**4. Conclusions**

The present study assessed the feasibility of performing soil flushing treatment with EDDS solution for remediating natural soils contaminated by Cu and Zn. An almost total removal of the bio-available fractions of these metals was achieved. Although the main factor that influenced the metals removal efficiency was the EDDS concentration in the extracting solution, this study proved that the effectiveness of the soil flushing was also dependent on several other factors, including contaminated soil depth as well as the different PTEs-EDDS complexes affinity. Moreover, tests under EDDS-deficiency conditions have further demonstrated that the Cu and Zn removal efficiency was lower due to their re-adsorption onto the soil and/or because of the occurrence of metal exchange processes. In order to take into account all of these aspects, it is essential to carry out bench-scale tests before performing a full-scale soil remediation treatment. Finally, the study proved that soil flushing, besides having a lower environmental impact and lower operative costs compared to soil washing (e.g., no soil excavation is required), is capable of achieving the same high removal efficiency, producing a smaller volume of contaminated spent solution and consuming a minor amount of chemical agents.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1660-4601/15/3/543/s1, Figure S1: Cu and Zn removal by soil flushing treatment at different times. 48 h; 96 h and at different values of bv—(a) Cu 4 bv; (b) Zn 4bv; (c) Cu 8 bv; (d) Zn 8 bv.

**Acknowledgments:** This study was carried out within the EUProject LIFE11 ENV/IT/000275 (ECOREMED).

**Author Contributions:** Marco Race and Alberto Ferraro performed all the tests, conducted all the analyses, and contributed to writing the manuscript. Massimiliano Fabbricino designed the study and participated in performing the tests as well as in writing the manuscript up to its final version. Antonio Panico supervised the whole work, edited the manuscript, and revised it critically. Alice Tognacchini participated in writing the manuscript and conducting analyses. Danilo Spasiano participated in performing the chemical analyses and edited the manuscript. Francesco Pirozzi participated in designing the study as well as in writing the manuscript and revising it critically.

**Conflicts of Interest:** The authors declare no conflict of interest.


**Meet the editors**

Dr. Shymaa Enany is an assistant professor of Microbiology and Immunology at the Suez Canal University, Egypt. She received her PhD degree from the School of Medical and Dental Sciences, Niigata University, Japan. Her dissertation focused on the molecular characterization of community-acquired methicillin-resistant *Staphylococcus aureus* (CA-MRSA), the leading cause of

many life-threatening illnesses. For her postdoctoral work, she collaborated with many laboratories in San Diego, California, USA, and in Niigata, Japan, analyzing the proteome of *Staphylococcus aureus* and monitoring the reactions of innate immunity and airway cytokines in response to MRSA infections.

Dr. Laura E. Crotty Alexander is a Pulmonary Critical Care physician at the University of California, San Diego, and VA San Diego Healthcare System. As a biomedical basic science researcher, she studies host-pathogen interactions at molecular, cellular, organ, and organism levels and recently defined pro-virulent effects of inhalants such as cigarette smoke and e-cigarette vapor on

Staphylococcal virulence. Dr. Crotty Alexander's clinical interests include bacterial pneumonia and sepsis, e-cigarette effects on innate immunity, and moderate-to-severe asthma.


**7. Recommendations**

In addition to the parameters to be used, there are some general recommendations when performing ULP measurements. First, as mentioned before, we proposed to perform contact angle measurements with the same liquids and substrates in order to find out whether capillary penetration is taking place for a liquid substrate combination. Measuring the contact angle will support interpretation of the ULP results.

Second, we wanted to point out the necessity of understanding the processes taking place during penetration in order to enable a sound interpretation of the results. If new combinations of liquids and substrates are used, it might not be clear which physical/chemical processes could influence ultrasound transmission during penetration. There might be cases in which the slope of a decreasing ultrasound curve cannot be simply interpreted as penetration speed. An example for such a case would be measurements with coagulating inks on primed surfaces, where the ink forms a coagulating layer once it gets into contact with the paper surface.

If ULP measurements of new combinations of liquids and substrates are to be performed, a comparison of measurements with a flat and a grooved sample holder might be useful. Comparing the two sample holders can reveal how strongly ultrasound transmission is influenced by entrapped air bubbles, which is an important information for the interpretation of results.

Furthermore, mostly 1 and 2 MHz have been used to perform ULP measurements and these frequencies have proved to be suitable for paper. However, other measurement frequencies could potentially reveal events on different spatial scales [10] and might be needed for substrates other than paper (e.g., membranes, nanoporous films, etc.). In this context it is important to remember that the bubble size critical for resonance also depends on the frequency of the ultrasound signal.


**4. Conclusions**

The present chapter assesses the impact of infrastructure on the urbanization by smart cities in India. Smart city urbanization is measured by population, density, and growth rate of the population of the 85 smart cities in India. On the other hand, smart city-wise availability of infrastructure is measured by the considering city level total road length, number of latrines, water supply capacities, number of electricity connections, hospitals, schools, colleges, universities, banks, and credit societies.

The factor analysis is used to create an infrastructure index by considering all the infrastructure variables. The OLS regression analysis is used to measure the impact of infrastructure on urbanization. The OLS regression results suggest that the availability of infrastructure has a positive and statistically significant effect on the urbanization measured by the smart city population and densities of the smart city population. This indicates that the smart city mission promotes India's urbanization.

India's cities and towns are having a serious lack of adequate infrastructure facilities. The Report on Indian Urban Infrastructure and Services [21] urged that urban India severely faces deficiency in the provision of urban public services such as street lights, solid waste management, roads, sewerage, and drinking water. The report estimated that Rs 39.2 lakh crores at 2009–10 prices are required over a 20-year period to achieve this growth. The outlay on urban roads accounts for Rs 17.3 lakh crore (or 44%) of this amount. In this perspective, the smart cities mission is appropriate for the promotion of urbanization in India by huge investment in infrastructure. It is very much important to indicate that India had a total of 7935 cities and towns in 2011. Therefore, smart cities initiatives only for 100 cities may not fulfill the urbanization dream for India. In the coming years, India should have more smart cities to explore the benefits of urbanization for higher economic growth.


*3.3. Analyses of Cis-Elements in Water Lily SOD Gene Promoters*

Retrieving cis-regulatory elements from the promoter regions of water lily *SODs* enabled the differentiation of gene functions and regulatory roles. By using the PlantCARE database, an analysis was conducted on the 2 kb upstream region from the start codon of individual *SOD* gene. Based on current findings, the cis-elements were classified into three categories: light related, stress related, and hormone response elements (Figure 3).

**Figure 3.** Exploration of abiotic-stress related cis-regulatory components in the water lily *SOD* promoter regions. (**a**) Various hormone-associated and stress-responsive elements are explored. (**b**) The size of pie chart corresponds to the ratio of the respective promoter element. Cis-elements that share functional similarity are represented by the same colors.

