process of heating and drying at high temperature, so for unstable vitamins such as VC, VB 1 and folic acid problems with detection may have occurred. In addition, part of fat-soluble vitamins in fish meal were lost during oil extraction.
The mineral composition analyses show that calcium accounts for a very large proportion of body content in the isopod, which is the relatively stable ubiquitous elements. However, the body concentration of metal elements in isopods is highly affected by the intertidal environment, and they have a high tolerance to heavy metal contaminants. In areas with severe anthropogenic contamination, heavy metal elements are concentrated through food chains and can accumulate in isopods 39 . For example, high concentrations of copper in Ligia from the Santa Rosalía area are consistent with mining activities at this location 40 . In addition, it has been reported that Ligia sp. can accumulate harmful organic chemicals, such as POPs 41 , TBT 42 , and even radioactive substances 43 . The sampled area in the present study, Jinsha Bay, Zhanjiang City (ID = 59, Fig. 7) is a hot spot for human activity and waste discharge, therefore it is likely that Ligia would be exposed to and accumulate many heavy metal pollutants. Since the intertidal zones are exposed to pollution from both marine and terrestrial sources, isopods could potentially be used as biomonitors of pollution in these habitats in a similar way to terrestrial isopods in soil ecotoxicology 44 . Indeed, Longo et al. 45 reported that Ligia italica play the role of bioindicator for heavy metals pollution in the supralittoral zone. Based on the nutritional analysis reported here, Ligia offers the potential as a natural additive for animal food, but Ligia should only be sourced from relatively clean environments or from artificial culture.

Conclusion
To conclude, given the rapid growth rate under culture, acclimatization ability and the fact that it can be cultivated either in or out of water, in addition to the nutritional analysis reported here, it suggests that Ligia extocia has potential to serve as an alternative natural food source in aquaculture even animal farming. It is rich in taurine and flavor amino acids and has been confirmed that especially suitable for cuttlefish which prefers to live on crustacean as diet. However, the unbalanced amino acid composition and lower content of PUFA may limit its practical value. Considering its unique semi-terrestrial ability and its role in the material cycle of the coastal zone, further study is warranted to elucidate its biological characteristics as a potential model species and on how Ligia diet translates into food quality in animal culture.

Data availability
The data sets used and analyzed during the current study are available from the corresponding author on reasonable request.