Diversity of 'Candidatus Liberibacter asiaticus' Strains in Texas Revealed by Prophage Sequence Analyses.

Prophages/phages are important components of the genome of 'Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

Lateral Flow Immunoassay Reader Technologies for Quantitative Point-of-Care Testing.

Due to the recent pandemic caused by coronavirus disease 2019 (COVID-19), the lateral flow immunoassay used for its rapid antigen test is more popular than ever before. However, the history of the lateral flow immunoassay is about 60 years old, and its original purpose of use, such as a COVID-19 rapid antigen test or a pregnancy test, was the qualitative detection of a target analyte. Recently, the demand for quantitative analysis of lateral flow immunoassays is increasing in various fields. Lateral flow immunoassays for quantitative detection using various materials and sensor technologies are being introduced, and readers for analyzing them are being developed. Quantitative analysis readers are highly anticipated for their future development in line with technological advancements such as optical, magnetic field, photothermal, and electrochemical sensors and trends such as weight reduction, miniaturization, and cost reduction of systems. In addition, the sensing, processing, and communication functions of portable personal devices such as smartphones can be used as tools for the quantitative analysis of lateral flow immunoassays. As a result, lateral flow immunoassays can efficiently achieve the goal of rapid diagnosis by point-of-care testing. Readers used for the quantification of lateral flow immunoassays were classified according to the adopted sensor technology, and the research trends in each were reviewed in this paper. The development of a quantitative analysis system was often carried out in the assay aspect, so not only the readers but also the assay development cases were reviewed if necessary. In addition, systems for quantitative analysis of COVID-19, which have recently been gaining importance, were introduced as a separate section.

Diversity of Citrus tristeza virus Strains in the Upper Gulf Coast Area of Texas.

Citrus tristeza virus (CTV) in Texas was first reported in the 1950s and has since been sporadically reported in the residential areas in the Upper Gulf Coast region. Because the major rootstock for commercial citriculture in South Texas is sour orange, which is susceptible to CTV decline, the spread of CTV into South Texas can pose a great threat to Texas citrus industry. Thirty-six CTV-positive samples, collected during surveys conducted in the Upper Gulf Coast area of Texas from 2013 to 2018, were first analyzed by strain-specific real-time PCR (RT-PCR) targeting various regions of CTV Open reading frame (Orf) 1a and then by amplicon sequencing derived from p25 and p20 region of CTV genome. Among 36 samples, 33 were successfully genotyped by strain-specific RT-PCR and by amplicon sequencing followed by phylogenetic analysis. Variability in the detection of CTV strains was observed over a 6-year period. In 2013, T3 and T30 were the only strains detected in the Upper Gulf Coast of Texas, but in further surveys until 2018, additional strains were detected, including T36, VT, and RB. Mixed infections were also detected in 14 samples comprising about 42% of CTV samples examined in the study. Although genotyping mixed infection samples by targeting Orf 1a and full-length p25, residing in the 5' and 3' region of the CTV genome, respectively, confirmed the presence of multiple strains in these samples, incongruent genotyping data were observed. These findings suggested that the current status of CTV strain diversity in Texas Upper Gulf Coast region might have been established by multiple introductions of CTV-infected plant materials for propagation and with a potential recombination in planta.

Optical Glucose Sensor Using Pressure Sensitive Paint.

A glucose sensor is used as an essential tool for diagnosing and treating diabetic patients and controlling processes during cell culture. Since the development of an electrochemical-based glucose sensor, an optical glucose sensor has been devised to overcome its shortcomings, but this also poses a problem because it requires a complicated manufacturing process. This study aimed to develop an optical glucose sensor film that could be fabricated with a simple process using commercial pressure sensitive paints. The sensor manufacturing technology developed in this work could simplify the complex production process of the existing electrochemical or optical glucose sensors. In addition, a photometric method for glucose concentration analysis was developed using the color image of the sensor. By developing this sensor and analysis technology, the basis for glucose measurement was established that enables two-dimensional, online, and continuous measurement. The proposed sensor showed good linearity at 0-4 mM glucose in an aqueous sample solution, its limit of detection was 0.37 mM, and the response time was 2 min.

