Effects of inbreeding on growth and survival rates, and immune responses of ridgetail white prawn Exopalaemon carinicauda against the infection of Vibrio parahaemolyticus

Journal Pre-proof Effects of inbreeding on growth and survival rates, and immune responses of ridgetail white prawn Exopalaemon carinicauda against the infection of Vibrio parahaemolyticus

Jiajia Wang, Qianqian Ge, Jitao Li, Jian Li PII:

S0044-8486(19)31300-6

DOI:

https://doi.org/10.1016/j.aquaculture.2019.734755

Reference:

AQUA 734755

To appear in:

aquaculture

Received date:

24 May 2019

Revised date:

15 November 2019

Accepted date:

18 November 2019

Please cite this article as: J. Wang, Q. Ge, J. Li, et al., Effects of inbreeding on growth and survival rates, and immune responses of ridgetail white prawn Exopalaemon carinicauda against the infection of Vibrio parahaemolyticus, aquaculture (2018), https://doi.org/ 10.1016/j.aquaculture.2019.734755

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© 2018 Published by Elsevier.

Journal Pre-proof Effects of inbreeding on growth and survival rates, and immune responses of ridgetail white prawn Exopalaemon carinicauda against the infection of Vibrio parahaemolyticus Jiajia Wang1, 2, Qianqian Ge1, 2, Jitao Li1, 2, Jian Li1, 2, * Authors' affiliations and addresses: 1

Key Laboratory for Sustainable Utilization of Marine Fisheries Resources, Ministry of Agriculture and

Rural, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, China 2

Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National

Laboratory for Marine Science and Technology, Qingdao, China

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*Corresponding author: Jian Li

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Tel.: +86 532 85826690; fax: +86 532 85826690

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E-mail: [email protected]

Address: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Nanjing Road 106,

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Qingdao 266003, P. R. China

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Authors' email:

E-mail: [email protected] (ORCID: http://orcid.org/0000-0003-2292-6528)

Qianqian Ge

E-mail: [email protected] (ORCID: http://orcid.org/0000-0003-4862-0615)

Jitao Li

E-mail: [email protected]

Jian Li

E-mail: [email protected]

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Jiajia Wang

Journal Pre-proof Abstract Inbreeding could lead to a decrease in genetic variation within a population and subsequent depression of performance traits. The purpose of this study was to investigate the effects of inbreeding on the growth-related traits, immune responses, antioxidant status and the acute hepatopancreatic necrosis disease (AHPND) resistant capacity of ridgetail white prawn (Exopalaemon carinicauda) using a new inbred line named EC5. The results demonstrated that the genetic polymorphisms of EST-SSR markers decreased with an increase in artificial inbreeding, and there was less growth traits and a lower survival rate among inbred populations than the control population. Inbreeding effects on the immune

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responses and antioxidant status were studied in experimental full-sibling inbred populations of E.

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carinicauda at five levels of inbreeding coefficient (F=0.785, F=0.816, F=0.859, F=0.886, F=0.908)

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under controlled laboratory conditions. Inbreeding affected the total haemocyte count, antibacterial activity and phenoloxidase (PO) activity, and the haemocyanin (HEM) concentration decreased after the

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ninth generation. Antioxidant status showed a similar pattern: superoxide dismutase (SOD) and catalase

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(CAT) activity in the cell-free haemolymph decreased. The effects of inbreeding on survival after infection with Vibrio parahaemolyticus were significant, as the cumulative mortality of shrimp in the

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control population (CP) was significantly lower than that in the F9 generation of EC5 inbred family during 24-72 h after infection with V. parahaemolyticus (P<0.05). Additionally, the levels of antibacterial

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activity, immune enzymes including lysozyme (LZM) activity, alkaline phosphatase (AKP) activity and PO activity in cell-free haemolymph were significantly higher in the control population than those in the F9 generation 6 h after infection with V. parahaemolyticus (P<0.05). Furthermore, the expression levels of anti-lipopolysaccharide factor (ALF), LZM, Tollip, evolutionarily conserved signaling intermediate in Toll pathway (ECSIT), Toll and signal transducer and activator of transcription (STAT) in the hepatopancreas, and Crustin, LZM and Tollip in haemocytes were significantly higher in the control population than those in the F9 generation after infection with V. parahaemolyticus (P<0.05). These results suggested that the F9 generation has a weaker disease-resistant capability than the control population when infected with V. parahaemolyticus. In conclusion, this study demonstrates that a high level of inbreeding has a negative effect on growth-related traits and physiological health of E. carinicauda, thus highlighting the need to maintain genetic diversity in selective breeding programs. Key words: Inbreeding, immune response, V. parahaemolyticus, disease-resistant capability

Journal Pre-proof Introduction Inbreeding, whether natural or artificially reared, is unavoidable in small populations, when genetically related individuals mate despite the occurrence of random mating (Charlesworth et al., 2009; Ott et al., 2009). Inbreeding is associated with a reduction in fitness traits among the offspring, and that called as inbreeding depression. Inbreeding depression arises because inbreeding typically accelerates the loss of genetic diversity, increases the proportion of homozygotes and correspondingly decreases the proportion of heterozygotes and these can result in reduced fitness including the occurrence of homozygous lethal recessives (Quaglietti et al., 2016). Such fitness reduction affects all aspects of the

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life histories of both plants and animals, as well as many different fitness-related traits including survival

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(Frommen et al., 2008), reproductive ability (Bickley et al., 2013; Feng et al., 2014) and disease

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susceptibility (Arkush et al., 2015; Smallbone et al., 2016).

