A comprehensive evaluation of utilization potential of semiterrestrial isopods, Ligia exotica in China: a new aquatic feed?

Background: 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 practical value for food & feed of Ligia, this study focused on growth rate under laboratory rearing conditions and detailed analysis of the overall nutrient content of the species in comparison to two other aquatic food media (krill and sh meal). Results: 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 but the proportion of avor amino acids was higher. In particular, the content of taurine was much higher. Amino acid score and chemical score show that the most restricted amino acids of isopod meal are methionine and cysteine. The extremely unbalanced amino acid composition may affect the absorption and utilization by consumers. In terms of fatty acids, the total polyunsaturated fatty acids in isopod is very low. A total of 12 vitamins were examined. The VK 1, VE, VB 2 , VB 3 , VB 5 content of isopod meal were signicantly higher than those of krill meal and sh meal. Similarly, most of the 11 mineral elements are the highest in isopod meal. Conclusions: Ligia offers potential as an alternative natural food source especially in aquaculture given the growth rate under culture and the overall nutrient content. But Ligia collected in most of the eld would be deemed unt for human consumption because of the relatively low nutritional value and heavy metal content exceeding the provided standard. Ligia isopods offer some potential to become a crustacean model animal for commercial aquaculture crustaceans, further study is warranted to elucidate its biological characteristics.

Our preliminarily study has con rmed that Ligia exotica can provide a high-quality natural diet for cultured cuttle sh Sepia pharaonis (Xu et al. 2018). Ligia isopods are also utilized in traditional Chinese medicine for the treatment of muscle injury, swelling and pain, or to overcome malnutrition in children (Sun et al. 2015).
Extracts from Ligia exotica were proven to have obvious proliferation inhibitory effects on a range of biochemical and cellular functions such as cervical cancer cells HeLa, stomach cancer cell SGC-7901 and NCI-60 human tumor cell in vitro and have an inhibitory effect on mouse sarcoma S180-induced transplantable cancers by intraperitoneal injection in tumors over 7d (Bian et al. 2007). A novel nucleoside, elucidated as 3'-O-(α-D-glucosyl) inosine, had been isolated from Ligia exotica but no bioactivity identi ed (Seong et al. 2000).
As far as we are aware, there is no published report on the nutritional analysis of Ligia isopods although there is evidence of potential nutritional as well as medicinal value. To explore this potential value further we established a 70-day culture experiment on juveniles of Ligia to evaluate their growth performance and compared the nutritional content of Ligia exotica meal with two other regular used marine feed sources, white sh meal and Antarctic krill (Euphausia superba) meal. The objectives included whether Ligia can be arti cially cultured on a large scale and to document their growth rate and how well the nutritional value compared with the reference feed sources. It is hoped that the approach adopted here can provide information on this potential as a feed source within animal culture as well as contributing to increasing our understanding of basic crustacean physiology.

Materials And Methods
1 Growth rate determination of juveniles Ligia exotica Ligia exotica (Chen et al. 1987. Figure.1) were collected at the embankment of Tiaoshun Island in Zhanjiang City, Guangdong Province, China (N21 °28, E110 °39'), and were cultured in a 40 cm × 20 cm × 30 cm aquarium, with oyster shells stacked on the right side and a plastic ba e with small holes through which seawater can pass was xed to the left 10 cm of the aquarium. Sea water reached half the height of the oyster stack and a lter pump was installed. Daily feeding of Ligia exotica was with tilapia sh pellet feed placed on dry oyster shells.

Isolated Culture Of Gravid Females
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. Tilapia pellet feed was spread on the paper as a food source. The cotton, gauze and seawater were changed every two days.

Culture Of Juveniles
When developed to a certain extent, the 50-60 juveniles would crawl out from the brooding female. The time of birth was recorded, and the mothers removed from the plastic box to avoid cannibalism. Juveniles were divided into groups of 10 and cultured in a constant temperature incubator at 28 ℃. The culture conditions are described above.