Poly(Pyridinium Salt)s Containing 2,7-Diamino-9,9'-Dioctylfluorene Moieties with Various Organic Counterions Exhibiting Both Lyotropic Liquid-Crystalline and Light-Emitting Properties.

A series of poly(pyridinium salt)s-fluorene main-chain ionic polymers with various organic counterions were synthesized by using ring-transmutation polymerization and metathesis reactions. Their chemical structures were characterized by Fourier Transform Infrared (FTIR), proton (1H), and fluorine 19 (19F) nuclear magnetic resonance (NMR) spectrometers. These polymers showed a number-average molecular weight (Mns) between 96.5 and 107.8 kg/mol and polydispersity index (PDI) in the range of 1.12-1.88. They exhibited fully-grown lyotropic phases in polar protic and aprotic solvents at different critical concentrations. Small-angle X-ray scattering for one polymer example indicates lyotropic structure formation for 60-80% solvent fraction. A lyotropic smectic phase contains 10 nm polymer platelets connected by tie molecules. The structure also incorporates a square packing motif within platelets. Thermal properties of polymers were affected by the size of counterions as determined by differential scanning calorimetry and thermogravimetric analysis measurements. Their ultraviolet-visible (UV-Vis) absorption spectra in different organic solvents were essentially identical, indicating that the closely spaced π-π* transitions occurred in their conjugated polymer structures. In contrast, the emission spectra of polymers exhibited a positive solvatochromism on changing the polarity of solvents. They emitted green lights in both polar and nonpolar organic solvents and showed blue light in the film-states, but their λem peaks were dependent on the size of the counterions. They formed aggregates in polar aprotic and protic solvents with the addition of water (v/v, 0-90%), and their λem peaks were blue shifted.

Effects of food availability on growth performance and immune-related gene expression of juvenile olive flounder (Paralichthys olivaceus).

Unfavorable environmental conditions and inappropriate culture practices have increased the vulnerability of cultured fish to disease infection. Up to date many studies have aimed to determine a feeding regimen to maximize productivity; however, very little information on immune responses of cultured fish in response to underfeeding or overfeeding is available. Therefore, a preliminary study was conducted to evaluate effects of graded feeding levels (i.e., food availability) on growth performance and immune-related gene expression of juvenile olive flounder (Paralichthys olivaceus). Six different feeding rates including 1, 4, 7, 10, 13, and 16% body weight per day (BW/d) were randomly assigned to three replicate tanks stocking 150 fish (average initial body weight: 0.27 ±â€¯0.02 g; mean ±â€¯SD) per tank. A feeding trial lasted for two weeks. Based on the results of the weight gain, nutrient gain, and whole-body compositions and energy content, the feeding rate of 10%, 13%, and 16% BW/d resulted in high nutritional status, whereas the feeding rate of 1% and 4% BW/d resulted in low nutritional status. Intermediate nutritional status was observed at the feeding rate of 7% BW/d. In the given rearing conditions the optimum feeding rate resulting in the maximum growth was estimated to be 11.9% BW/d based on the quadratic broken-line regression model, chosen as the best-fit model among the tested models. Expression of immune-related genes including IL-8 and IgM was significantly down-regulated in the flounder fed at 1% BW/d in comparison to those fed at 7% BW/d. Interestingly, expression of these genes in the flounder fed at 10%, 13%, and 16% BW/d was relatively down-regulated in comparison to that of the flounder fed at 7% BW/d. Although no statistical difference was detected, overall response patterns of other immune-related genes, including TLR3, polymeric Ig receptor, lysozyme C-type, GPx, SOD, and Trx followed what IL-8 and IgM exhibited in response to the various feeding rates. Given the current challenges in aquaculture of the flounder our findings suggest to prohibit underfeeding or overfeeding (i.e., ad-libitum feeding) when culturing the young flounder.