In this study, five phytohormone-associated elements (Salicylic acid (SA), Gibberellin, Auxin, Abscisic acid (ABA), and Methyl jasmonate (MeJA), were detected, including ABRE, TCA-components, TGACG-and CGTCA-motif, TATC-box, P-box, etc. Moreover, four stress-responsive components (drought, low-temperature, light, and anaerobic) were recognized, such as LTR, ARE, TCT-motif, LAMP-element, MBS, etc (Figure 3). Generally many light reactive components were detected to be extensively dispersed in same group species, and demonstrating the important part of water lily *SODs* in response to light stress. Comparative analysis of the findings revealed that the *SOD* promoter cis-elements in water lily species can exhibit a significant response to abiotic stresses and can play a role in regulating plant growth and development and stress response.


2.2.1. *L*1/2-NMF

Sparsity is an inherent property of hyperspectral data. To reduce the solution space and derive results with expected sparsity levels, some sparseness regularizations are added to constrain the sparseness of abundances. The *L*1 regularizer is popular for generating sparse solutions. However, the *L*1 regularizer may not enforce a sufficiently sparse solution while preserving the additivity constraint over the abundances since the sum-to-one constraint is a fixed *L*1 norm. In [35], Qian et al. propose the *L*1/2-NMF, based on the *L*1/2 regularizer. The *L*1/2 regularizer possesses two advantages over the *L*1 regularizer. It can still enforce sparsity with the full additivity constraint imposed. Another advantage is that the *L*1/2 regularizer can obtain sparser solutions than the *L*1 regularizer does [36]. The model of NMF with the *L*1/2 regularizer is as follows: [35]

$$f(\mathcal{W}, H) = \frac{1}{2} \left\| X - \mathcal{W}H \right\|\_F^2 + \lambda \left\| H \right\|\_{1/2} \tag{5}$$

where

$$\|\|H\|\|\_{1/2} = \sum\_{p,n=1}^{P,N} H\_{pn}^{1/2} \tag{6}$$

and *Hpn* denotes the (*p*, *n*)-th element of *H*.

The objective in (5) is nonincreasing under the multiplicative update rules:

$$\mathcal{W} = \mathcal{W}.\*(\mathcal{X}H^T)./\mathcal{W}HH^T\tag{7}$$

$$H = H.\*(\mathcal{W}^T\mathcal{X})./(\mathcal{W}^T\mathcal{W}H + \frac{\lambda}{2}H^{-\frac{1}{2}})\tag{8}$$

where *H*− 12 denotes the reciprocal element-wise square root for each element in *H*.


**1. Introduction**

Collagen is one of the most frequently applied biomaterials for biomedical research as well as clinical applications [1,2]. The main industrial sources of collagen are bovine and porcine tissues; however, there is increasing demand for alternative sources. Marine collagens which can be obtained from both invertebrates and vertebrates [3,4] show promising features and have the potential to overrule mammalian collagens in biomedical applications for several reasons. Marine collagens do not bear the risk of disease translation and are not allergy-causing; they are not subjected to ethical or religious concerns, show low inflammatory response and can be obtained with high yield [1]. From the beginning of the 21st century research on marine collagens has continuously emerged [5,6]. Scaffolds for tissue engineering applications are increasingly developed from collagens of marine origin, such as from fish collagen, collagen of marine sponges, jellyfish collagen and collagen from marine gastropods [5]. In two own studies we applied marine collagens for the fabrication of porous scaffolds: first we adapted the procedure of biomimetic mineralization of bovine collagen to collagen of the Atlantic salmon *Salmo salar* and prepared mineralized porous scaffolds

from salmon collagen for the application in bone tissue engineering [7]. A second study applied collagen of the jellyfish *Rhopilema esculentum* which is structurally similar to human collagen II [8] for the preparation of porous scaffolds to be used for chondral tissue engineering [9]. In the present study, we combined fibrillized jellyfish collagen with biomimetically mineralized salmon collagen to a biphasic scaffold suitable for osteochondral defect regeneration. The applied technique was already described in 2007 for the generation of biphasic, but monolithic scaffolds from mineralized bovine tendon collagen and fibrillized collagen from calf skin/hyaluronic acid composite [10]. Joint freeze-drying and chemical crosslinking of the two different phases resulted in a scaffold material which overcame the risk of delamination of the mineralized and non-mineralized phases, since the scaffolds consisted of a unified whole [10]. Challenge in the fabrication of biphasic scaffolds for the regeneration of both cartilage and the subchondral bone layer is the mechanically stable conjunction of the different phases, which have different mechanical properties to mimic the chemical nature of elastic, water-rich chondral ECM, and the stiff, mineralized bone ECM. The main challenge, however, is the simultaneous chondrogenic and osteogenic differentiation, guided by the scaffold properties which should recapitulate the native milieu of bone and cartilage development [11–14]. Furthermore, when osteochondral tissue engineering constructs are differentiated in vitro, a suitable osteochondral medium should be developed [15,16]. The aim of the present study was to generate biphasic, but monolithic porous scaffolds from fibrillized jellyfish collagen for the chondral part and biomimetically mineralized salmon collagen for the bony part. Multipotent human mesenchymal stromal cells (hMSC) were differentiated simultaneously into chondrocytes and osteoblasts, respectively, in this biphasic scaffold in vitro. To generate a microenvironment, which guides the cells to the two differentiation lineages, we inserted the cells into the chondral part with a tenfold higher cell density compared to the bony part and, furthermore, used an alginate hydrogel for embedding the cells in the porous chondral part. Furthermore, an osteochondral medium was developed and sequential seeding of the scaffold phases with pre-differentiated MSC was performed.

### **2. Results**


*Edited by Rajamanickam Rajkumar*

Cervical Cancer is one of the leading cancers among women, especially in developing countries. Prevention and control are the most important public health strategies. Empowerment of women, education, "earlier" screening by affordable technologies like visual inspection, and treatment of precancers by cryotherapy/ LEEP are the most promising interventions to reduce the burden of cervical cancer.Dr Rajamanickam Rajkumar had the privilege of establishing a rural population based cancer registry in South India in 1996, as well as planning and implementing a large scale screening program for cervical cancer in 2000. The program was able to show a reduction in the incidence rate of cervical cancer by 25%, and reduction in mortality rate by 35%. This was the greatest inspiration for him to work on cerrvical cancer prevention, and he edited this book to inspire others to initiate such programs in developing countries. InTech - Open Access Publisher plays a major role in this crusade against cancer, and the authors have contributed to it very well.

