Abstract

Simple Summary

   Identification of differentially expressed genes (DEGs) between
   disease-resistant and -susceptible animals is a method to screen genes
   contributing to anti-disease. Previously, we found that there were few
   genes differentially expressed in hemocytes between V.
   parahaemolyticus-resistant and -susceptible shrimp. The present study
   hypothesized that these shrimp had different immune responses against
   pathogen infection and tested it using comparative transcriptome
   analysis.

Abstract

   Vibrio parahaemolyticus is one of the main causative agents leading to
   acute hepatopancreatic necrosis disease, the severe bacterial disease
   that occurs during shrimp aquaculture. Hemocytes play important roles
   during Vibrio infection. Previously, we found that there were few
   differentially expressed genes (DEGs) between hemocytes from V.
   parahaemolyticus-resistant and -susceptible shrimp before infection. We
   considered that there should be different immune responses between them
   after a pathogen infection. Here, the transcriptome data of hemocytes
   from V. parahaemolyticus-resistant and -susceptible shrimp before and
   after a pathogen infection were compared. The results showed that there
   were 157 DEGs responsive to infection in V. parahaemolyticus-resistant
   shrimp, while 33 DEGs in V. parahaemolyticus-susceptible shrimp. DEGs
   in V. parahaemolyticus-resistant shrimp were mainly related to immune
   and glycolytic processes, while those in V.
   parahaemolyticus-susceptible shrimp were mainly related to metabolism,
   with only two DEGs in common. A further analysis of genes involved in
   glucose metabolism revealed that GLUT2, HK, FBP, and PCK1 were lowly
   expressed while PC were highly expressed in hemocytes of the V.
   parahaemolyticus-resistant shrimp, indicating that glucose metabolism
   in shrimp hemocytes was related to a V. parahaemolyticus infection.
   After the knockdown of PC, the expression of genes in Toll and IMD
   signaling pathways were down-regulated, indicating that glucose
   metabolism might function through regulating host immunity during V.
   parahaemolyticus infection. The results suggest that the immune
   responses between V. parahaemolyticus-resistant and -susceptible shrimp
   were apparently different, which probably contribute to their different
   V. parahaemolyticus resistance abilities.

   Keywords: penaeid shrimp, hemocytes, Vibrio parahaemolyticus,
   glycolysis, immunity

1. Introduction

   The frequent outbreak of shrimp diseases leads to a large amount of
   production losses in global shrimp culture, which seriously affects its
   economic value. The sustainable development of shrimp culture is
   closely related to the control of shrimp diseases. In addition to the
   pathogen, the disease resistance of the host is also an important
   factor in disease control [[28]1,[29]2]. The acute hepatopancreatic
   necrosis disease (AHPND), a disease caused by bacteria [[30]3], can
   lead to serious mortality of cultured shrimp and cause substantial
   economic losses to the shrimp culture industry. Vibrio
   parahaemolyticus, one of the main pathogens of AHPND, carries an
   extrachromosomal plasmid that encodes a binary toxin PirA^Vp and
   PirB^Vp, homologous to the Photorhabdus insect-related (Pir) toxins
   [[31]4]. These toxins have been identified as the primary contributors
   to AHPND toxicity [[32]5].

   Understanding the mechanisms of disease resistance is an important
   theoretical basis to solve the problem of shrimp disease. At present, a
   large number of genes or regulatory processes related to shrimp disease
   resistance have been reported. In Litopenaeus vannamei, a comparison of
   gene expression in cephalothoraxes of shrimp between resistant and
   susceptible families against VP[AHPND] revealed that immune response
   and energy metabolism might contribute to the resistance of shrimp
   against VP[AHPND] [[33]6]. A comparative transcriptome of gills from
   shrimp with varying resistances against VP[AHPND] revealed that some
   genes participating in endocytosis, protein synthesis and immune
   responses were related to Vibrio-resistance of L. vannamei [[34]7].
   Comparative transcriptome analysis of the hepatopancreas from shrimp in
   two families with different resistances to VP[AHPND] revealed that some
   immune-related genes were highly expressed in shrimp from the resistant
   family while metabolic-related genes were highly expressed in shrimp
   from the susceptible family [[35]8]. An integrated analysis of the
   hepatopancreas between transcriptome and metabolome in L. vannamei
   suggested that the relative prosperous metabolic state in
   Vp[AHPND]-susceptible shrimp might benefit bacterial proliferation,
   while the activation of NF-κB and cAMP pathways induced by arachidonic
   acid metabolism in Vp[AHPND]-resistant shrimp after infection might
   contribute to host resistance ability [[36]9]. These studies revealed
   that metabolism and immunity in shrimp jointly participated in the
   defense process against pathogen infection.