Inbreeding and loss of genetic diversity are predicted to decrease the resilience of environmental

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change and increase mortality (Spielman et al., 2004). Inbreeding could render populations more

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vulnerable to pathogens due to the depletion of variation in genes responsible for immunity against pathogens. Both major histocompatibility complex (MHC) genes involved in parasite recognition and

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the initiation of adaptive immune system responses and neutral diversities were lost after several generations of inbreeding in Kryptolebias marmoratus (Ellison et al., 2012). Additionally, inbreeding

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influenced the response of Gila topminnow to Listonella anguillarum (Giese et al., 2003), and also increased disease susceptibility in Poecilia reticulata (Smallbone et al., 2016). This phenomenon was also found to occur in invertebrate, inbreeding significantly reduced the resistance of Drosophila melanogaster to both the thuringiensin toxin and Serratia marcescens (Spielman et al., 2004). However, Luo et al (2014) found that there was no significant difference in Fenneropenaeus chinensis survivorship among the control population, and populations subjected to low levels of inbreeding groups after challenge with White Spot Syndrome Virus (WSSV) (Luo et al., 2014). The ridgetail white prawn Exopalaemon carinicauda is one of the major commercial mariculture species naturally distributed in China (Xu et al., 2010), which belongs to the Palaemonidae family of Crustacea. Due to the merits of fast growth, high reproductive ability and short reproductive cycle, the culture scale of E. carinicauda has expanded in recent years (Zhang et al., 2015). Similar to other invertebrates, E. carinicauda lacks an adaptive immune system and their defense depends entirely on various innate immune responses as primary mechanisms to fight against invading pathogens (Armitage

Journal Pre-proof et al., 2017; Cerenius et al., 2010). The growth, disease resistance and survival of an organism are partly determined by the capability of the immune system (Tumburu et al., 2012). Therefore, immune function is important to assess the effects on the fitness traits of inbreeding. Numerous pathogens have caused disease outbreaks in both vertebrate and invertebrate populations and therefore threaten the survival of organisms globally. The previous studies have shown that Vibrio parahaemolyticus infection caused a new emerging shrimp disease called acute hepatopancreatic necrosis disease (AHPND) that results in great loss of global shrimp production (Han et al., 2015; Lai et al., 2015). It had been demonstrated that V. parahaemolyticus infection could result in severe cellular

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damage and necrosis of hepatopancreas (Maralit et al., 2018). Our previous studies investigated the

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immune response of E. carinicauda infected with V. parahaemolyticus and explored some parameters as

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potential disease-resistant indicators for evaluating the physiological status and potential diseaseresistant capability of shrimp when infected with V. parahaemolyticus, including some pivotal immune

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enzymes and immune related genes (Ge et al., 2018; Ge et al., 2017). These indicators were used in this

parahaemolyticus.

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study to evaluate the disease-resistant capability of inbreeding E. carinicauda when infected with V.

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Understanding the effects of inbreeding on fitness traits in crustacean is important because of their economic value and the constraints imposed on aquaculture by limited brood stock, high stocking

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densities and infectious disease. Although many studies have demonstrated that inbreeding can affect immune (Luo et al., 2014; Ren et al., 2017), morphological (Moss et al., 2008; Ríos-Pérez et al., 2015), physiological (Wang et al., 2017), and reproductive traits (Ott et al., 2009) in crustaceans, to our knowledge, no information is available on the effects of inbreeding on V. parahaemolyticus tolerance in crustacean. Inbreeding in animals can increase their susceptibility to pathogens, but it is unclear whether all pathogens affected equally. This study aims to determine whether inbreeding reduces the immunocompetence of E. carinicauda by analyzing the possible effects of inbreeding on the growthrelated traits, immune responses and antioxidant status of E. carinicauda of EC5 inbred family from different inbreeding generations and investigating the relationship between inbreeding and susceptibility to V. parahaemolyticus infection under controlled laboratory conditions. 2. Materials and methods 2.1 Study population Since 2009, our laboratory has begun to research the selection of inbred lines of E. carinicauda as

Journal Pre-proof a crustacean laboratory animal at the Yellow Sea Fisheries Research Institute using two wild and geographically distinct Chinese populations (Jiaozhou Gulf and Laizhou Gulf). A strain shall be regarded as inbred when it has been mated brother×sister for twenty or more consecutive generations, and the way of full sibling mating has been handed down to the eleventh generation of inbred lines of E. carinicauda. The establishment process of full sibling family for E. carinicauda is depicted in Fig. 1 and by repeating the process, 11 generations of full sibling families were obtained simultaneously until the 2018. The F1 generation of full-sibling families were established by brother×sister mating, and the F 2 generation fullsibling families were obtained from the F1 generation with brother×sister mating, and the F3, F4, F5, F6,

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F7, F8, F9, F10 and F11 generations were obtained in a similar way. The control group was established by

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strict mating of males and females sampled from a base population of known pedigree, and all had an

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average inbreeding coefficient (F) of below 1%. Five generations (F7, F8, F9, F10 and F11) with different levels of inbreeding coefficient (F=0.785, F=0.816, F=0.859, F=0.886 and F=0.908) were established.

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2.2 Growth-related traits measurement and analysis

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A standardized procedure for family production was used in larvae rearing. Each inbred full-sibling and the control population of fertilized eggs were hatched in separate tanks. Hatched larvae passed

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through six zoea stages to post-larvae in 2 weeks at 25.0-26.0℃. A random sample of almost 100 postlarvae from each generation was transferred into separate 5 larger tanks for rearing. We assessed the

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growth of the animals by recording the body weight and body length of all individuals alive, when the animals had reached ages of 60 days, 80 days, 100 days and 120 days. The survival rate was also assessed during each of the four grow-out stages by dividing the number of shrimps stocked in each concrete pond at the beginning and the number found alive at the four grow-out stages. The inbreeding depression coefficient (IDC) for all growth-related traits were evaluated according to (Ritland, 1990) as follows: G (1- inbred )

IDC=(F

Gcontrol

control -Finbred )

×100%

where Gcontrol and Ginbred represent the mean body weight of the control shrimps and the inbreeding shrimps, and Fcontrol and Finbred represent the inbreeding coefficient of the control population and the inbreeding population, respectively. 2.3 EST-SSR polymorphism examination analysis The 46 EST-SSR markers from our previous study (Wang et al., 2018) were used to analyze the