ISBN 978-953-51-0183-3

Topics on Cervical Cancer With an Advocacy for Prevention

Photo by weisschr / iStock


*Article* **Transcriptomics Analysis of the Toxicological Impact of Enrofloxacin in an Aquatic Environment on the Chinese Mitten Crab (***Eriocheir sinensis***)**

**Qiaona Wang 1,2, Ziling Xu 1,2, Ying Wang 1,2, Guangming Huo 2 , Xing Zhang 3 , Jianmei Li 3 , Chun Hua 2 , Shengjie Li 1,2,\* and Feng Zhou 2,\***

	- **\*** Correspondence<EMAIL_ADDRESS>(S.L<EMAIL_ADDRESS>(F.Z.)

**Abstract:** Enrofloxacin is an important antimicrobial drug that is widely used in aquaculture. Enrofloxacin residues can have negative effects on aquatic environments and animals. The toxicological effects of different concentrations of enrofloxacin residues in cultured water on Chinese mitten crabs (*Eriocheir sinensis*) were compared. A histological analysis of the *E. sinensis* hepatopancreas demonstrated that the hepatopancreas was damaged by the different enrofloxacin residue concentrations. The hepatopancreas transcriptome results revealed that 1245 genes were upregulated and that 1298 genes were downregulated in the low-concentration enrofloxacin residue group. In the high-concentration enrofloxacin residue group, 380 genes were upregulated, and 529 genes were downregulated. The enrofloxacin residues led to differentially expressed genes related to the immune system and metabolic processes in the hepatopancreas of the Chinese mitten crab, such as the genes for alkaline phosphatase, NF-kappa B inhibitor alpha, alpha-amylase, and beta-galactosidase-like. The gene ontology terms "biological process" and "molecular function" were enriched in the carboxylic acid metabolic process, DNA replication, the synthesis of RNA primers, the transmembrane transporter activity, the hydrolase activity, and the oxidoreductase activity. A Kyoto Encyclopedia of Genes and Genomes pathway analysis determined that the immune and metabolic signal transduction pathways were significantly enriched. Furthermore, the nonspecific immune enzyme (alkaline phosphatase) and the metabolic enzyme system played a role in the enrofloxacin metabolism in the *E. sinensis* hepatopancreas. These findings helped us to further understand the basis of the toxicological effects of enrofloxacin residues on river crabs and provided valuable information for the better utilization of enrofloxacin in aquatic water environments.

**Keywords:** enrofloxacin; *Eriocheir sinensis*; transcriptome

#### **1. Introduction**

The Chinese mitten crab (*Eriocheir sinensis*) is a commonly farmed crustacean species typically found in benthic aquatic environments [1]. The Yangtze River Delta is a primary breeding area for crabs in China [2]. Generally, farmers adopt intensive and high-density farming methods to obtain high returns and profits. This breeding practice leads to a gradual deterioration in water quality and increases the incidence of infectious diseases [3]. To prevent disease outbreaks during crab farming, large amounts of antibiotics are used and, as such, are released into the natural water environment.

Enrofloxacin is a third-generation fluoroquinolone antibacterial drug [4,5] which is often used to treat bacterial infections in crab breeding operations in China because of its wide antibacterial spectrum and high potency [6,7]. Although enrofloxacin is approved for aquaculture in China [8], when used in large quantities, the antimicrobial remains in the aquaculture water and sediment accumulates in aquatic animals. Pharmacokinetic

**Citation:** Wang, Q.; Xu, Z.; Wang, Y.; Huo, G.; Zhang, X.; Li, J.; Hua, C.; Li, S.; Zhou, F. Transcriptomics Analysis of the Toxicological Impact of Enrofloxacin in an Aquatic Environment on the Chinese Mitten Crab (*Eriocheir sinensis*). *Int. J. Environ. Res. Public Health* **2023**, *20*, 1836. https://doi.org/10.3390/ ijerph20031836

Academic Editors: Xun Wang, Zhiyuan Wang and Xin Zhao

Received: 19 October 2022 Revised: 7 January 2023 Accepted: 8 January 2023 Published: 19 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

studies on enrofloxacin in crustaceans, including the *Eriocheir sinensis* [9–11], Atlantic horseshoe crab [12], and giant mud crab [13], have been reported. Enrofloxacin can be biotransformed in vivo into its major metabolite, ciprofloxacin, in crabs. More importantly, Roca et al. reported that quinolone residues are not degraded during processing and that their presence in food poses a risk to human health [14]. The risk of enrofloxacin and ciprofloxacin residues in aquatic products and the aquatic environment has become an important issue that has attracted increasing attention.

Many recent studies on aquaculture animals have focused on the residual characteristics of enrofloxacin and ciprofloxacin; the rate of biotransformation; and the health risks of consuming residual enrofloxacin and ciprofloxacin in *Procambarus clarkia* [15], *Exopalaemon carinicauda* [16], and *Pangasianodon hypophthalmus* [17]. Su et al. reported that enrofloxacin has significant effects on the gene expression of the cytochrome P450 3 (CYP3), glutathione S-transferases (GST), and phosphoenolpyruvate carboxykinase (PEPCK) in crabs [18], which are key molecules that affect the metabolism, immunity, and antitumor activity of organisms [19–22]. However, there are still few enrofloxacin toxicology reports on Eriocheir sinensis in cultured water.

In this study, we built a microcosm that authentically simulated aquaculture conditions and used it as a research tool to determine the mechanisms underlying the toxicological effects exerted on crabs through exposure to different concentrations of enrofloxacin. In crustaceans, the hepatopancreas has multiple functions, including nutrient absorption and metabolization, the storage of minerals and energy reserves, the synthesis of lipoproteins, the detoxification of heavy metals, and the excretion of uric acid [23]. In addition, the hepatopancreas is an important organ and is the primary site for the synthesis, excretion, and regulation of immune and metabolic molecules [24]. Su et al. demonstrated that enrofloxacin regulated immunity- and metabolism-related gene expression, such as that of CYP3, GST, and PEPCK [18]. Here, we continued to explore the effect of enrofloxacin on the hepatopancreas by using RNA sequencing. Thus, we collected the hepatopancreatic tissue from *E. sinensis* following 10 days of exposure to two different concentrations of enrofloxacin. Histomorphological observations and transcriptome analyses revealed the toxicological effects of enrofloxacin as well as the mechanisms underlying these effects.

We further analyzed the toxicological effects of different concentrations of enrofloxacin on *E. sinensis* using hepatopancreas transcriptome analyses. These results aided in understanding their effect on the immune system and metabolic process disorders of *Eriocheir sinensis* and laid the foundation for further research on *Eriocheir sinensis* by developing an understanding of the defense mechanism essential for maintaining healthy mitten crabs in aquaculture.