An Optimized Colorimetric Readout Method for Lateral Flow Immunoassays.

Despite its broad penetration of various markets, the quantitative lateral flow immunoassay (LFIA) suffers from sensitivity issues in some cases. To solve this problem, an optimized colorimetric readout method for LFIA quantification is proposed in this study. An assay reader device utilizing a color camera and an analysis method using a Bayer filtered image were developed. Spectrometric measurements of the assay test line were performed to determine the color channel that contains the test line information and effectively minimizes noise. The change in the intensity ratio with increasing concentration of the target substance in the sample was largest in the green channel. The linear range of the output curve ranged from 0 to 10 ng/mL, and the detection limit was 2 ng/mL. The suggested instrumentation and analysis methods are expected to effectively resolve the low-sensitivity problems of the former LFIA systems and to offer other prospective functionalities for LFIA quantification.

Road Surface Classification Using a Deep Ensemble Network with Sensor Feature Selection.

Deep learning is a fast-growing field of research, in particular, for autonomous application. In this study, a deep learning network based on various sensor data is proposed for identifying the roads where the vehicle is driving. Long-Short Term Memory (LSTM) unit and ensemble learning are utilized for network design and a feature selection technique is applied such that unnecessary sensor data could be excluded without a loss of performance. Real vehicle experiments were carried out for the learning and verification of the proposed deep learning structure. The classification performance was verified through four different test roads. The proposed network shows the classification accuracy of 94.6% in the test data.

Microenvironmental Interaction Between Hypoxia and Endothelial Cells Controls the Migration Ability of Placenta-Derived Mesenchymal Stem Cells via α4 Integrin and Rho Signaling.

Mesenchymal stem cells (MSCs) are a powerful source for cell therapy in degenerative diseases. The migration ability of MSCs is an important factor that enhances the therapeutic effect of the cells when they are transplanted into target tissues or organs. Hypoxia and the endothelial barrier, which are representative migration microenvironmental factors, are known to be regulated by the integrin-mediated pathway in several cancers. However, their regulatory mechanisms in MSCs remain unclear. Here, the objectives of the study were to compare the expression of markers related to integrin-mediated signaling in placenta-derived MSCs (PDMSCs) dependent on hypoxia and co-cultured with human umbilical vein endothelial cells (HUVECs) and to evaluate their correlations between migration ability and microenvironmetal factors including hypoxia and endothelial cells. The migration abilities of PDMSCs exposed to hypoxic conditions were significantly increased compared with normal fibroblasts (WI-38) and control (P < 0.05). Interestingly, decreased integrin α4 in PDMSCs under hypoxia induce to increase migration abilities of PDMSCs. Also, Rho family-related markers were significantly increased in PDMSCs under hypoxic conditions compared with normoxia (P < 0.05). Furthermore, the migration ability of PDMSCs was decreased by Rho kinase inhibitor treatment (Y-27632) and co-culturing with HUVECs in an ex vivo system. ROCK activity was increased by inhibiting integrin α4 with HUVECs and hypoxia compared with the absence of HUVECs and under normoxia. The findings suggest microenvironment event by hypoxia and the interaction with endothelial cells may be useful as a regulator of MSC migration and provide insight into the migratory mechanism of MSCs in stem cell-based therapy.