Sampling
Each group of juveniles were sampled every 5 days and the culture experiment lasted for a total of 70 days, providing 15 temporal samples. After being frozen at -20 ℃, they were placed at room temperature for 20 minutes to volatilize the water on the body surface and were weighed with a high-precision electronic balance.
The replicate groups of juveniles were weighed on the same days after hatching from different mothers.
2 Analysis of nutritional components of Ligia exotica and comparative substrates

Analysis Method
The frozen Ligia exotica contained individuals caught in the eld, were subsequently dried at 75℃ for one day in an oven, ground into powder and stored at -20 ℃ until analysis.
Two readily available aquatic food substrates were used for comparative purposes. Antarctic krill (Euphausia superba) meal was purchased from China National Fisheries Corporation. It was rapidly cooked at 80 ~ 95℃ for 20 ~ 25 min, dehydrated and dried on board when caught at sea and transported to the laboratory to be stored at -20 ℃.
Fishmeal was white sh meal (degreased) imported from Russia, which was mainly composed of the paci c cod Gadus macrocephalus. When delivered to the laboratory, the samples were divided into several bags, stored at -20 ℃ and sampled at random during the experiment.
A range of nutrient components were analyzed from Ligia and the comparative substrates as described in Table 1. Based on the amino acid scoring standard model recommended by FAO & WHO (1985) 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 (Pellett & Yong, 1980). 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.
where aa is the amino acid content of the sample (%); AA FAO&WHO is the content of the same amino acid recommended by FAO & WHO ( ) (shown in Table 5); 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).

Statistic Analysis
Following the Chinese national determination standard method to analyze the nutritional components of substrates, 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. Before analyzing all the data statistically, their normality was tested by Shapiro-Wilk test method in SPSS 24.0 software. It indicated that all the original data follow the normal distribution.
Levene's test was adopted to deal with nutritional parameters with speci c values for Analysis of Variance (ANOVA, two tailed) in three raw materials (isopod meal, antarctic krill meal and shmeal). If the data followed the assumption of homogeneity of variances, Duncan's multiple range test (multiple F test) was used to identify the difference in means. Meanwhile Fisher's least signi cant difference (LSD) would be employed as references to con rm 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 signi cance or otherwise of the differences. Statistical signi cance is p < 0.05. Mean ± standard deviation was used to describe the statistical data.
By employing the analytic hierarchy process (AHP) technique, the following structural analysis model was established for evaluating the nutritional value of sh meal, isopod meal and krill powder: 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 one researcher with expertise in aquaculture nutrition (Ocean University of China, China). According to the scoring criteria in Table 2, each of them rated the nutritional components of the substrates and assessed the accuracy of the nutritional value judged by the four nutritional indicators. The judgment matrices of each 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 form 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 obtained through arithmetical average, which calculated from the matrices given by experts.

Results
Growth performance of juvenile Ligia exotica As shown in Fig. 3, the average body weight of the new-hatched juveniles of Ligia exotica is 0.24 ± 0.005 mg. The increase of body weight (BW) in the early stages is not signi cant until 15 days after hatching (DAH), after that BW increases from 0.85 ± 0.02 mg to 6.37 ± 0.04 mg at 45 DAH. From then on, the weight gain of individuals begins to accelerate signi cantly, reaching 12.69 ± 0.01 mg at 55 DAH and 16.37 ± 0.41 mg at 60 DAH. The exponential growth equation of juveniles Ligia is y = 0.3485e 0.3119x , and the correlation index R 2 is 0.93, which demonstrate a good positive correlation of body weight gain over time. The nal BW value of Ligia at 70 DAH is 31.06 ± 1.06 mg. Weight gain rate of juvenile Ligia is ( nal BW-initial BW) / initial BW × 100% is 13026.76%. Speci c growth rate per day is (ln nal BW -ln initial BW ) / DAH × 100% is 6.97%.