   Hemocytes play an important role in the immune defense system of
   shrimp, not only as the executors of the cellular immune process but
   also for the synthesis and release of a variety of immune factors,
   providing a material basis for humoral immunity [[37]10]. Previously,
   we established a method to extract hemocytes from uninfected shrimp
   with different resistances to V. parahaemolyticus. Transcriptome
   analysis found that several genes were differentially expressed in
   hemocytes between V. parahaemolyticus-resistant and -susceptible shrimp
   at the background level [[38]11], leading to a speculation that the
   differences in shrimp resistance ability against V. parahaemolyticus
   infection might be attributed to the variations in their immune
   responses. In the present study, shrimp from the same batch as
   previously reported [[39]11] were used to analyze whether there were
   different immune responses between hemocytes from V.
   parahaemolyticus-resistant and -susceptible shrimp after pathogen
   infection. Hemocytes were collected from shrimp before infection and at
   three hours post-infection, and comparative transcriptome analysis was
   conducted to identify genes related to immune responses in V.
   parahaemolyticus-resistant and -susceptible shrimp, respectively. The
   data showed apparently different immune responses between them, which
   might contribute to the variation in their resistance abilities against
   V. parahaemolyticus infection.

2. Materials and Methods

2.1. Experimental Animals

   The experimental animals Litopenaeus vannamei that were from the same
   batch of shrimp, population 2, in our previously study [[40]11], were
   acquired from Rizhao breeding farm in Shandong and cultured in aerated
   seawater at 25 °C. Shrimp used in the experiment weighed 19.5 ± 1 g.
   Before the experiments, shrimp were confirmed to be free of Vibrio
   pathogens as described in our previous study [[41]11].

2.2. Experiment Design and Sampling

   A total of 140 individuals were used for immune challenge. The
   experiment design workflow was illustrated in [42]Figure 1. Each
   individual was labeled with vibration-induced emission (VIE) through
   injecting into the third segment and continued to be temporarily
   cultured in the aerated seawater at 25 ± 1 °C for seven days and fed
   twice a day. Two groups, group 0 h (G0h) and group 3 h (G3h), with 70
   individuals each, were set. For G0h, 300 μL hemolymph was extracted
   from each individual with a sterile syringe containing equal volume of
   ice-cold anticoagulant containing 27 mM trisodium citrate, 336 mM
   sodium, 115 mM glucose, and 9 mM EDTA·Na[2]·2H[2]O, at pH 7.4. Then,
   the shrimp were infected with V. parahaemolyticus under immersion
   infection with a final concentration of 5 × 10^6 CFU/mL. The infection
   experiments were performed using strains isolated from our laboratory
   [[43]12]. For G3h, the shrimp were directly infected under the same
   condition. At 3 h post-infection (hpi), 300 μL hemolymph was extracted
   from each individual and then put back for immersion infection. The
   hemolymph was centrifuged at 4 °C and at 1000× g for 10 min. The
   supernatant was removed, and the hemocytes were frozen in liquid
   nitrogen and stored at −80 °C. The mortality was recorded at 6, 9, 12,
   21, 24, 27, 30, 33, 36, 45, and 52 hpi. The hepatopancreases of
   moribund shrimp at each time point (D0h and D3h), shrimp before
   infection (0 hpi) and 3 h post-infection (3 hpi), and survival shrimp
   after 52 hpi (L0h and L3h) were collected for Vibrio load detection.

Figure 1.

   [44]Figure 1
   [45]Open in a new tab

   Workflow of the experimental design.