Journal Pre-proof genetic structure and genetic diversity of the control and inbred population. The EST-SSR were detected according to the method of Wang’s study (Wang et al., 2018). The EST-SSR markers diversity was estimated using POPGENE version 1.32 (Yeh et al., 1997), which included the following parameters: the number of alleles (Na), the observed and expected heterozygosities (Ho and He). The polymorphism information content (PIC) of each SSR marker was calculated (Botstein et al., 1980). 2.4 Sample collections for the immune responses and antioxidant status analysis The post-larvae shrimps were transferred into 150 L polyvinyl chloride polymer (PVC) tanks containing 120 L aerated sand-filtered seawater with a temperature 25.0-26.0℃ and initial salinity

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31±0.5 for rearing with the same feeding. Shrimps in the intermolt phase were sampled from the control

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population and each generation at 120 days, respectively. Ten shrimps (including 5 males and 5 females)

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were collected to sample haemolymph at each generation and growth stage. Meanwhile, haemolymph of ten shrimps were collected into a 2 mL sterile syringe containing an equal volume of anticoagulant

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solution (30 mM trisodium citrate, 0.34 M sodium chloride and 10 mM EDTA-Na2, pH=7.55), and then

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gently mixed in a sterile tube. A small part was immediately used to count the haemocytes and analyze phagocytic activity of the haemocytes, and the remainder was centrifuged at 1000× g for 10 min at 4°C,

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and the supernatant was dispensed into 2 mL Eppendorf tubes as cell-free haemolymph samples and stored at -80°C for analysis of other immune and antioxidant parameters. All assays for analyzing

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immune parameters were conducted in triplicate. 2.5 Experimental animals and bacterial culture for V. parahaemolyticus infected The shrimps from the control population were hatched in the same day as the F9 generation, and divided into two batches at 120 days, respectively. After the acclimatization, 90 shrimps were collected and divided into three groups of thirty, and used to record cumulative mortality after challenged with 10 7 cfu ml-1 V. parahaemolyticus. The 120 shrimps were used to investigate changes of immune parameters in E. carinicauda after challenged with 107 cfu ml-1 V. parahaemolyticus. The ten shrimps were collected to sample at 6 h and 48 h after injected with V. parahaemolyticus, respectively. In the control population, the same experimental design was implemented in the same experimental conditions. The V. parahaemolyticus strain (no. 20130629002S01) separated from the AHPND-infected L. vannamei was kindly provided by the Mariculture Disease Control and Pathogenic Molecular Biology Laboratory, Yellow Sea Fisheries Research Institute. The bacteria were grown overnight at 28℃ in tryptic soy agar (TSA) supplemented with 2% NaCl. One single V. parahaemolyticus bacterial colony was

Journal Pre-proof picked, then inoculated into tryptic soy broth (TSB) containing 2% NaCl and cultured by incubation 16 h with shaking at 200 rpm at 28℃. The V. parahaemolyticus concentrations were performed by bacterial plate count on thiosulfate citrate bile salts (TCBS) agar. 2.6 Determination of immune parameters There were six immune parameters (the total haemocyte counts, the antibacterial activity, the haemocyanin (HEM) concentration, Phenoloxidase (PO), lysozyme (LZM) and alkaline phosphatase (AKP) activity) and two antioxidant parameters (superoxide dismutase (SOD) and catalase (CAT) activity) were measured for analyzing the effect of inbreeding in different generations according to the

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previous study (Ge et al., 2018; Ren et al., 2017). The same immune parameters were measured to assess

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the potential disease-resistant capability of the control and inbred population when infected with V.

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parahaemolyticus. All assays for analyzing the parameters were conducted in triplicate. The haemocyte counts were totaled using Neubauer haemocytometer under a light microscope. A

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total of 200 μl anticoagulant haemolymph was placed on the haemocytometer and the haemocytes were

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counted and expressed as cells ml-1 haemolymph.

The antibacterial activities of the haemolymph were measured according to the method of Ge et al

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(2018) (Ge et al., 2018). A total of 300 μl bacterial suspension and 10 μl cell-free haemolymph sample were pipetted into 96-well ELISA plate, which was put into microplate reader and shaken for a little

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while. The OD570 nm was read and recorded as A0. Then the plate was incubated in the microplate reader in the dark at 37°C for 30 min and OD570 nm was recorded (A). The antibacterial activity was defined as Ua, then calculated as follows: Ua=(A0−A)/A. The absorbance of 100 μl haemolymph mixed with 900 μl sterile water was measured at 335 nm using Multiskan spectrum (Thermo, USA) to determine the haemocyanin (HEM) concentration, and that was calculated using an extinction coefficient of 17.26. Phenoloxidase (PO) activity of the haemolymph was measured spectrophotometrically at 490 nm by recording the formation of dopachrome produced from L-3,4-dihydroxyphenylalanine (L-DOPA, Sigma), according to the procedure described by Hernández-López et al (1996) (Hernández-López et al., 1996). One unit of PO activity was defined as an increase in absorbance of 0.001 min -1 ml-1 cell-free haemolymph. The activities of lysozyme (LZM), alkaline phosphatase (AKP), superoxide dismutase (SOD) and catalase (CAT) in cell-free haemolymph were measured using commercial kits (Jiancheng

Journal Pre-proof Bioengineering Institute, Nanjing, China) according to manufacturer's protocols. All assays for analyzing the above immune parameters were conducted in triplicate. 2.7 Quantitative RT-PCR for the expression of immunity-related genes Total RNA from haemocytes and hepatopancreas were extracted from different tissues using TRIzol Reagent (Invitrogen, USA) according to the manufacturer’s standard protocol and the contaminated genomic DNA was eliminated using RNase-free DNaseI (TaKaRa, China), respectively. The quantity, purity and integrity of extracted RNA was estimated by RNA electrophoresis on 1.5 % agarose gel and detected using Scientific NanoDrop 2000 (Thermo, USA) at an absorbance of 260 and 280 nm. For

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quantitative real-time PCR (qPCR) expression analysis, total RNA was reverse transcribed using the

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PrimeScriptTM Real-time PCR Kit (TaKaRa).