### **2. Materials and Methods**

#### *2.1. Experimental Design and Sampling*

To authentically simulate the culture environment of mitten crabs, three Chinese mitten crab culture drums with the same conditions were selected. Each drum had a diameter of 2 m, height of 1.2 m, and water depth of approximately 1 m. Twelve Chinese mitten crabs of similar sizes and weights were placed in each barrel. The amounts of enrofloxacin, 1.875 and 3.750 g, were determined according to the national drug standard, and the enrofloxacin was evenly sprinkled into two different Chinese mitten crab culture drums. After standing for 24 h, the concentration of enrofloxacin in the culture water was 0.63 mg/L in the low-concentration enrofloxacin residue group and 1.25 mg/L in the high-concentration enrofloxacin residue group. After 10 days of sprinkling, crabs were anesthetized on ice and then sampled from the control, low-concentration, and high-concentration enrofloxacin residue groups. The drugs in the aquaculture water were not completely degraded by the time of sampling. The remaining hepatopancreas samples were stored at −20 ◦C.

#### *2.2. Histopathological Analysis of the Hepatopancreas*

Hepatopancreatic tissue samples were immediately fixed in 4% paraformaldehyde for 24 h, dehydrated in gradient concentration of ethanol, and embedded in paraffin wax. Using a microtome, 4–5 µm thick sections were obtained and then stained with hematoxylin and eosin (HE). Histopathological changes were observed under a Nikon 50i optical microscope (Nikon Corporation, Tokyo, Japan).

#### *2.3. Total RNA Extraction and Sequencing*

The transcriptome sequencing and analyses were conducted by Novogene Co., Ltd. (Beijing, China). Total RNA was extracted using a TRIzol® Reagent Kit (Invitrogen, California, USA) according to the manufacturer's protocol. The RNA quality and quantity were examined using 1% Tris–acetate (TAE) agarose gel electrophoresis. Equal quantities (0.5 µg) of RNA from *E. sinensis* hepatopancreas samples were separately pooled to eliminate sample variation and to create two main samples. The samples were used for RNA-seq library construction using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA) according to the protocol. The AMPure XP system (Beckman Coulter, Beverly, MA, USA) was used to purify library fragments for selecting complementary DNA (cDNA). After the library was constructed, a Qubit2.0 (Thermo Fisher Scientific, Waltham, MA, USA) Fluorometer was used for initial quantification, and the library was diluted to 1.5 ng/µL. The Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA) was used to determine the insert size of the library. For ensuring the library quality, quantitative real-time PCR (qRT-PCR) was used to accurately quantify an effective library concentration of higher than 2 nM. Mixed DNA libraries were diluted to 4–5 pM for sequencing using an Illumina NovaSeq 6000 instrument (Illumina Inc, San Diego, CA, USA).

#### *2.4. De Novo Transcriptome Assembly*

We removed low-quality adapter sequences by filtering raw reads. The resulting clean reads were assembled to produce complete reference sequences using the Trinity program (v2.4.0; min\_kmer\_cov:3). Longer contigs were assembled until they could not be extended to either side. The unigenes were obtained by removing redundant transcripts. Using BLASTx (2.2.28+; threshold E-value=1 × 10−5 ), the assembled transcripts were aligned with the following National Center for Biotechnology Information protein databases: nonredundant (NR), nucleotide sequence (NT), protein family (PFAM), gene ontology (GO), and protein sequence (Swiss-Prot). The best hits were used for the functional annotation of the unigenes. Blast2GO (b2g4pipe\_v2.5, threshold E-value=1 × 10−6 ) was used to obtain and analyze GO annotations for the uniquely assembled transcripts.

### *2.5. Differentially Expressed Gene (DEG) Analysis*

DEGs between the control and residue groups were identified using the DESeq package (http://bioconductor.org/packages/release/bioc/html/DESeq.html, accessed on 30 October 2021). An absolute log2-fold change > 1 and FDR < 0.05 were used as thresholds to define DEGs. DEGs were then subjected to GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.

#### *2.6. Enzymatic Analysis*

Acid phosphatase (ACP), alkaline phosphatase (AKP), glutathione sulfotransferase (GSH-ST), and acetylcholinesterase (AchE) activities were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

#### *2.7. Statistical Analyses*

All data were analyzed using SPSS statistics 20 (IBM Inc., Chicago, IL, USA) through one-way analysis of variance (ANOVA), and differences between groups were analyzed using Student's *t*-test. Statistical significance was set at *p* < 0.05.

#### **3. Results 3. Results**

*2.6. Enzymatic Analysis*

facturer's instructions.

*2.7. Statistical Analyses*

#### *3.1. Enrofloxacin Residues Induced Hepatopancreas Injury in E. sinensis 3.1. Enrofloxacin Residues Induced Hepatopancreas Injury in E. sinensis*

using Student's *t*-test. Statistical significance was set at *p* < 0.05.

*Int. J. Environ. Res. Public Health* **2023**, *20*, x FOR PEER REVIEW 4 of 15

The degree of hepatopancreatic damage was a direct reflection of the intensity of the toxicity of the external stimuli as observed with the aid of HE staining [25]. In the hepatopancreases of the Chinese mitten crabs in the control group, the basement membranes were complete and clear, and the nuclei were arranged in an orderly manner. The absorbing cells, alveolar cells, fibroblasts, and embryonic cells were clearly distinguished (Figure 1A). However, relative to the control group, in the low-concentration enrofloxacin residue group, the hepatopancreases demonstrated an enlarged space in their lumens and deformed basement membranes; the components in the cells were loosely arranged, the number of nuclei was significantly reduced, and this was accompanied by inflammatory cell infiltration (Figure 1B). In the high-concentration enrofloxacin residue group, the damage to the basement membranes was more severe, the internal structure of the cell membranes was deformed, the internal arrangement of the cells was disordered, the cell structure was lost, secretion in the hepatopancreatic duct cavity was increased, and hepatopancreatic duct atrophy was observed (Figure 1C). The degree of hepatopancreatic damage was a direct reflection of the intensity of the toxicity of the external stimuli as observed with the aid of HE staining [25]. In the hepatopancreases of the Chinese mitten crabs in the control group, the basement membranes were complete and clear, and the nuclei were arranged in an orderly manner. The absorbing cells, alveolar cells, fibroblasts, and embryonic cells were clearly distinguished (Figure 1A). However, relative to the control group, in the low-concentration enrofloxacin residue group, the hepatopancreases demonstrated an enlarged space in their lumens and deformed basement membranes; the components in the cells were loosely arranged, the number of nuclei was significantly reduced, and this was accompanied by inflammatory cell infiltration (Figure 1B). In the high-concentration enrofloxacin residue group, the damage to the basement membranes was more severe, the internal structure of the cell membranes was deformed, the internal arrangement of the cells was disordered, the cell structure was lost, secretion in the hepatopancreatic duct cavity was increased, and hepatopancreatic duct atrophy was observed (Figure 1C).