2.3. DNA Extraction and Bacteria Load Detection

   Genomic DNA was extracted from the above collected the hepatopancreas
   using the Genomic DNA Extraction Kit following the instructions
   (TIANGEN, Beijing, China). NanoDrop 2000 (Thermo Fisher Scientific,
   Waltham, MA, USA) was used to measure the concentration and purity of
   DNA, and 1% agarose gel electrophoresis was used to test the integrity
   of DNA. PirA^Vp copy numbers in extracted DNA were detected by TaqMan
   probe fluorescent quantitative PCR using Ependorf MasterCycler EP
   Realplex (Ependorff, Hamburg, Germany), and each assay was carried out
   in four replicates. Primers and procedures used for RT-qPCR are shown
   in [46]Table S1.

2.4. RNA Extraction and Transcriptome Sequencing

   According to the survival data and Vibrio loads, shrimp were divided
   into V. parahaemolyticus-resistant and -susceptible individuals.
   Hemocytes from V. parahaemolyticus-resistant and -susceptible shrimp in
   G0h and G3h groups were mixed, respectively. In G0h, the samples were
   named as L0h for V. parahaemolyticus-resistant shrimp and D0h for V.
   parahaemolyticus-susceptible shrimp. In G3h, the samples were named as
   L3h for V. parahaemolyticus-resistant shrimp and D3h for V.
   parahaemolyticus-susceptible shrimp. Each sample included three
   replicates. Total RNA was extracted from each sample with RNAiso Plus
   (TaKaRa, Kyoto, Japan) according to the manufacturer’s instructions.
   The concentration, purity, and integrity of the total RNA was measured
   by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and 1%
   agarose gel electrophoresis, respectively. About 1 μg total rRNA from
   each sample was pre-treated with gDNA Eraser (TaKaRa, Kyoto, Japan),
   and the mRNA was enriched with a conventional kit (New England Biolabs,
   Ipswich, MA, USA). The mRNA was fragmented and used as template to
   synthesize the first-strand cDNA using random hexamers. The
   second-strand cDNA was synthesized after adding dNTPs, RNaseH, and DNA
   polymerase I. The purified double-stranded cDNA was repaired at the
   end, and the ploy(A) was added to connect the Illumina sequencing
   adapter. Finally, the sequencing was performed using the Illumina
   HiSeq2500 platform at Guangzhou Gene Denovo Biotech Co., Ltd.
   (Guangzhou, China).

2.5. Reads Mapping and Annotation

   Considering the quality of the data, only reads with a mass fraction
   greater than 10% were used for further analysis. Clean reads were
   mapped to the reference genome [[47]13] using HISAT2.2.4 [[48]14]. The
   mapped reads were assembled with StringTie v1.3.1 [[49]15,[50]16] in a
   reference-based approach. The reconstructed transcripts were compared
   to the reference genome, and new genes were annotated functionally by
   comparison with nucleotide sequences (NTs), non-redundant protein
   sequences (NRs), Swiss-Prot, protein orthologous clusters (COG), and
   the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

2.6. Differential Expression and Enrichment Analysis

   The expression level of each transcript was determined using its FPKM
   (fragments per million mapped reads per thousand base transcripts)
   values. DEGs were analyzed using the edgeR software package
   ([51]http://www.rproject.org/, accessed on 13 June 2023) with a false
   discovery rate (FDR) of <0.05 and the absolute multiple change of ≥2.
   In addition, a correlation analysis among samples was performed to
   confirm the reliability of the data. GO function and KEGG pathway
   enrichment analysis of DEGs were carried out using the online OmicShare
   tool ([52]http://www.omicshare.com/tools, accessed on 15 June 2023).
   Statistical significance was confirmed with Q values of <0.05.

2.7. RNA Interference

   Primers with T7 promoter sequences were designed to amplify the DNA
   template of EGFP and LvPC for dsRNA synthesis. The PCR programs were
   performed according to the product size and primer annealing
   temperature ([53]Table S1). PCR products were detected by
   electrophoresis on a 1% agarose gel and purified with the MiniBEST DNA
   Fragment Purification Kit (TaKaRa, Kyoto, Japan). The dsRNA was
   synthesized using Transcriptaid T7 High Yield Transcription Kit (Thermo
   Fisher Scientific, Waltham, MA, USA) and purified using a mixture of
   phenol and chloroform. The concentration of dsRNA was checked using
   NanoDrop 2000, and the quality of dsRNA was detected using 1% agarose
   electrophoresis.