In our previous study on the transcriptome analysis of the hepatopancreas in E. carinicauda infected

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with V. parahaemolyticus (SRA accession number: SRP096646) (Ge et al., 2017). The eight innate

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immune-related genes, the Tollip, Toll and ECSIT genes in Toll signal pathway, the STAT gene in JAK/STAT signal pathway, three antimicrobial peptides genes including anti-lipopolysaccharide factor

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(ALF), crustin and LZM, and β-1,3-glucan binding protein (LGBP), as important pattern recognition receptor were used for investigation. The 18S rRNA of E. carinicauda (GenBank accession number:

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GQ369794) was used as an internal control for expression. The 18S rRNA gene is abundant and stable in cells, and hardly affected by the external regulation. The expression of 18S rRNA gene is invariant in the

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present experimental conditions, and was used as an internal control in gene expression analysis of E. carinicauda (Ge et al., 2015). Each sample was done in triplicate. Primers for qPCR were designed using Primer5 software (Table 1) (Lalitha, 2000). The efficiency of qPCR was in the optimal range of 90-110% for all of the primer pairs used. All analyzed were based on the CT values of the PCR products. The expression was calculated with 2-∆∆CT methods. The qPCR assays were performed using the SYBR Premix Ex Taq™ II (TaKaRa) with an ABI PRISM 7500 Sequence Detection System (Applied Biosystems, USA). The qPCR was carried out in a total volume of 20 μl, containing 10 μl SYBRR Premix Ex TaqTM II (TaKaRa, China), 50-100 ng of cDNA, 0.8 μl each of Forward and Reverse primer, 0.4 μl ROX Reference Dye II (50×), and added sterile ddH 2O to total volume of 20 μl. The PCR used the following conditions: one cycle at 95℃ for 30 s, then 40 cycles of 95℃for 5 s, 60℃ for 34 s followed by 1 cycle of 95℃ 15 s, 60℃ for 1 min and 95℃for 15 s. 2.8 Statistical analysis

Journal Pre-proof All data presented were the means with standard errors (S.E.) of three independent replicates. Significant differences between the groups were determined using one-way ANOVA and Tukey’s multiple comparison test. Figure 4 and 5 present the results of an independent t-test. Differences were considered significant at P<0.05. Statistical computations were performed with IBM SPSS Statistics v22 (Holzer et al., 1992). 3 Result 3.1 Effects of inbreeding on the growth traits and survival rate of E. carinicauda Effects of inbreeding on the growth traits of E. carinicauda are shown in Table 2. The body weight

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of the control population was significantly higher than that of the EC5 inbred line of F7, F8, F9, F10 and

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F11 at four growth stages (P<0.05). The body length of the control population was significantly higher

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than that of the EC5 inbred line of F8, F10 and F11 at four growth stages (P<0.05). The body weight and body length of F7 were significantly higher than those of F10 and F11 at all growth stages (P<0.05), and

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no statistically significant differences existed between F10 and F11 were found at all growth stages.

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The survival rate of the control population was higher than all generations in the EC5 inbred line. However, no statistically significant differences were found between the F7, F8 generation and the control

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population at all growth stages except for 60 day growth stage. Meanwhile, the survival rate of the control population was significantly higher than that of the F9, F10 and F11 at the 100 and 120 days growth stage

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(P<0.05). There were no statistically significant differences between F 10 and F11 in the EC5 inbred line at all growth stages.

As shown in Table 3, the estimate d average inbreeding depression coefficient of body weight, body length and survival rate ranged from -13.25% to -29.92%, -4.50% to -11.95% and -8.36% to -32.83%, respectively, per 10% increase of inbreeding coefficient of F at the 120 day growth stage. 3.2 The genetic features of F7-F11 detected with EST-SSR loci A total of 46 EST-SSR loci were used to detect the dynamic change of genetic characteristics during the E. carinicauda inbreeding process in the F7 to F11 generations of the EC5 inbred line. The results showed that 16 of the EST-SSR loci (34.78%, 16/46) became genetically homozygous in the F11 of EC5 inbred line. The number of alleles gradually decreased with the increase of inbreeding generations, same as observed heterozygosity (Ho), expected heterozygosity (He) and polymorphism information content (PIC) in Table 4. 3.3 Effect of inbreeding on the immune responses and antioxidant status of E. carinicauda

Journal Pre-proof Effects of inbreeding on the immune parameters and antioxidant status of E. carinicauda are shown in Fig. 2. The total haemocyte count, antibacterial activity, PO activity and HEM concentration showed a decreasing trend as the inbreeding level increased. Inbreeding had no remarkable effect on AKP and LZM activities. The total haemocyte count, antibacterial activity, PO activity and HEM concentration in the F9-F11 generations were significantly lower than those in the control population (P<0.05). The total haemocyte count, PO activity and HEM concentration in the F9, F10 and F11 generations were lower than those in the F7 and F8 generations (P<0.05). The SOD activities in the F7-F11 generations were significantly lower than that in the control

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population (P<0.05), but there were no significant difference in SOD activity among the five generations.

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The CAT activities in the F10 and F11 generations were significantly lower than that in the control

in the F10 and F11 generations (P<0.05).

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population (P<0.05), and the CAT activities in the F7-F9 generations were significantly higher than that

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3.4 Cumulative mortalities of shrimp after injection with V. parahaemolyticus

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The cumulative mortalities of shrimp in the control population and the F9 generation after injection with 107 cfu ml-1 V. parahaemolyticus are shown in Fig. 3. The E. carinicauda suffered a peak of deaths

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during 24-48 h. The mortality of shrimp in the F9 generation was 3.33% at 12 h, whereas no shrimp in the control population died at this time under the same condition. In the F9 generation, the mortality

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quickly increased to 23.33% at 24 h, while there was only 13.33% in the control population. Afterwards, 73.33% of the shrimp in the F9 generation died at 72 h, while only 56.67% death of shrimp in the control population. The mortalities of shrimp in the F9 generation during 12-72 h was significantly higher than that in the control population (P<0.05). 3.5 Variation of the total haemocyte count, HEM concentration and antibacterial activity after infection with V. parahaemolyticus The total haemocyte count, HEM concentration and antibacterial activity in the F9 generation and the control population after injection with 107 cfu ml-1 V. parahaemolyticus are shown in Fig. 4. The total haemocyte count in the F9 generation and the control population initially decreased, reaching its minimum at 6 h, respectively. Afterwards, the total haemocyte count gradually increased to the normal level in the F9 generation and the control population at 48 h. In the F9 generation, the shrimp had a significantly higher total haemocyte count than that in the control population at 6 and 48 h (P<0.05), although total haemocyte count in the F9 generation was lower at the start of the experiment.