Acid phosphatase (ACP), alkaline phosphatase (AKP), glutathione sulfotransferase (GSH-ST), and acetylcholinesterase (AchE) activities were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manu-

All data were analyzed using SPSS statistics 20 (IBM Inc., Chicago, IL, USA) through

**Figure 1.** Hematoxylin and eosin staining of *E. sinensis* hepatopancreas specimens following enrofloxacin exposure at different doses. (**A**) CON represents the control group, (**B**) ENR-L represents the low-concentration enrofloxacin residue group, and (**C**) ENR-H represents the high-concentration enrofloxacin residue group. **Figure 1.** Hematoxylin and eosin staining of *E. sinensis* hepatopancreas specimens following enrofloxacin exposure at different doses. (**A**) CON represents the control group, (**B**) ENR-L represents the low-concentration enrofloxacin residue group, and (**C**) ENR-H represents the high-concentration enrofloxacin residue group.

#### *3.2. Enrofloxacin Residues Led to Multiple Gene Expression Disorders in the Hepatopancreases of Crabs 3.2. Enrofloxacin Residues Led to Multiple Gene Expression Disorders in the Hepatopancreases of Crabs*

To elucidate the molecular mechanism underlying the toxicological effects of enrofloxacin on crabs, a de novo assembled transcriptome analysis of the hepatopancreatic samples was performed. The data on the success rate of gene annotation were analyzed in seven databases, including NR, GO, KOG, KO, NT, SwissProt, and PFAM. The annotation success rate in NR was 24604, accounting for 25.62%; that in GO was 26538, accounting for 27.64%; that in KOG was 7494, accounting for 7.8%; that in KO was 9251, accounting for 9.63%; that in NT was 17269, accounting for 17.98%; that in SwissProt was 14294, To elucidate the molecular mechanism underlying the toxicological effects of enrofloxacin on crabs, a de novo assembled transcriptome analysis of the hepatopancreatic samples was performed. The data on the success rate of gene annotation were analyzed in seven databases, including NR, GO, KOG, KO, NT, SwissProt, and PFAM. The annotation success rate in NR was 24,604, accounting for 25.62%; that in GO was 26,538, accounting for 27.64%; that in KOG was 7494, accounting for 7.8%; that in KO was 9251, accounting for 9.63%; that in NT was 17,269, accounting for 17.98%; that in SwissProt was 14,294, accounting for 14.88%; and that in PFAM was 26,542, accounting for 27.64% (Table S1). Through the analysis of the transcriptomic data of the three groups of crab hepatopancreas samples, we obtained DEGs corresponding to the two doses of enrofloxacin. The DEG analysis of the RNA sequence revealed 1245 upregulated and 1298 downregulated genes in the hepatopancreases of the low-concentration enrofloxacin residue group relative to the control group (Figure 2A). Meanwhile, compared with the control group, the DEG analysis demonstrated 380 upregulated and 529 downregulated genes in the hepatopancreases of the high-concentration enrofloxacin residue group (Figure 2B).

**Figure 2.** Volcano plot of the differences in the expression profiles of *E. sinensis* samples in the control and residue groups, respectively. CON represents control, (**A**) ENR-L represents low-concentration enrofloxacin residue group, and (**B**) ENR-H represents high-concentration enrofloxacin residue group. The x-axis represents logFC (fold change), while the y-axis represents −log10 (*p*-value). Red represents significantly upregulated genes, blue represents significantly downregulated genes, and each circle represents a single gene. **Figure 2.** Volcano plot of the differences in the expression profiles of *E. sinensis* samples in the control and residue groups, respectively. CON represents control, (**A**) ENR-L represents low-concentration enrofloxacin residue group, and (**B**) ENR-H represents high-concentration enrofloxacin residue group. The x-axis represents logFC (fold change), while the y-axis represents −log10 (*p*-value). Red represents significantly upregulated genes, blue represents significantly downregulated genes, and each circle represents a single gene.

accounting for 14.88%; and that in PFAM was 26542, accounting for 27.64% (Table S1). Through the analysis of the transcriptomic data of the three groups of crab hepatopancreas samples, we obtained DEGs corresponding to the two doses of enrofloxacin. The DEG analysis of the RNA sequence revealed 1,245 upregulated and 1,298 downregulated genes in the hepatopancreases of the low-concentration enrofloxacin residue group relative to the control group (Figure 2A). Meanwhile, compared with the control group, the

pancreases of the high-concentration enrofloxacin residue group (Figure 2B).

#### *3.3. GO Analysis of DEGs Found Significant Enrichment of Biological Processes Related to Metabolism Process in Enrofloxacin Residue Groups 3.3. GO Analysis of DEGs Found Significant Enrichment of Biological Processes Related to Metabolism Process in Enrofloxacin Residue Groups*

To investigate the function of these DEGs in the hepatopancreases of crabs exposed to enrofloxacin, the 2543 The DEGs corresponding to a low dose of enrofloxacin were entered into the GO database, and the results included the biological processes (BP), cellular components (CC), and molecular function (MF). The DEGs in the low-concentration enrofloxacin residue group, relative to the control group, demonstrated an obvious enrichment of the biological processes chiefly related to the metabolic processes, including the carbohydrate metabolic process (37 DEGs, *p* = 0.000136), tyrosine metabolic process (13 DEGs, *p* = 0.001826), and glycerolipid metabolic process (9 DEGs, *p* = 0.006136; Figure 3A; Table 1). Similarly, the 909 DEGs corresponding to a high dose of enrofloxacin were entered into the GO database, and an enrichment of the biological processes was again evident, including the carbohydrate metabolic process (17 DEGs, *p* = 0.000478), purine nucleobase metabolic process (13 DEGs, *p* = 0.029346), and tricarboxylic acid cycle (3 DEGs, *p* = 0.008759; Figure 3B; Table 1). It is worth noting that the high concentration of enrofloxacin residues led to DNA and RNA damage, and the obvious enrichment in the DEGs involved in translation, DNA-templates (12 DEGs, *p* = 0.029357), mRNA processing (8 DEGs, *p* = 0.032291), and so on (Figure 3B). Metabolic abnormalities were closely related to DNA and RNA damage as well as transcription and translation errors. DNA damage can impair metabolic organ functions and induce tissue inflammation, which disrupts the homeostasis of the systemic metabolism [26]. Moreover, the Venn diagram illustrates that, in the BP To investigate the function of these DEGs in the hepatopancreases of crabs exposed to enrofloxacin, the 2543 The DEGs corresponding to a low dose of enrofloxacin were entered into the GO database, and the results included the biological processes (BP), cellular components (CC), and molecular function (MF). The DEGs in the low-concentration enrofloxacin residue group, relative to the control group, demonstrated an obvious enrichment of the biological processes chiefly related to the metabolic processes, including the carbohydrate metabolic process (37 DEGs, *p* = 0.000136), tyrosine metabolic process (13 DEGs, *p* = 0.001826), and glycerolipid metabolic process (9 DEGs, *p* = 0.006136; Figure 3A; Table 1). Similarly, the 909 DEGs corresponding to a high dose of enrofloxacin were entered into the GO database, and an enrichment of the biological processes was again evident, including the carbohydrate metabolic process (17 DEGs, *p* = 0.000478), purine nucleobase metabolic process (13 DEGs, *p* = 0.029346), and tricarboxylic acid cycle (3 DEGs, *p* = 0.008759; Figure 3B; Table 1). It is worth noting that the high concentration of enrofloxacin residues led to DNA and RNA damage, and the obvious enrichment in the DEGs involved in translation, DNA-templates (12 DEGs, *p* = 0.029357), mRNA processing (8 DEGs, *p* = 0.032291), and so on (Figure 3B). Metabolic abnormalities were closely related to DNA and RNA damage as well as transcription and translation errors. DNA damage can impair metabolic organ functions and induce tissue inflammation, which disrupts the homeostasis of the systemic metabolism [26]. Moreover, the Venn diagram illustrates that, in the BP of the DEGs, the enrichment between the low- and high-concentration residue groups relative to the control group affected three common biological processes: the carboxylic acid metabolic process, carbohydrate metabolic process, and DNA replication and synthesis of RNA primers (Figure 3C). Therefore, these results illustrated that enrofloxacin residues may affect the hepatopancreatic metabolic processes of *E. sinensis*.