   After dosage optimization, 2 μg/g shrimp was used for RNA interference.
   Hemocytes of five shrimp in each group were collected as one sample at
   48 h after dsRNA interference. Three replicates were set for each
   treatment. Total RNA was extracted according to [54]Section 2.4.

2.8. Quantitative Real-Time PCR

   About 1 μg total RNA was used to synthesize the first strand of cDNA
   with the PrimeScript™ RT kit with gDNA Eraser (TaKaRa, Kyoto, Japan).
   The product was diluted 20 times using nuclease-free water. qRT-PCR was
   carried out with THUNDERBIRD^® SYBR^® qPCR Mix in Eppendorf
   Mastercycler ep realplex (Eppendorf, Hamburg, Germany) at a total
   volume of 10 μL (5 μL qPCR mixture, 1 μL diluted cDNA, 0.3 μL forward
   and reverse primers, and 3.4 μL nuclease-free water). The amplification
   program was set as follows: 95 °C for 2 min; 40 cycles of 95 °C for 15
   s, annealing temperature for 15 s and 72 °C for 30 s; and followed by a
   melting curve. Each sample was assessed in three technical replicates.
   In order to standardize expression levels, 18S rRNA was used as an
   internal control. The expression profile of each gene was calculated by
   2^−∆∆CT and shown with log2 fold change values. Primers used for the
   detection of genes in Toll and IMD pathways are listed in [55]Table S1.

2.9. Statistical Analysis

   All data were displayed in the form of mean ± S.E. and calculated using
   one-way analysis of variance (ANOVA) and Ducan’s Multiple Comparisons.
   Differences between comparisons were considered statistically
   significant at p < 0.05 and statistically highly significant at p <
   0.001.

3. Results

3.1. The Loads of V. parahaemolyticus in Hepatopancreas of Treated Shrimp

   The cumulative mortality rates for two shrimp groups G0h and G3h were
   24.2% and 22.9%, respectively ([56]Figure 2A). The standard curve
   equation for PirA^Vp was y = 3.0362x + 34.723, where x was the
   logarithm value of the Vibrio copy number per μL standard plasmid, and
   y was the Ct value. The correlation coefficient R^2 = 0.9263, which was
   a good linear relationship between the concentration of the standard
   sample and the Ct value. As shown in [57]Figure 2B, the loads of V.
   parahaemolyticus in hepatopancreas of shrimp in L0h, D0h, and D3h were
   151, 302, and 268 copy per ng hepatopancreatic DNA, respectively, while
   those in L3h, 0 hpi, and 3 hpi were very low, which were less than one
   copy per ng hepatopancreatic DNA. The data suggest that V.
   parahaemolyticus did not proliferate substantially at 3 hpi.

Figure 2.

   [58]Figure 2
   [59]Open in a new tab

   The cumulative mortality rates and the load of V. parahaemolyticus in
   the hepatopancreas of infected shrimp from two groups, G0h and G3h. (A)
   shows the accumulative mortality rates of shrimp in two groups. (B)
   shows the copy number of V. parahaemolyticus per ng hepatopancreatic
   DNA in different samples. L0h, the sample of V.
   parahaemolyticus-resistant shrimp in G0h. L3h, the sample of V.
   parahaemolyticus-resistant shrimp in G3h. D0h, the sample of V.
   parahaemolyticus-susceptible shrimp in G0h. D3h, the sample of V.
   parahaemolyticus-susceptible shrimp in G3h. 0 hpi, the sample of shrimp
   before V. parahaemolyticus infection. 3 hpi, the sample of shrimp at 3
   h post V. parahaemolyticus infection.