Journal Pre-proof The HEM concentration in the F9 generation and the control population decreased to the minimum at 6 h, respectively, meanwhile the HEM concentration in the F9 generation and the control population recovered to the initial level at 48 h, while it was significantly higher in the control population than that in the F9 generation at 48 h (P<0.05). The antibacterial activities in the F9 generation and the control population initially increased after V. parahaemolyticus injection, reaching their peak at 6 h, respectively, and the antibacterial activity in the F9 generation was significantly lower than that in the control population at 6 h after infection with V. parahaemolyticus (P<0.05).

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3.6 Variation of immune enzyme activities after infection with V. parahaemolyticus

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The LZM, AKP and PO activities in the F9 generation and the control population after injection with

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107 cfu ml-1 V. parahaemolyticus are shown in Fig. 5. The shrimp in the F9 generation had significantly lower LZM activity than that in the control population at 6 h and 48 h after infection with V.

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parahaemolyticus (P<0.05). Additionally, the shrimp in the F9 generation had significantly lower AKP

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and PO activities than that in the control population after infection with V. parahaemolyticus at 6 h (P<0.05), whereas, there was no significant difference between the F9 generation and the control

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population regarding AKP and PO activities after infection with V. parahaemolyticus. at 48 h 3.7 Expression profiles of immune-associated genes in haemocytes and hepatopancreas of shrimp

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after infection with V. parahaemolyticus

The relative mRNA expression of immune-associated genes in haemocytes and hepatopancreas of shrimp in the F9 generation and the control population after infection with V. parahaemolyticus are shown in Fig. 6. As shown in Fig. 6A, the mRNA expression of Crustin, LZM, Tollip, ECSIT, Toll and STAT were significantly downregulated in the haemocytes of the F9 generation compared to the control population after infection with V. parahaemolyticus at 6 h (P<0.05). Meanwhile, there were no significant differences between the F9 generation and the control population in the relative mRNA expression of ALF and SOCS in haemocytes at 6 h after infection with V. parahaemolyticus. The mRNA expression of Crustin, LZM and Tollip were significantly downregulated in the haemocytes of the F9 generation compared to the control population after infection with V. parahaemolyticus at 48 h (P<0.05). Meanwhile, there were no significant differences between the F9 generation and the control population in the relative mRNA expression of ALF, ECSIT, Toll, STAT and SOCS in haemocytes at 48 h after infection with V. parahaemolyticus.

Journal Pre-proof As shown in Fig. 6B, the mRNA expression of ALF, LZM, Tollip, ECSIT, Toll and STAT were significantly downregulated in the hepatopancreas of the F9 generation compared to the control population after infection with V. parahaemolyticus at 6 h (P<0.05). Meanwhile, there were no significant differences between the F9 generation and the control population in the relative mRNA expression of Crustin and SOCS in the hepatopancreas after infection with V. parahaemolyticus at 48 h. The mRNA expression of ALF, Crustin, LZM, Tollip, ECSIT, Toll and STAT were significantly downregulated in the hepatopancreas of the F9 generation compared to the control population after infection with V. parahaemolyticus at 6 h (P<0.05). Meanwhile, there was no significant difference

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hepatopancreas after infection with V. parahaemolyticus at 48 h.

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between the F9 generation and the control population in the relative mRNA expression of SOCS in the

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4 Discussion

Normally, in outbred species inbreeding usually leads to reductions in the mean for fitness

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performance, termed inbreeding depression. Not all species showed inbreeding depression for all

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characters studied, but virtually showed inbreeding depression for most characters (Frankham, 2005). The previous studies have suggested that inbreeding affect the physiological status of invertebrates

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(Wang et al., 2017), and other studies have shown that inbreeding could weaken immune functions and cause oxidative stress (Luo et al., 2014; Ren et al., 2017). However, there was no report on changes in

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the immune and antioxidant defense systems of shrimps, particularly with respect to V. parahaemolyticus tolerance. Therefore, we aimed to evaluate the effects of inbreeding on growth-related traits, the immune responses and antioxidant defense systems, and V. parahaemolyticus tolerance in E. carinicauda, then collect important information for understanding inbreeding depression. We selected only one population in each generation to compare the immune and antioxidant defense systems with the control population for the reliability of the inbreeding coefficient of inbred population and to avoid the confusion of the genetic background. The crustaceans lack an adaptive immune system and rely entirely on an efficient innate immune system to defend themselves from pathogen invasions (Jayanthi et al., 2018; Zhou et al., 2018). The invertebrate innate immune system comprises cellular and humoral responses, antibacterial peptides, phenoloxidase cascade and other important components of defense (Malagoli et al., 2017). Haemocytes play essential roles in the innate immune system of crustaceans, and the total haemocyte count in the haemolymph has been considered as a functional indicator of immune capability (Zhou et al., 2017). The

Journal Pre-proof antibacterial activity can reflect humoral responses due to bacterial killing, and the shrimp could resist foreign pathogens by improving the antibacterial activity (Jiang et al., 2015). The haemocyanin is a novel important type of non-specific innate immune defense molecule, especially participating in multiple roles of immune defense in shrimp (Zhang et al., 2009). In this study, the total haemocyte count, antibacterial activity and haemocyanin concentration in the F9, F10 and F11 generation were significantly lower than those in the control population of E. carinicauda (P<0.05). Ren et al (2017) also found that inbreeding could affect antibacterial activity in the haemolymph of crabs (Ren et al., 2017). As a non-self-recognition system, the prophenoloxidase (proPO) system could participate in the

of

innate immune responses of invertebrate through accompanying with the cellular responses via hemocyte

ro

attraction and inducing melanization, phagocytosis, cytotoxic reactant production, and the formation of