may affect the hepatopancreatic metabolic processes of *E. sinensis*.

**Figure 3.** Gene ontology (GO) analysis of the differentially expressed genes (DEGs) in the *E. sinensis* hepatopancreases following different exposure doses of enrofloxacin. Colors represent *p*-values. The x-axis represents the gene ratio. The y-axis represents GO terms involved in biological processes. Venn diagrams showing number of biological processes in *E. sinensis* hepatopancreas samples following (**A**) low and (**B**) high enrofloxacin exposure doses. (**C**) Enrichment of DEGs of the three common biological processes affected by both low (blue) and high (yellow) enrofloxacin exposure doses. **Figure 3.** Gene ontology (GO) analysis of the differentially expressed genes (DEGs) in the *E. sinensis* hepatopancreases following different exposure doses of enrofloxacin. Colors represent *p*-values. The x-axis represents the gene ratio. The y-axis represents GO terms involved in biological processes. Venn diagrams showing number of biological processes in *E. sinensis* hepatopancreas samples following (**A**) low and (**B**) high enrofloxacin exposure doses. (**C**) Enrichment of DEGs of the three common biological processes affected by both low (blue) and high (yellow) enrofloxacin exposure doses.

of the DEGs, the enrichment between the low- and high-concentration residue groups relative to the control group affected three common biological processes: the carboxylic acid

#### *3.4. Molecular Function in GO Analysis of DEGs*

The molecular function in the GO analysis reflected the biological function that could be affected by the DEGs. Among the molecular functions, the transmembrane transporter activity was most significantly affected in the low-concentration enrofloxacin residue group relative to the control group (44 DEGs, *p* = 0.000256) followed by the hydrolase activity (34 DEGs, *p* = 0.004765), the adenosine triphosphate enzyme (ATPase) activity (27 DEGs, *p* = 0.012104), sequence-specific DNA binding (26 DEGs, *p* = 0.002681), heme binding (25 DEGs, *p* = 2.19 × 10−5 ), the hydrolase activity, hydrolyzing O-glycosyl compounds (22 DEGs, *p* = 4.31 × 10−6 ), iron ion binding (21 DEGs, *p* = 0.001086), chitin binding (18 DEGs, *p* = 2.02 × 10−5 ), the sulfuric ester hydrolase activity (15 DEGs, *p* = 7.41 × 10−9 ), and the aminoacyl-transfer RNA ligase activity (2 DEGs, *p* = 0.036552; Figure 4A). In the high-concentration enrofloxacin residue group, compared with the control, the transmembrane transporter activity was also the most significantly affected (25 DEGs, *p* = 6.42 × 10−6 ) followed by the hydrolase activity (19 DEGs, *p* = 0.000301), the oxidoreductase activity (19 DEGs, *p* = 0.002669), the structural molecule activity (17 DEGs, *p* = 0.009975), chitin binding (14 DEGs, *p* = 2.81 × 10−8 ), the catalytic activity (14 DEGs, *p* = 0.022347), the hydrolase activity and hydrolyzing O-glycosyl compounds (9 DEGs, *p* = 0.048616), and the RNA-directed 50 -30 RNA polymerase activity (7 DEGs, *p* = 0.004283; Figure 4B). Notably, the molecular functions, including the sulfuric ester hydrolase activity, the hydrolase activity, hydrolyzing O-glycosyl compounds, chitin binding, the metallocarboxypeptidase activity, the transmembrane transporter activity, the catalytic activity, the 1-alkyl-2-acetylglycerophosphocholine esterase activity, the oxidoreductase activity, and the phosphatase activity, occurred repeatedly in both the low- and high-concentration enrofloxacin residue groups (Figure 4C).

 Control group, 2 low-concentration enrofloxacin residue group, and 3 high-concentration enrofloxacin residue group. groups (Figure 4C).

*3.4. Molecular Function in GO Analysis of DEGs*

The molecular function in the GO analysis reflected the biological function that could be affected by the DEGs. Among the molecular functions, the transmembrane transporter activity was most significantly affected in the low-concentration enrofloxacin residue group relative to the control group (44 DEGs, *p* = 0.000256) followed by the hydrolase activity (34 DEGs, *p* = 0.004765), the adenosine triphosphate enzyme (ATPase) activity (27 DEGs, *p* = 0.012104), sequence-specific DNA binding (26 DEGs, *p* = 0.002681), heme binding (25 DEGs, *p* = 2.19 × 10−5), the hydrolase activity, hydrolyzing O-glycosyl compounds (22 DEGs, *p* = 4.31 × 10−6), iron ion binding (21 DEGs, *p* = 0.001086), chitin binding (18 DEGs, *p* = 2.02 × 10−5), the sulfuric ester hydrolase activity (15 DEGs, *p* = 7.41 × 10−9), and the aminoacyl-transfer RNA ligase activity (2 DEGs, *p* = 0.036552; Figure 4A). In the highconcentration enrofloxacin residue group, compared with the control, the transmembrane transporter activity was also the most significantly affected (25 DEGs, *p* = 6.42 × 10−6) followed by the hydrolase activity (19 DEGs, *p* = 0.000301), the oxidoreductase activity (19 DEGs, *p* = 0.002669), the structural molecule activity (17 DEGs, *p* = 0.009975), chitin binding (14 DEGs, *p* = 2.81 × 10−8), the catalytic activity (14 DEGs, *p* = 0.022347), the hydrolase activity and hydrolyzing O-glycosyl compounds (9 DEGs, *p* = 0.048616), and the RNA-directed 5′-3′ RNA polymerase activity (7 DEGs, *p* = 0.004283; Figure 4B). Notably, the molecular functions, including the sulfuric ester hydrolase activity, the hydrolase activity, hydrolyzing O-glycosyl compounds, chitin binding, the metallocarboxypeptidase activity, the transmembrane transporter activity, the catalytic activity, the 1-alkyl-2-acetylglyc-