General nutritional components of Ligia
As shown in Table 3, the crude protein of isopod meal is less than both the krill meal and shmeal (F = 225.18, df = 2). The crude fat (F = 224.02, df = 2) and cholesterol content (F = 1430.29, df = 2) of the isopod meal is lower whereas, the crude ash content (F = 237.64, df = 2) of isopod meal is higher than krill and shmeal. Amino Acids Composition Table 4 shows the composition and content of 18 amino acids and taurine in isopod meal, antarctic krill meal and sh meal. ΣAA is total amino acids,ΣEAA is total essential amino acids Σ NEAA is total nonessential amino acids ΣFAA is total avor amino acids.
Amino acids marked e means essential amino acids, while f means avor amino acid.
Numerical values marked with the same letter a, b or c are not statistically signi cantly different. Note: Values are means of triple determination ± standard error ΣAA is total amino acids,ΣEAA is total essential amino acids Σ NEAA is total nonessential amino acids ΣFAA is total avor amino acids.
Amino acids marked e means essential amino acids, while f means avor amino acid.
Numerical values marked with the same letter a, b or c are not statistically signi cantly different.
The total amino acid content (ΣAA) of isopod meal is higher than that of krill meal but signi cantly lower than the total amino acids of sh meal (F = 989.81, df = 2). The contents of the nine essential amino acids (ΣEAA) differ with the lowest values in Ligia (F = 181.36, df = 2). While the ratio of essential amino acids to the total amount of amino acids (ΣEAA / ΣAA) of isopod meal is the lowest in those three substrates (F = 17.92, df = 2).
Surprisingly however, the content of taurine, a bene cial non-protein amino acid, is much greater in Ligia than that of krill meal and sh meal (F = 784.36, df = 2). In terms of the content of ve avored amino acids, the amino acid content of isopod meal is higher than that of krill powder (F = 91.28, df = 2), and its proportion to the total amino acid (ΣFAA / ΣAA) is also higher than both of krill meal and sh meal (F = 51.10, df = 2).

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 4 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 amino acid pattern of whole egg protein as described later.
Amino Acid Score (AAS) and Chemical Score (CS) re ect the relationship of protein composition and utilization ratio from different perspectives. As can be seen from Table 5, the superscript letters indicate clearly that in most of the case, the lowest scores of amino acids are in isopod category, while Ile and Met + Cys may be the most distinct that less than half of those of krill meal and shmeal (F = 3024.7, F = 2392.19 respectively for AAS, and F = 2935.5, F = 2392.19 for CS. All df = 2). Methionine and cystine are therefore the main limiting amino acids of isopod as suggested by their content. EAAI index re ects how close the essential amino acid content of material is to the standard protein (egg protein). Comparing the values of EAAI of isopod meal, krill meal and shmeal, indicates that the protein quality of the isopod is worse than that of krill meal and shmeal (F = 1294.4, df = 2). Most of the highest AAS and CS scores of shmeal demonstrate that shmeal is rich in essential amino acids and it is well-balanced in composition, therefore easy to be digested and absorbed by human.
Nutritional Composition Of Fatty Acid Table 6 shows the fatty acid composition of isopod meal, krill meal and sh meal. 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. 14 fatty acids were detected in krill meal, including 4 SFA,4 MUFA,6 PUFA, while all 4 SFA,6 MUFA and 7 PUFA were found in sh meal. The content of saturated fatty acid (SFA) in isopod meal is like that of krill but higher than that in sh meal (F = 18.00, df = 2), which may show that its fatty acid characteristics are closer to those of terrestrial animals. The content of monounsaturated fatty acids (SFA, F = 71.11, df = 2), and EPA and DHA are the lowest in the isopod (F = 367.63 and F = 311.70 respectively, while df = 2). Although the content of n-6 PUFA is slightly higher in the isopod than that of krill meal (F = 55.69, df = 2), the total content of PUFA is far lower than either krill meal or shmeal (F = 117.81, df = 2). As shown in Table 7 the vitamin composition of isopod meal is relatively comprehensive. Among the four fatsoluble vitamins, the content of VA is lower in the isopod than in sh meal, while the contents of VK1 and VE (F = 1.81 × 10 5 , df = 2) are much higher than those in krill and sh meal. In addition, the contents of watersoluble vitamin VB 2 (F = 1.30 × 10 5 , df = 2),VB 3 (t = 19.13, df = 2)and VB 5 are the highest in isopod meal among the three materials. Values are means of triple determination ± standard error.
Values with the same letter a, b or c indicates that the differences are not statistically signi cant between mean values at the p < 0.05 level.