3.2. Differential Immune Responses of Hemocytes against Pathogen Infection
between V. parahaemolyticus-Resistant and -Susceptible Shrimp

   Differential analysis identified 24 differentially expressed genes
   (DEGs) between L0h and D0h, including 18 up-regulated DEGs and 6
   down-regulated DEGs in D0h, 157 DEGs between L0h and L3h, including 56
   up-regulated DEGs and 101 down-regulated DEGs in L3h, and 33 DEGs
   between D0h and D3h, including 10 up-regulated DEGs and 23
   down-regulated DEGs in D3h ([60]Figure 3A,B, [61]Table S2). Among the
   DEGs from two groups, only five genes were shared by at least two
   comparisons. Particularly, only two genes were shared by L0h vs. L3h
   comparison and D0h vs. D3h comparison, including aromatic-L-amino-acid
   decarboxylase-like gene and transmembrane protease serine 9-like gene
   ([62]Figure 3C, [63]Table S2), suggesting quite different immune
   responses of hemocytes against pathogen infection between V.
   parahaemolyticus-resistant and -susceptible shrimp.

Figure 3.

   [64]Figure 3
   [65]Open in a new tab

   Differentially expressed genes (DEGs) from L0h vs. D0h comparison, L0h
   vs. L3h comparison, and D0h vs. D3h comparison. A total of 212 DEGs,
   including 24 DEGs from L0h vs. D0h comparison, 157 DEGs from L0h vs.
   L3h comparison, and 33 DEGs from D0h vs. D3h comparison, were
   identified and displayed in the histogram (A), the heatmap (B), and the
   Venn diagram (C).

   Gene ontology (GO) analysis showed that DEGs from L0h vs. L3h
   comparison were mainly enriched in the biological processes including
   cellular processes, single organism processes, metabolic processes,
   biological processes, biological process regulation, and stimulus
   response processes. In the molecular function category, most DEGs were
   involved in binding and catalytic activity. Among cellular components,
   most DEGs were enriched in cells, cell parts, organelles, and
   membranes. DEGs from D0h vs. D3h comparison were mainly enriched in the
   biological processes including metabolic processes, single organism
   processes, biological regulation, biological process regulation, and
   multicellular biological processes. In the molecular function category,
   most DEGs were involved in binding and catalytic activity. Among
   cellular components, most DEGs were enriched in cells, cell parts,
   organelles, and membranes ([66]Figure 4). KEGG analysis showed that no
   DEGs from either comparison were significantly enriched in signaling
   pathways ([67]Figure 5).

Figure 4.

   [68]Figure 4
   [69]Open in a new tab

   Gene ontology (GO) enrichment analysis of DEGs from L0h vs. L3h
   comparison (A) and D0h vs. D3h comparison (B).

Figure 5.

   [70]Figure 5
   [71]Open in a new tab

   KEGG enrichment analysis of DEGs from L0h vs. L3h comparison (A) and
   D0h vs. D3h comparison (B).

3.3. The Glycolysis Process Showed Different Responses to Pathogen Infection
in Hemocytes between V. parahaemolyticus-Resistant and -Susceptible Shrimp

   Although no pathway was significantly enriched for DEGs by KEGG
   analysis, functional annotation showed that several genes in the
   glycolysis process had apparently different expression patterns against
   pathogen infection in hemocytes between V. parahaemolyticus-resistant
   and -susceptible shrimp. In hemocytes of V. parahaemolyticus-resistant
   shrimp, solute carrier family 2 promoting glucose transporter member
   1-like isomer X1 (Glut2), hexokinase type 2-like (HK),
   6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (FBP), and
   phosphoenolpyruvate carboxykinase 1 (PCK1) were all down-regulated
   after pathogen infection ([72]Figure 6A–D). In addition, the trehalose
   transporter (facilitated trehalose transporter, Tret1) was also
   down-regulated after pathogen infection in the hemocytes of V.
   parahaemolyticus-resistant shrimp ([73]Figure 6F). However, the
   expression profiles of these genes were not apparently affected by
   pathogen infection in the hemocytes of V. parahaemolyticus-susceptible
   shrimp. Furthermore, the expression level of pyruvate carboxylase (PC)
   was only down-regulated after pathogen infection in the hemocytes of V.
   parahaemolyticus-susceptible shrimp ([74]Figure 6E). The data suggested
   that the glycolysis process in hemocytes responded to pathogen
   infection differently between V. parahaemolyticus-resistant and
   -susceptible shrimp.