Low-dose mycophenolate mofetil in tablet form or capsule form combined with tacrolimus in the early period after kidney transplantation: a prospective randomized trial
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AIMS: The tablet form (500 mg) of mycophenolate mofetil (MMF) provides more convenience of taking drugs and cost-effectiveness than the capsule form (250 mg). We examined the efficacy and safety of MMF in its different forms combined with tacrolimus in kidney transplant recipients. METHODS: This multicenter, 26-week, randomized trial was performed to compare the efficacy and safety of the tablet form of MMF versus the capsule form of MMF in 156 kidney transplant recipients. Allograft function, the incidence of efficacy failure (biopsy-proven acute rejection (BPAR), death, graft loss, or loss to follow-up), and adverse events were compared. RESULTS: The mean dose (mg/day) of MMF at 26 weeks was comparable: 1,052.6 ± 194.2 in the tablet group vs. 1,155.6 ± 298.1 in the capsule group (p = 0.063). Trough levels of tacrolimus at 26 weeks were comparable. The mean estimated glomerular filtration rate of the tablet group at 26 weeks post-transplant was not inferior to that of the capsule group. The incidence of efficacy failure was similar in the two groups: tablet group, 5.2% and capsule group, 7.7% (difference -2.5%; 95% confidence interval -5.22 - 10.21%). The incidence of BPAR until 26 weeks post-transplant in the tablet group was 3.9%, compared to 7.7% in the capsule group (p = 0.346). There was no significant difference in the incidence of discontinuations and serious adverse events between the groups. CONCLUSION: Low-dose MMF in tablet form combined with tacrolimus can be considered as an efficacious and safe immunosuppressive regimen in the early period after kidney transplantation.
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Interleukin-6 is associated with obesity, central fat distribution, and disease severity in patients with acute pancreatitis.

BACKGROUND/OBJECTIVE: Acute pancreatitis (AP) is a systemic inflammatory disease, and cytokines are suggested to be related to the course of AP. Obesity and central fat distribution are considered to have been associated with severe AP. This study investigated the profile of inflammatory cytokines in AP to determine how they are related to obesity, central fat distribution, and AP severity. METHODS: Fifty-nine patients with AP were prospectively enrolled in the study. Body mass index and waist circumference were obtained at admission. Serum levels of inflammatory cytokines, IL-Iß, IL-1ra, IL-6, TNF-α, sTNFR-I, and sTNFR-II, were measured on day 1 and 2 of AP. RESULTS: Of the patients included in the study, 19 (32%) were overweight, 23 (39%) had central fat distribution, and 23 (39%) had moderate AP. IL-1ra and IL-6 were significantly higher in overweight patients compared with non-overweight patients. IL-1ra, IL-6, TNF-α, and sTNFR-I were significantly higher in patients with central fat distribution compared with patients with non-central fat distribution. IL-6, sTNFR-I, and sTNFR-II were significantly higher in patients with moderate pancreatitis compared to those with mild pancreatitis. Among the six cytokines, IL-6 was commonly elevated in patients with central fat distribution, overweight, and moderate AP. The areas under the receiver operating characteristic curves of IL-6 for predicting the association with overweight, central fat distribution, and AP severity were 0.678, 0.716, and 0.801, respectively (P < 0.05). CONCLUSIONS: IL-6 is a good marker for AP severity and is associated with obesity and central fat distribution in AP patients.

Synthesis and validation of novel cholesterol-based fluorescent lipids designed to observe the cellular trafficking of cationic liposomes.

Cholesterol-based fluorescent lipids with ether linker were synthesized using NBD (Chol-E-NBD) or Rhodamine B (Chol-E-Rh), and the usefulnesses as fluorescent probes for tracing cholesterol-based liposomes were validated. The fluorescent intensities of liposomes containing these modified lipids were measured and observed under a microscope. Neither compound interfered with the expression of GFP plasmid, and live cell images were obtained without interferences. Changes in the fluorescent intensity of liposomes containing Chol-E-NBD were followed by flow cytometry for up to 24h. These fluorescent lipids could be useful probes for trafficking of cationic liposome-mediated gene delivery.

Structural study of citrate layers on gold nanoparticles: role of intermolecular interactions in stabilizing nanoparticles.