Comparison Of Mineral Composition
The mineral composition of Ligia exotica is shown in Table 8. Ubiquitous mineral elements such as calcium (F = 3995.94, df = 2), potassium (F = 590.06, df = 2) and magnesium (F = 658.60, df = 2) are most abundant in the isopod. Trace mineral element ferrum (F = 13185.74, df = 2) is also the richest in the isopod, whilst copper content (F = 160.04, df = 2) in both isopod and krill meal is higher than that in shmeal. Chromium (F = 33.59, df = 2) and selenium (F = 406.02, df = 2) are also the highest in the isopod. Numerical values marked with the same letter a, b or c are not statistically signi cant different at the p < 0.05 level.

Comprehensive comparison of amino acids, fatty acids, vitamins and minerals
Based on the group decision hierarchy process software YAAHP (Yet Another Analytic Hierarchy Process Software, V10.0) to summaries of expert judgment, the weighted index nutritional value was calculated, as shown in Fig. 4.
For human or animal consumption, the importance of amino acids, fatty acids, vitamins and minerals are different (blue numbers in the middle of Fig. 4 show the weight of the elements, the larger the number, the greater the importance of the index, and the following blue numbers in the bottom of Figure mean the same).
On this basis, shmeal is the most preferable substrate, and isopod meal is the worst, due largely to its imbalance in nutritional elements.
In order to better visualize the differences of nutrient composition of the three food materials, a radar chart ( Fig. 5) was constructed including essential amino acids, avor amino acids, essential amino acid index, Σ PUFA, vitamins (8 parameters) and minerals (9 parameters). The rank order of the three food materials for each of the main nutrient category is based on the top rank having the highest value in the number of parameters, followed by the second and third respectively, considering that numerous parameters are involved.
For example, in the mineral category, isopod substrate has six the highest parameters, followed by krill with two and shmeal one (Table 8), so they are ranked rst, second and third respectively. To facilitate comparison with other factors in the radar chart, numbers "50", "40", "30" were assigned to the rst, second and third ranked materials respectively, while factors of ΣFAA / ΣAA(%),ΣEAA / ΣAA(%), EAAI andΣPUFA were scored based on the actual values from Table 4, Table 5 and Table 6.
As can be seen from Fig. 5, there are clear differences between the assessed nutritional value of isopod, krill and sh meal. Isopod substrate scores better in minerals and vitamin content, and has a certain avor stimulating effect (view from ΣFAA / ΣAA(%)). However, fatty acid content, especially ΣPUFA, is far lower than that of krill meal and sh meal. Fish meal scores best in EAAI andΣPUFA. Unsaturated fatty acids are known to have bene cial physiological functions such as improving blood microcirculation and increasing the activity of brain cells. The closer the protein composition is to the egg protein, the easier it absorbed and utilized by humans (view from EAAI). To sum up, the nutritional value of isopod is inferior to that of krill and sh meal. L. exotica represents one of the oldest documented introductions of marine organisms (Hurtado et al, 2018), originally described by Roux (1828) at the docks in Marseille, France (ID = 61, Fig. 6). This is the northernmost location, while the southernmost location is Sunday Island, Australia (Green, 1962. ID = 58, Fig. 6). They are widely distributed in tropical and temperate regions, including the Seychelles archipelago and Hawaiian