Figure 6.

   [75]Figure 6
   [76]Open in a new tab

   Expression profiles of several glucose metabolism-related genes in the
   hemocytes of V. parahaemolyticus-resistant and -susceptible shrimp.
   (A–F) show the expression of solute carrier family 2 promoting glucose
   transporter member 1-like isomer X1 (Glut2), hexokinase type 2-like
   (HK), 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (FBP),
   phosphoenolpyruvate carboxykinase 1 (PCK1), and facilitated trehalose
   transporter (Tret1), respectively. L0h, the sample of V.
   parahaemolyticus-resistant shrimp in G0h. L3h, the sample of V.
   parahaemolyticus-resistant shrimp in G3h. D0h, the sample of V.
   parahaemolyticus-susceptible shrimp in G0h. D3h, the sample of V.
   parahaemolyticus-susceptible shrimp in G3h. Significant differences
   between PC knockdown and control groups are shown with an asterisk at p
   < 0.05, two asterisks at p < 0.01, and three asterisks at p < 0.001.

3.4. Knockdown of PC Inhibited the Expression of Genes in the NF-κB Signaling
Pathway

   Previous study revealed that the down-regulation of PC could cause
   lactate accumulation, which then inhibited host immunity in mammals
   [[77]3,[78]15]. Therefore, PC was knocked down by RNAi, leading to 92%
   down-regulation of the gene ([79]Figure 7). After PC knockdown, several
   genes in the NF-κB pathway, including Toll1, Doral, Relish, Cactus,
   nuclear factor interleukin 3 (NFIL3), and ALF, were all down-regulated
   by 71%, 78%, 86%, 61%, 84%, and 91%, respectively ([80]Figure 7). The
   results suggested that the knockdown of PC led to the inhibition of the
   NF-κB signaling pathway in shrimp hemocytes.

Figure 7.

   [81]Figure 7
   [82]Open in a new tab

   Expression changes of several genes in the NF-κB signaling pathway in
   hemocytes after PC knockdown. Significant differences between PC
   knockdown and control groups are shown with three asterisks at p <
   0.001.

4. Discussion

   Shrimp usually have different resistance abilities against specific
   pathogens. In the present study, we found that although the main
   immune-related tissues, hemocytes, from shrimp with different
   resistances against V. parahaemolyticus have similar transcriptional
   profiles at the background level, it showed apparently different immune
   responses at the early infection stage. As the bacterial loads remained
   at a low level in all tested shrimp at 3 hpi, the differences in immune
   response should reflect the genetic variation among individuals rather
   than those caused by different proliferation speeds of Vibrio in
   shrimp. Therefore, different immune responses in the tested shrimp
   contributed to their distinct resistance against V. parahaemolyticus
   infection. However, it still needs to be verified in more groups of
   shrimps whether this phenomenon is generally accepted.

   In the V. parahaemolyticus-susceptible shrimp, the immune response
   genes in hemocytes were mainly related to metabolic processes. This was
   similar to our previous reports on the hepatopancreas of V.
   parahaemolyticus-susceptible shrimp, in which many metabolic processes
   were responsive to Vibrio infection [[83]8]. During infection, bacteria
   could manipulate host metabolism for their survival and replication. In
   the mice macrophages, Salmonella typhimurium promoted its intracellular
   replication through the up-regulation of host glycolysis and a decrease
   in host serine synthesis process, which would provide carbon sources
   for bacteria and increase the production of bacterial virulence factors
   [[84]17]. Host lipids can be hijacked by bacteria to meet their energy
   needs or to hide themselves from the host cell [[85]18]. Notably, the
   potential regulatory tandem of inflammatory response and amino acid
   metabolism, such as the biosynthesis of phenylalanine, tyrosine, and
   tryptophan and the metabolism of phenylalanine, has important functions
   in adaptive and innate immunity [[86]19]. In V.
   parahaemolyticus-susceptible shrimp, there were significantly fewer
   immune-related genes in hemocytes responsive to Vibrio infection when
   compared with those in V. parahaemolyticus-resistant shrimp. These data
   suggested that changes in metabolic processes in V.
   parahaemolyticus-susceptible shrimp might reduce the resistance ability
   of V. parahaemolyticus-susceptible shrimp to the pathogen. However, the
   underlying mechanism needs to be further investigated.