The structure of citrate adlayers on gold nanoparticles (AuNPs) was investigated. Infrared (IR) and X-ray photoelectron spectroscopy (XPS) analyses indicate citrate anions are adsorbed on AuNPs through central carboxylate groups. A unique structure of adsorbed citrate is determined, and a pH-induced structural transition is presented. IR analysis probes dangling dihydrogen anions (H2Citrate(-)) and hydrogen bonding of carboxylic acid groups between adsorbed and dangling citrate anions. A contribution of steric repulsion between citrate layers to particle stability is characterized. Structure-based modeling, which is consistent with scanning tunneling microscopy (STM) and transmission electron microscopy (TEM) images in the literature, suggests organization details relating to the formation of self-assembled layers on (111), (110), and (100) surfaces of AuNPs. Adsorption characteristics of the citrate layer include the interaction between hydrogen-bonded citrate chains, bilayer formation, surface coverage, and chirality. The enthalpic gain from intermolecular interactions and the importance of molecular structure/symmetry on the adsorption are discussed. Combining the enthalpic factor with surface diffusion and adsorption geometry of (1,2)-dicarboxyl fragments on Au(111), H2Citrate(-) anions effectively stabilize the (111) surface of the AuNPs. The detailed understanding of intermolecular interactions in the molecular adlayer provides insight for nanoparticle formation and stabilization. We expect these findings will be relevant for other nanoparticles stabilized by hydroxy carboxylate-based amino acids and have broad implications in NP-based interfacial studies and applications.

Chromosome-Scale, De Novo, Phased Genome Assemblies of Three Australian Limes: Citrus australasica, C. inodora, and C. glauca.

Huanglongbing (HLB) is a severe citrus disease worldwide. Wild Australian limes like Citrus australasica, C. inodora, and C. glauca possess beneficial HLB resistance traits. Individual trees of the three taxa were extensively used in a breeding program for over a decade to introgress resistance traits into commercial-quality citrus germplasm. We generated high-quality, phased, de novo genome assemblies of the three Australian limes using PacBio long-read sequencing. The genome assembly sizes of the primary and alternate haplotypes were determined for C. australasica (337 Mb/335 Mb), C. inodora (304 Mb/299 Mb), and C. glauca (376 Mb/379 Mb). The nine chromosome-scale scaffolds included 86-91% of the genome sequences generated. The integrity and completeness of the assembled genomes were estimated to be at 97.2-98.8%. Gene annotation studies identified 25,461 genes in C. australasica, 27,665 in C. inodora, and 30,067 in C. glauca. Genes belonging to 118 orthogroups were specific to Australian lime genomes compared to other citrus genomes analyzed. Significantly fewer canonical resistance (R) genes were found in C. inodora and C. glauca (319 and 449, respectively) compared to C. australasica (576), C. clementina (579), and C. sinensis (651). Similar patterns were observed for other gene families associated with potential HLB resistance, including Phloem protein 2 (PP2) and Callose synthase (CalS) genes predicted in the Australian lime genomes. The genomic information on Australian limes developed in the present study will help understand the genetic basis of HLB resistance.

Maternal exercise during pregnancy affects mitochondrial enzymatic activity and biogenesis in offspring brain.

The present study addresses whether exercise during pregnancy in mouse alters mitochondrial function in the brains of the resultant offspring. We divided pregnant mice into four groups: a control group and groups of mice that exercised for 20 (E20m), 30 (E30m) and 40 min/d (E40m). The pregnant mice ran on a treadmill at 12 m/min, 5 d/week for a duration of 3 weeks. The protein expression of cytochrome c oxidase subunit Va (CVa) was downregulated in the offspring of the E20m group, unlike that in the control animals, whereas CVa expression was reserved in the E40m neonates. The F1-ATPase catalytic core (Core) protein expression levels were the highest in the E40m group neonates. Complex I, IV and ATPase activities were significantly lower in the E20m group than that in the control group neonates and were reserved in the E30m and E40m group neonates. The activities of citrate synthase and pyruvate dehydrogenase were consistent with those of complex I, IV and ATPase. Peroxisome proliferator-activated receptor-gamma coactivator 1-alpha, mitochondrial transcription factor A, nuclear respiratory factor-1 and mitochondrial DNA showed high levels of expression in the E40m neonates compared with the other groups. Malondialdehyde (MDA) levels in E40m neonates were higher than that in the controls but were lower than that in the E20m neonates. Finally, 40 min/d of maternal exercise improved mitochondrial function in the resultant pups and was concomitant with brain-derived trophic factor induction in the hippocampus, thereby functionally improving short-term memory.