-p

nodules and capsules (Ananda et al., 2017). Numerous studies have found that PO plays a critical function in invertebrate immune defense (Sritunyalucksana et al., 2000). Jiang et al (2017) believed that PO was

re

a fundamental immune and physiological factor in Apostichopus japonicus (Jiang et al., 2017), Fagutao

lP

et al (2017) found that PO not only regulate other immune-related genes and defend against pathogenic microorganisms but also maintain normal functioning, and is thus essential for survival in Marsupenaeus

na

japonicus (Fagutao et al., 2010). Our result showed that PO activity in F9, F10 and F11 generations were significantly lower than that in the control population (P<0.05). These results indicated that inbreeding

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may affect the function of the proPO system even the capability of immune defense in E. carinicauda. Aquatic animals are susceptible to oxidative stress, which has an efficient antioxidant defense system to prevent oxidative stress and maintain a balanced cellular redox state (Cardona et al., 2017). The antioxidant defense system consists of a cascade of enzymes (including SOD and CAT) and non‐ enzymatic small antioxidant molecules (Wang et al., 2009; Xu et al., 2017). The SOD protects living cells against oxidative stress from superoxide radicals, but in invertebrates, SOD also has a role in the innate immune system. Mai et al (2014) found that SOD have important antibacterial and antiviral function in M. japonicus (Mai et al., 2014). In this study, SOD activities in the F7~F11 generations were significantly lower than that in the control population, and the CAT activities in the F10 and F11 generations were significantly lower than that in the control population (P<0.05). Overall, our data suggests that the immune and antioxidant defense systems of the inbred E. carinicauda (with inbreeding coefficient: F=0.859, F=0.886 and F=0.908) was less effective compared to the control population (with F=0.25), except AKP, LZM and CAT activities. The previous studies have

Journal Pre-proof shown that inbreeding has a significant effect on the immunity to parasitic infection significantly in a species. The inbred hosts (F=0.5) have a weaker parasite resistance than the control breeding regime in P. reticulata, and inbred fish were slower in purging their gyrodactylid infections, suggesting that inbreeding reduces the immunocompetence of guppies and increases their susceptibility to gyrodactylid infection (Smallbone et al., 2016). Moreover, the higher inbreeding coefficient (F=0.908) significantly reduced resistance of D. melanogaster to both the thuringiensin toxin and live Serratia marcescens, which suggested that individuals with higher inbreeding coefficient have reduced parasite resistance (Spielman et al., 2004). Furthermore, inbreeding compromises immune defense in invertebrate, Rantala et al (2007

of

and 2011) found that inbreeding significantly reduced encapsulation response against nylon

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monofilament in female Epirrita autumnata (Rantala et al., 2007) and inbreeding reduced the realized

-p

immune response in Tenebrio molitor (Rantala et al., 2011). Ren et al (2017) found that a high level of inbreeding could severely reduce the immune responses and antioxidant status of P. trituberculatus (Ren

re

et al., 2017). Therefore, we compared the immune response of the F9 generation and the control

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population infected with V. parahaemolyticus which is known to be caused acute hepatopancreatic necrosis disease by investigating the immune parameters in our previous study.

na

Numerous pathogens threaten the survival of organisms, and that can cause an outbreak of diseases in the vertebrates and invertebrates (Zhang et al., 2019). As a member of invertebrate, E. carinicauda is

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usually infected by several pathogens which causes enormous economic losses in aquaculture production (Duan et al., 2014; Fei et al., 2016). The previous studies have shown that V. parahaemolyticus infection cause acute hepatopancreatic necrosis disease and thus leads to high mortality rate in cultured E. carinicauda (Ge et al., 2017). We have confirmed the potential disease-resistance indicators to assess the physiological status and potential disease-resistance capability of E. carinicauda when infected with V. parahaemolyticus in our previous study (Ge et al., 2018). In this study, we compared the physiological status and disease-resistance capability of the F9 generation of EC5 inbred family with the control population using these potential disease-resistant indicators including the cumulative mortality of E. carinicauda during 72 h after injection of V. parahaemolyticus, the total haemocyte count, HEM concentration, antibacterial activity, and the activities of four immune enzymes (LZM, AKP and PO) in cell-free haemolymph, and the expression of eight immune related genes (ALF, Crustin, LZM, Tollip, ECSIT, Toll, STAT and SOCS) in haemocytes and hepatopancreas of E. carinicauda. The disease resistance of shrimp is always measured by the cumulative mortality or survival rate

Journal Pre-proof after pathogens infection (Tepaamorndech et al., 2018). In this study, the cumulative mortality of the F9 generation (73.33%) was significantly higher than that of the control population (56.67%) 72 h after infected with V. parahaemolyticus. In crustaceans, an important immune defense reaction of haemocytes is phagocytosis, and haemocyte count reduction is often caused by pathogenic infection (Lee et al., 2002). For example, depletion of haemocytes was reported in the tiger shrimp Penaeus monodon when it was injected with a Vibrio anguillarum after 6 h (Braak et al., 2002), and a decreased haemocyte count was also observed in the white shrimp Litopenaeus vannamei injected with Vibrio alginolyticus after 6 h (Li et al., 2010). In

of

this study, the total haemocyte count decreased in the F9 generation and the control population injected

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with V. parahaemolyticus at 6 h and then returned to the normal at 48 h. The lowered haemocyte count

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in the F9 generation could mean that animals had lower resistance than that in the control population. A similar study reported in P. trituberculatus that the total haemocyte count in resistant crabs was lower

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the more resistant crabs (Fu et al., 2016).

re

than that in normal crabs, which may be attributed to the haemocytes playing a more important role in

Haemocyanin (HEM), the multifunctional glycoprotein in the haemolymph of invertebrates, was

na

found to play important roles in shrimp defense response to pathogens (Qin et al., 2018; Zhang et al., 2004). Haemocyanin concentration decreased by approximately 33.3% in Pacific white shrimp (L.