**Figure 4.** Gene ontology (GO) analysis of the DEGs in *E. sinensis* hepatopancreas samples following different exposure doses of enrofloxacin. Colors represent *p*-values. The x-axis represents the gene **Figure 4.** Gene ontology (GO) analysis of the DEGs in *E. sinensis* hepatopancreas samples following different exposure doses of enrofloxacin. Colors represent *p*-values. The x-axis represents the gene ratio. The y-axis represents GO terms involved in molecular functions. Venn diagrams showing number of molecular functions in *E. sinensis* hepatopancreas samples following (**A**) low and (**B**) high exposure doses. (**C**) Enrichment of DEGs of the common molecular functions affected by both low (blue) and high (yellow) enrofloxacin exposure doses.

### *3.5. Results of KEGG Analysis of DEGs*

The abundant signaling pathway information in the KEGG database helped elucidate the system-level biological functions, such as the metabolic and inflammatory pathways, oxidative stress, protein modification, and cell death, among others [27]. Relative to the control group, a KEGG enrichment analysis of the DEGs in the low- and high-concentration enrofloxacin residue groups was performed. The KEGG annotation analysis demonstrated that, following the low-dose administration of enrofloxacin, the DEGs were significantly enriched in multiple basic pathways; the abundantly significant pathways were related to the starch and sucrose metabolism, the lysosome metabolism, the sphingolipid metabolism, the two-component system, other glycan degradation, the galactose metabolism, nonalcoholic fatty liver disease, folate biosynthesis, the thiamine metabolism, and the tryptophan metabolism, among others (Figure 5A). In addition, in the high-concentration enrofloxacin administration group, relative to the control group, DEGs were significantly enriched in ribosome biogenesis in eukaryotes; the starch and sucrose metabolism; carbohydrate digestion and absorption; lysosome, pantothenate, and CoA biosynthesis; folate biosynthesis; the galactose metabolism; the thiamine metabolism; the nitrogen metabolism; and the fatty acid metabolism, among others (Figure 5B). The Venn diagram illustrates that 12 pathways were enriched by KEGG following exposure to low or high doses of enrofloxacin, including the starch and sucrose metabolism, the lysosome metabolism, the sphingolipid metabolism, the two-component system, the folate metabolism, the Toll and Imd signaling pathways, the thiamine metabolism, pantothenate and CoA biosynthesis, carbohydrate digestion and absorption, aminobenzoate degradation, and protein digestion and absorption (Figure 5C). Consistent with the GO analysis, the KEGG enrichment analysis also revealed that enrofloxacin exposure may affect the metabolic processes of the crab hepatopancreas.

ratio. The y-axis represents GO terms involved in molecular functions. Venn diagrams showing number of molecular functions in *E. sinensis* hepatopancreas samples following (**A**) low and (**B**) high exposure doses. (**C**) Enrichment of DEGs of the common molecular functions affected by both low

The abundant signaling pathway information in the KEGG database helped elucidate the system-level biological functions, such as the metabolic and inflammatory pathways, oxidative stress, protein modification, and cell death, among others [27]. Relative to the control group, a KEGG enrichment analysis of the DEGs in the low- and high-concentration enrofloxacin residue groups was performed. The KEGG annotation analysis demonstrated that, following the low-dose administration of enrofloxacin, the DEGs were significantly enriched in multiple basic pathways; the abundantly significant pathways were related to the starch and sucrose metabolism, the lysosome metabolism, the sphingolipid metabolism, the two-component system, other glycan degradation, the galactose metabolism, nonalcoholic fatty liver disease, folate biosynthesis, the thiamine metabolism, and the tryptophan metabolism, among others (Figure 5A). In addition, in the high-concentration enrofloxacin administration group, relative to the control group, DEGs were significantly enriched in ribosome biogenesis in eukaryotes; the starch and sucrose metabolism; carbohydrate digestion and absorption; lysosome, pantothenate, and CoA biosynthesis; folate biosynthesis; the galactose metabolism; the thiamine metabolism; the nitrogen metabolism; and the fatty acid metabolism, among others (Figure 5B). The Venn diagram illustrates that 12 pathways were enriched by KEGG following exposure to low or high doses of enrofloxacin, including the starch and sucrose metabolism, the lysosome metabolism, the sphingolipid metabolism, the two-component system, the folate metabolism, the Toll and Imd signaling pathways, the thiamine metabolism, pantothenate and CoA biosynthesis, carbohydrate digestion and absorption, aminobenzoate degradation, and

(blue) and high (yellow) enrofloxacin exposure doses.

*3.5. Results of KEGG Analysis of DEGs*

processes of the crab hepatopancreas.

**Figure 5.** Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs in *E. sinensis* hepatopancreas samples following different exposure doses of enrofloxacin. Colors represent *p*-values. The x-axis represents the number of genes annotated in a KEGG pathway. The y-axis represents the KEGG pathway categories. Venn diagrams showing categories of KEGG pathways in *E. sinensis* hepatopancreas samples following both (**A**) low and (**B**) high exposure doses. (**C**) Enrichment, following KEGG analysis, of DEGs of the system-level biological functions affected by both low (blue) and high (yellow) enrofloxacin exposure doses.