Discussion
Islands suggesting signi cant colonization ability, but the species has 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 (Yin, 2013), including Taiwan (ID = 35, 36. Figure 6). 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 form of wharves and dams. Ligia sp. plays an important role as a scavenger/detritivore, feeding on a large range of organic matter plant debris and animal corpses brought by tides.
The only published study on Ligia growth we are aware of to date is that of  on the eld population and growth of Ligia pallasii Brandt. Further work examined the nutritional requirements of Ligia pallasiii using arti cial feed and demonstrated that this species is able to grow from 56.5 mg to 111.6 mg over 40 weeks of culture (Carefoot, 1984). The resulting speci c growth rate (SGR) is only 0.24%. In contrast, for the Paci c white shrimp Litopenaeus vannamei, the SGR of genetically selected high growth lines could reach 29.25% (Rosenberry, 2013). In present study, juvenile Ligia exotica have an SGR of 6.97% after 70 days of culture.
The primary aim of this 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 con rmed that L. exotica provide a good diet for juvenile cuttle sh (Xu et al. 2013). However, it is still unknown how long this feed could support the growth of juvenile cuttle sh if be used as a sole diet. In comparison to both krill and sh 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 shmeal. 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 sh 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 in some mammals, e.g., as an essential dietary requirement for cats, and a critical supplement for marine sh feed (Salze & Davis. 2015) Ligia contains more saturated fatty acid (SFA) than shmeal and krill, but carries few polyunsaturated fatty acid (PUFA), which is important to humans and other animals. 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 in krill and shmeal. It should be noted that the vitamin content in substrate is highly variable, in uenced by several factors, such as origin and composition of the animal, meal processing method, and product freshness (Miles & Chapman. 2012). 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 sh 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.
Isopods have a high tolerance to heavy metals contaminants in intertidal environment and their body concentration of metal elements are highly affected by the environment. In areas with severe anthropogenic contamination, heavy metal elements are transmitted through food chains and can accumulate in isopods (Hopkin et al. 2013). For example, high concentrations of copper in Ligia from the Santa Rosalía area are consistent with mining activities at this location (Garciahernandez et al. 2015). The area we sampled Jinsha Bay, Zhanjiang City (ID = 59, Fig. 6) is a hot spot for human activity and waste discharge, therefore it is likely that Ligia would accumulate any heavy metal pollution. The body tissues of Ligia are also very high in iron content. According to Chinese national food safety standards (GB 2762 − 2017) and the standard for the use of food forti ers (GB14880-2012), the mineral content of L. exotica is outside of the standards as food for human consumption. 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 (Gestel et al. 2017).
In China, crustacean farming for food (represented by species such as Paci c white shrimp and American cray sh) is a signi cant 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. Ligia extocia, given its abundance and large geographic range therefore has the potential to become a model animal for crustacean studies related to aquaculture, to better understand some of the physiological properties of crustacea such as the shrimp 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 (Cheng et al. 2006). This undoubtedly has great theoretical and applied value.

Conclusions
In conclusion, Ligia extocia has potential to serve as an alternative natural food source in aquaculture or animal farming given the growth rate under culture, acclimatization ability and the fact that it can be cultivated either in or out of water. It is especially suitable for cuttle sh which prefer live crustacean as diet.
However, the unbalanced amino acid composition and lower content of PUFA may limit its practical value. Ligia collected in the eld are deemed un t for human consumption because of the heavy metal content exceeding the provided standard. 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.

List Of Abbreviations
Amino Acids include  Growth curve (body weight gain over time) of juvenile Ligia exotica (n=3 groups, each contain 10 individuals from the same female. ±Bar means standard error ) Figure 4 Nutritional value of three substrates based on group decision analytic hierarchy process of expert judgement Figure 5