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vannamei) after injection with Taura syndrome virus (TSV) (Song et al., 2003). In this study, the HEM concentration decreased in the F9 generation and the control population injected with V. parahaemolyticus after 6 h. Moreover, the HEM concentration in the F9 generation was significantly lower than in the control population 48 h after injection. Similar study found in L. vannamei that the haemocyanin concentration decreased to the lowest level at 3 h and then recovered to the initial value after 48 h after injection with Vibrio harveyi, meanwhile, the haemocyanin concentration of the resistant shrimp was significantly higher than that of normal shrimp during 24-120 h after injection with Vibrio harveyi (Huang et al., 2013). The immune enzymes play an important role in the control of systemic bacterial infections in the shrimps. Chen et al (2009) reported that the LZM activity of P. trituberculatus was induced to increase after V. alginolyticus injection (Chen et al., 2009). Mu et al (2012) reported that the AKP activity in the high V. alginolyticus-resistant P. trituberculatus family was higher than that in the no-selected crab (Mu et al., 2012). In this study, the activities of immune enzymes, such as LZM, and AKP, increased in the

Journal Pre-proof cell-free haemolymph of shrimp following V. parahaemolyticus infection. Moreover, the LZM and AKP activities of the F9 generation was significantly lower than those of the control population at the early stage (6 h) after V. parahaemolyticus infection. Preliminary data in V. parahaemolyticus infection studies showed that genes of Toll, IMD and JAK/STAT pathways and their downstream antimicrobial peptides (AMPs), such as antilipopolysaccharide factor, crustin and lysozyme, are overexpressed in the hepatopancreas which is one of the major AHPND targets (Maralit et al., 2018). Ge et al (2018) found that the early stage (6 h) and the late stage (48 h) were the critical period to compared the resistant to V. parahaemolyticus infection,

of

and that the expression of ALF, LZM, ECSIT and Tollip was up-regulated in the disease-resistance

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capability of shrimp when infected with V. parahaemolyticus (Ge et al., 2018). In this study, we used the

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eight representative immune-related genes as the potential disease-resistant indicators to evaluate the disease-resistant capability of the control population and the F9 population. The expressions of Crustin,

re

LZM and Tollip were more downregulated in haemocytes of the F9 generation than that of the control

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population, and the expressions of ALF, LZM, Tollip, ECSIT, Toll and STAT were downregulated in hepatopancreas of the F9 generation compared to the control population.

na

Inbreeding is unavoidable in closed population if not managed properly and can cause inbreeding depression in commercial traits due to the increased expression of recessive deleterious alleles. This

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study showed that inbreeding effected the growth-related traits, antioxidant defense and immune system of E. carinicauda. The genetic diversity and the physiological status of E. carinicauda gradually declined with an increase in inbreeding generations. In summary, this study provides a specific degree of inbreeding depression with different levels of inbreeding in the different generations. Acknowledgments

This project was financially supported by the earmarked fund for National Key R & D Program of China (2018YFD0901302), Modern Agro-industry Technology Research System (No. CARS-48), The Program of Shandong Leading Talent (No. LNJY2015002), National Natural Science Foundation of China (No. 31472275) and Qingdao Industrial Development Program Science and Technology Benefit Special Project (17-3-3-62-nsh), Central Public-interest Scientific Institution Basal Research Fund, CAFS (NO.2019ZD0603).

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Journal Pre-proof Table 1. Primers used in the experiment Primer sequence (5′-3′)

18s-F

TATACGCTAGTGGAGCTGGAA

18s-R

GGGGAGGTAGTGACGAAAAAT

ALF-F

GGTTCATCCTGCTGTCCTG

ALF-R

GAGCCCCATAATCCAACAAT

Crustin-F

CCCAACGAGATCGAAGTGAT

Crustin-R

GCGTGAGTGGAGTGTAGCAA

LZM-F

TGAACTTGCTACTGTGCTGGA

LZM-R

GTTGATGGCTTCCGTGTTG

Tollip-F

CGTGTAGGCCGTATTCAGGT

Tollip-R

GAGGATTTTTGGCTCCGTTA

ECSIT-F

TCTTGCTCGTGTACGCTACG

ECSIT-R

AAAGCGCGAAGAAATTGATG

Toll-F

GACTATTTCCCTCTGGCTCG

Toll-R

TGAAGGTTAGGTCATGGTGG

STAT-F

TGTTGGTTGGTGGCAAGTTA

STAT-R

CCGGACTGTTCACCTTTGTT

SOCS-F

GAGGGCAAGACATTCAGTCC

SOCS-R

GTAAGCCACTGGAGCTCGTC

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Primer

Journal Pre-proof Table 2 Comparative analysis of growth traits and survival rate between EC5 inbred line and the control population Trait

Growth stages

Group

60 days

80 days

a

100 days

120 days

a

1.73±0.57a

Body weight

Control population

0.60±0.27

(g)

F7

0.46±0.19b

0.86±0.22b

1.18±0.32b

1.55±0.52b

F8

0.41±0.15c

0.77±0.24bc

1.15±0.33b

1.41±0.35bc

F9

0.43±0.17c

0.82±0.19bc

1.16±0.22b

1.47±0.28bc

F10

0.41±0.15c

0.66±0.19cd

0.96±0.31bc

1.28±0.25c

F11

0.39±0.13c

0.58±0.17d

0.85±0.23c

1.26±0.22c

Body length

Control population

33.70±4.58a

40.69±4.43a

45.05±5.84a

48.74±5.83a

(mm)

F7

32.26±3.47ab

38.93±3.97ab

42.77±4.26b

47.02±5.21ab

F8

30.87±3.58c

36.73±3.53b

42.56±4.73b

45.28±3.64bc

F9

31.74±3.64bc

38.17±3.28ab

42.88±4.47b

46.55±3.88b

c

43.58±4.02c

c

1.44±0.51

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1.03±0.32

a

F10

29.77±3.84

F11

29.11±3.60c

34.06±3.37c

38.63±4.10c

43.99±3.69c

Survival rate

Control population

95.00±1.73a

82.00±2.00a

68.00±2.83a

55.00±3.32a

(%)