Co-Occurrence Analysis of Citrus Root Bacterial Microbiota under Citrus Greening Disease.

Candidatus Liberibacter asiaticus (CLas) is associated with Citrus Huanglongbing (HLB), a devastating disease in the US. Previously, we conducted a two-year-long monthly HLB survey by quantitative real-time PCR using root DNA fractions prepared from 112 field grapefruit trees grafted on sour orange rootstock. Approximately 10% of the trees remained CLas-free during the entire survey period. This study conducted 16S metagenomics using the time-series root DNA fractions, monthly prepared during twenty-four consecutive months, followed by microbial co-occurrence network analysis to investigate the microbial factors contributing to the CLas-free phenotype of the aforementioned trees. Based on the HLB status and the time when the trees were first diagnosed as CLas-positive during the survey, the samples were divided into four groups, Stage H (healthy), Stage I (early), II (mid), and III (late) samples. The 16S metagenomics data using Silva 16S database v132 revealed that HLB compromised the diversity of rhizosphere microbiota. At the phylum level, Actinobacteria and Proteobacteria were the predominant bacterial phyla, comprising >93% of total bacterial phyla, irrespective of HLB status. In addition, a temporal change in the rhizosphere microbe population was observed during a two-year-long survey, from which we confirmed that some bacterial families differently responded to HLB disease status. The clustering of the bacterial co-occurrence network data revealed the presence of a subnetwork composed of Streptomycetaceae and bacterial families with plant growth-promoting activity in Stage H and III samples. These data implicated that the Streptomycetaceae subnetwork may act as a functional unit against HLB.

The complete chloroplast genome sequence of the medicinal plant, Dracocephalum rupestre (Lamiaceae).

Dracocephalum rupestre (tribe Mentheae; Lamiaceae) is a perennial herb from Korea and China with high ornamental and medicinal value. Here, we report its complete chloroplast genome to provide insight into the phylogenetic relationships of Dracocephalum. The genome is 151,230 bp long, with two inverted repeat regions (25,643 bp each) that separate a large single-copy region (82,536 bp) and a small single-copy region (17,408 bp). It contains 133 genes that encode 88 proteins, eight rRNAs, and 37 tRNAs. The maximum-likelihood phylogenetic analysis strongly supported Dracocephalum monophyly, showing that the genus forms a sister group with the subtribe Menthinae in the tribe Mentheae.

A Field Deployable Real-Time Loop-Mediated Isothermal Amplification Targeting Five Copy nrdB Gene for the Detection of 'Candidatus Liberibacter asiaticus' in Citrus.

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

High water temperature-mediated immune gene expression of olive flounder, Paralichthys olivaceus according to pre-stimulation at high temperatures.

Increased ocean temperature due to global warming affects the health and immunity of fish. In this study, juvenile Paralichthys olivaceus were exposed to high temperature after pre-heat (Acute: Acute heat shock at 32 °C, AH-S: Acquired heat shock at 28 °C & short recovery (2 h) and heat shock at 32 °C, AH-L: acquired heat shock at 28 °C and long recovery (2 days), AH-LS: acquired heat shock at 28 °C & long (2 days) + short (2 h) recovery). Heat shock after pre-heat significantly upregulated various immune-related genes, including interleukin 8 (IL-8), c-type lysozyme (c-lys), immunoglobulin M (IgM), Toll-like receptor 3 (tlr3), major histocompatibility complex IIα (mhcIIα) and cluster of differentiation 8α (cd8α) in the liver and brain of P. olivaceus. This study showed pre-exposure to high temperatures below the critical temperature can activate fish immunity and increase tolerance to high temperatures.