   In the hemocytes of V. parahaemolyticus-resistant shrimp, there were
   many more immune-related genes responsive to Vibrio infection. A
   comparative transcriptome analysis of the hepatopancreas from V.
   parahaemolyticus-resistant and -susceptible shrimp showed that, at the
   background level, the immune-related genes were highly expressed in V.
   parahaemolyticus-resistant shrimp and the metabolism-related genes were
   highly expressed in V. parahaemolyticus-susceptible shrimp, and the
   high expression profile was maintained after Vibrio infection [[87]8].
   Although there were few DEGs in hemocytes between V.
   parahaemolyticus-resistant and -susceptible shrimp at the background
   level, the immune responsive genes in hemocytes at 3 hpi exhibited
   similar characteristics as in the hepatopancreas. In the hemocytes of
   V. parahaemolyticus-resistant shrimp, some immune-related genes such as
   ankyrin repeat domain-containing protein, ficolin-1, venom protease,
   etc. were up-regulated after Vibrio infection. Previous studies have
   shown that these genes positively regulate host immunity
   [[88]20,[89]21,[90]22]. Therefore, expression changes in the
   immune-related genes in the hemocytes after Vibrio infection might
   contribute to shrimp resistance ability to V. parahaemolyticus.

   Some metabolism-related genes were also responsive to the infection in
   the hemocytes of V. parahaemolyticus-resistant shrimp. Among them,
   several glycolysis-related genes including GLUT2, HK, FBP, and PCK1
   were all down-regulated after Vibrio infection. In the V.
   parahaemolyticus-resistant shrimp, the down-regulation of GLUT2 and HK
   could limit the cell uptake of glucose and its inflow into the process
   of glycolysis, which could reduce the level of glycolysis. In
   arthropods, trehalose acts as the principal hemolymph sugar, which can
   be degraded into glucose [[91]23,[92]24,[93]25]. In the hemocytes of V.
   parahaemolyticus-resistant shrimp, a gene encoding trehalose
   transporter was also down-regulated after Vibrio infection, which would
   also lead to the low level of cellular glucose. Meanwhile, the
   down-regulation of FBP and PCK1 can limit the gluconeogenesis process,
   which promotes glucose entering glycolysis to effectively maintain the
   normal energy supply through glycolysis and the TCA cycle.

   In contrast, the expression level of these genes was not affected by
   Vibrio in the hemocytes of the V. parahaemolyticus-susceptible shrimp.
   This could allow sufficient glucose to enter the glycolytic process.
   Meanwhile, the expression of pyruvate carboxylase (PC) was
   significantly down-regulated, which may inhibit the TCA cycle and cause
   lactate accumulation [[94]26]. In mammals, lactate is regarded as a
   negative regulator of RLR signaling pathway and many important immune
   cells [[95]5]. In the present study, we found that the knockdown of PC,
   which might lead to lactate accumulation, suppressed the expression of
   genes in the classical NF-κB signaling pathway. Notably, as an
   inhibitor of the NF-κB signaling pathway, the expression of Cactus was
   also inhibited after PC knockdown. This might be due to the knockdown
   of PC that can cause the overall inhibition of the NF-κB signaling
   pathway. It indicated that the host immune defense might be decreased
   under this condition. Therefore, the differential responses of genes in
   the glycolytic process in hemocytes might partially contribute to the
   distinct resistance abilities of shrimp to V. parahaemolyticus. Further
   investigation will be conducted to reveal the underlying regulatory
   mechanism.

5. Conclusions

   In the present study, we illustrated that the immune responses in the
   hemocytes of shrimp with different V. parahaemolyticus resistance
   abilities vary greatly. The metabolic process, glycolysis, might affect
   host resistance to a Vibrio infection by directly influencing host
   immunity. The present data provide new insights into understanding the
   genetic mechanisms of shrimp disease resistance.

Acknowledgments