EC5-F7

88.33±2.36b

77.78±9.07a

62.96±2.62ab

48.15±2.62ab

EC5-F8

90.21±3.54b

75.68±7.14a

66.25±3.78ab

51.25±6.78ab

EC5-F9

91.67±2.36ab

78.79±2.14a

60.61±2.14b

43.94±2.14b

EC5-F10

90.00±2.00b

66.00±2.00b

50.00±4.47c

39.00±3.32c

EC5-F11

87.00±1.73b

64.00±2.83b

48.00±2.83c

40.00±4.47c

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33.89±3.60

c

39.63±4.55

Journal Pre-proof Table 3 Inbreeding depression on growth-related traits of five levels of inbreeding at 120 days stage IDC (Body weight)

IDC (Body length)

IDC (Survival rate)

F7

-13.25%

-4.50%

-15.87%

F8

-22.67%

-8.70%

-8.36%

F9

-17.50%

-5.23%

-23.41%

F10

-29.36%

-11.95%

-32.83%

F11

-29.92%

-10.73%

-30.04%

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Journal Pre-proof Table 4 Genetic dynamic characteristics during the E. carinicauda inbreeding process from F7 to F11 detected with 46 microsatellite loci Na

Ho

He

PIC

Control population

6.435

0.545

0.652

0.667

F7

2.261

0.258

0.337

0.270

F8

2.391

0.278

0.350

0.234

F9

2.217

0.261

0.296

0.214

F10

1.913

0.230

0.237

0.174

F11

1.870

0.203

0.231

0.151

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Generation

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Fig. 1. The inbred population establishment process for E. carinicauda

0.6 a

a

CP

F7

a

a

b

b

b

F8 F9 generation

b

c

c

4 2 0 F8 F9 generation

a

a

a

a

lP

0.20 0.10

a

b

6 4 2

b

F7

8 7 6 5 4 3 2 1 0

a

F7

a

F8 F9 generation

b

a

F10

F11

a

a

F7

F8 F9 F10 generation

F11

b

b

F8 F9 generation

F10

a

0.4

a

a

b c

0.3

b

0.2 0.1 0 CP

F7

F8 F9 F10 generation

F11

20 b

0 CP

0

F10 F11

F11

CAT activity (U/ml)

8

F7 F8 F9 generation

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CP

SOD activity/U

a

0.30

0.00

b

0.5

a

PO activity/U

0.40

c

0.1

CP

na

LZM activity (U/ml)

0.50

F10 F11

b

0.2

-p

F7

b

0.3

re

CP

b

CP

AKP activity (U/ml)

HEM concentration (mg/ml)

a

a

0.4

F10 F11

8 6

0.5

of

a

ro

7 6 5 4 3 2 1 0

Antibacterial activity

Total haemocyte count (×107/mL)

Journal Pre-proof

a 16

a

a

a b

12

b

8 4 0 CP

F7

F8 F9 generation

F10 F11

Fig. 2 The immune parameters of control population (CP) and different inbreeding generations in E. carinicauda

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Cumulative mortality (%)

90

CP

80

*

F9

70 *

60 50 40 *

30 20 10 0 6

12

24 48 Hours after infection (hpi)

72

of

Fig. 3. The cumulative mortality of E. carinicauda in the control population (CP) and the F9 generation after injection with 107 cfu/ml V. parahaemolyticus.

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Asterisk indicates the significant difference between the control population and the F9 generation at

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same time. (P<0.05)

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6 * 4

* *

2

12

1.6

CP F9

10

Antibacterial activity/Ua

CP F9

HEM concentration (mmol/L)

Total haemocyte count (×107/ml)

8

8 6

*

*

4 2 0

0

1.2

*

0.8 *

0.4 0

0

0 6 48 Hours after infection (hpi)

CP F9

6

48

0

Hours after infection (hpi)

6

48

Hours after infection (hpi)

Fig. 4. The total haemocyte count, HEM concentration and antibacterial activity in cell-free haemolymph

0.2

8 6 4

*

2 0

0

0 6 48 Hours after infection (hpi)

lP

0 6 48 Hours after infection (hpi)

PO activity (mmol/L)

0.4

*

10

0.6

ro

*

CP F9

-p

0.6

12

re

CP F9

AKP activity (U/ml)

LZM activity (U/ml)

0.8

of

of control population (CP) and F9 generation after injection with V. parahaemolyticus.

CP F9

0.5 0.4

*

0.3 *

0.2 0.1 0

0 6 48 Hours after infection (hpi)

Fig. 5. The immune enzymes activities in cell-free haemolymph of control population (CP) and F9

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generation after injection with V. parahaemolyticus.

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Relative mRNA expression

CP-6

a

8

F9-6

7

CP-48

6

F9-48

2

a

a

4 3

a

a

5

b

b a a

a

a

c

c

b

a

b

c

a

b

c

c

c

c

bb

cc aa

d

1

bb

ALF

Crustin

LZM

of

0 Tollip

ECSIT

CP-6 F9-6

-p

12

re

10

6

lP

8

4

b

cc

d

c

ALF

Crustin

LZM

c

F9-48 a b b a

b

b

c

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0

a

a

b

CP-48

a

a

a

na

Relative mRNA expression

SOCS

a

14

2

STAT

ro

16

B

Toll

cc

cc

bb

b aaaa

c Tollip

ECSIT

Toll

STAT

SOCS

Fig. 6 The relative mRNA expression of immune-associated genes in haemocytes (A) and hepatopancreas (B) of the control population (CP) and F9 generation (F9) at 6 hpi and 48 hpi after infection with V. parahaemolyticus.

Journal Pre-proof Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Ethical approval and informed consent statement The collection and handling of the animals in this study was approved by the Animal Care and Use Committee at the Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, and all experimental animal protocols were carried out in accordance with national and institutional guidelines for the care and use of laboratory animals at the Yellow Sea Fisheries Research Institute,

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Chinese Academy of Fishery Sciences.

Journal Pre-proof We newly established an inbred strain of Exopalaemon carinicauda, restricting breeding and keeping to F11. Inbreeding reduced the immune responses and antioxidant status of Exopalaemon carinicauda. Higher level inbreeding generation has a weaker disease-resistant capability when infected

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with Vibrio parahaemolyticus.