#### *3.6. Enrofloxacin Residues Led to Immune System and Metabolic Process Disorders in the Hepatopancreases of Chinese Mitten Crabs*

By analyzing the transcriptomic data of the three groups of crab hepatopancreas samples, we obtained DEGs corresponding to the two doses of enrofloxacin. The results showed that low-concentration enrofloxacin exposure produced 2543 DEGs between the control and experimental groups of which 1245 were upregulated and of which 1298 were downregulated (Figure 2A). In the meantime, high-concentration enrofloxacin exposure yielded a total of 909 DEGs between the control and experimental groups with 380 upregulated and 529 downregulated genes (Figure 2B). Compared with the control group, we found that the DEGs related to the immune system and metabolic processes showed significant changes in both the low- and high-concentration enrofloxacin residue groups. In the immune system, the DEGs, including those for alkaline phosphatase (AKP), dual oxidase 1, and nuclear factor-κB (NF-κB) inhibitor alpha, changed significantly of which NF-κB inhibitor alpha was upregulated in both the low- and high-concentration enrofloxacin residue groups, while the other DEGs were downregulated. In the metabolic processes, the DEGs changed significantly, such as those for venom phosphodiesterase 2-like, beta 1, 4-endoglucanase, alpha-amylase, arylsulfatase A-like, the ecdysteroid receptor (EcR) gene, beta-galactosidase-like, pantothenate kinase 3-like, carboxypeptidase B-like, trypsin-like serine proteinase, chitinase 3, and juvenile hormone esterase-like carboxylesterase 1, and all of them were downregulated in both the low- and high-concentration enrofloxacin residue groups (Table 2). Thus, these results demonstrated that immune system and metabolic process disorders may be key factors in *E. sinensis* hepatopancreatic damage.

*Int. J. Environ. Res. Public Health* **2023**, *20*, x FOR PEER REVIEW 11 of 15

**Table 2.** Summary of DEGs related to immune system and metabolic processes in the *E. sinensis* transcriptome.

1 Control group, 2 low-concentration enrofloxacin residue group, and 3 high-concentration enrofloxacin residue group.

## *3.7. Immune Responses and Metabolic Enzymatic Activities following Exposure to Enrofloxacin Residues*

Our analysis revealed that damage to the *E. sinensis* hepatopancreas was primarily associated with immune responses and metabolic processes. By analyzing DEGs, we found that enrofloxacin residues led to immune system and metabolic process disorders in the hepatopancreases of Chinese mitten crabs. The widely used indicators ACP and AKP are potential indicators for evaluating the impact of pollutants on the immune defense of biological organisms [28,29]. The other important indicators GSH-ST and AchE are indicators of the metabolic processes of the liver and hepatopancreas in animals [30,31]. Therefore, we selected those genes from the target DEGs for the qPCR analyses. The results demonstrated that the enzymatic activities of ACP and AKP in the hepatopancreases of the specimens were significantly decreased in both the low- and high-concentration enrofloxacin residue groups (Figure 6A). Notably, the results demonstrated that the enzymatic activities of GSH-ST and AchE were also significantly decreased in both the low- and high-concentration enrofloxacin residue groups (Figure 6B). *3.7. Immune Responses and Metabolic Enzymatic Activities following Exposure to Enrofloxacin Residues*  Our analysis revealed that damage to the *E. sinensis* hepatopancreas was primarily associated with immune responses and metabolic processes. By analyzing DEGs, we found that enrofloxacin residues led to immune system and metabolic process disorders in the hepatopancreases of Chinese mitten crabs. The widely used indicators ACP and AKP are potential indicators for evaluating the impact of pollutants on the immune defense of biological organisms [28,29]. The other important indicators GSH-ST and AchE are indicators of the metabolic processes of the liver and hepatopancreas in animals [30,31]. Therefore, we selected those genes from the target DEGs for the qPCR analyses. The results demonstrated that the enzymatic activities of ACP and AKP in the hepatopancreases of the specimens were significantly decreased in both the low- and high-concentration enrofloxacin residue groups (Figure 6A). Notably, the results demonstrated that the enzymatic activities of GSH-ST and AchE were also significantly decreased in both the low- and high-concentration enrofloxacin residue groups (Figure 6B).

Our results indicated that exposure to low or high doses of enrofloxacin resulted in hepatopancreatic damage in *E. sinensis*. The RNA sequencing results demonstrated that enrofloxacin-induced hepatopancreatic damage was closely related to disorders of the

Enrofloxacin, a third-generation fluoroquinolone antibacterial drug, is commonly used to treat bacterial infections in crab breeding; however, the side effects of this antimicrobial treatment deserve attention. For example, the expression level of key genes and enzymes in the hepatopancreases of the *E. sinensis* specimens treated with enrofloxacin were disturbed [18]. The enrofloxacin biotransformation product, ciprofloxacin, at low

\*\*\* *p* < 0.001. (**A**) Pollution impact indicators, acid phosphatase (ACP) and alkaline phosphatase (AKP), indicating the impact of low- and high-concentration enrofloxacin residues on the immune defense of *E. sinensis*. (**B**) Metabolic process indicators, glutathione sulfotransferase (GSH-ST) and

metabolic processes and the immune system.

is "no significance".

**4. Discussion**

enrofloxacin residue group, and ENR-H represents the high-concentration enrofloxacin residue group. \*\*\* *p* < 0.001. (**A**) Pollution impact indicators, acid phosphatase (ACP) and alkaline phosphatase (AKP), indicating the impact of low- and high-concentration enrofloxacin residues on the immune defense of *E. sinensis*. (**B**) Metabolic process indicators, glutathione sulfotransferase (GSH-ST) and acetylcholinesterase (AchE), indicating the impact of low- and high-concentration enrofloxacin residues on metabolic processes of the liver and pancreas in *E. sinensis*. The meaning of "ns" in figure is "no significance".

#### **4. Discussion**

Our results indicated that exposure to low or high doses of enrofloxacin resulted in hepatopancreatic damage in *E. sinensis*. The RNA sequencing results demonstrated that enrofloxacin-induced hepatopancreatic damage was closely related to disorders of the metabolic processes and the immune system.

Enrofloxacin, a third-generation fluoroquinolone antibacterial drug, is commonly used to treat bacterial infections in crab breeding; however, the side effects of this antimicrobial treatment deserve attention. For example, the expression level of key genes and enzymes in the hepatopancreases of the *E. sinensis* specimens treated with enrofloxacin were disturbed [18]. The enrofloxacin biotransformation product, ciprofloxacin, at low and high doses affected the expression of many genes in the hepatopancreases of the *E. sinensis* specimens, and these were primarily enriched in the metabolic processes and the immune system.

The metabolism involves a series of reactions that occur in living cells to sustain life [32]. Metabolic disorders can lead to the occurrence and development of various disorders and diseases in organisms that seriously affect the quality of life of these organisms and that may even threaten their lives. In addition, the metabolic disorders of crabs seriously affect the yield and environment of the aquaculture [33–35]. In eukaryotes, the metabolic processes are involved in various interconnected cellular pathways, molecular signaling pathways, and metabolic materials and products [36–38]. At the molecular level, metabolic changes depend on the configuration of the metabolic pathways, which are regulated by key metabolic enzymes, transcription factors, protein modifications, and the metabolite clearance status; many of these pathways are closely related to the mitochondrial and lysosomal digestive functions [39,40]. In this study, we determined that enrofloxacin exposure affected the metabolism of many substances, including carbohydrates, lipids, starch, and sucrose, as determined by the GO and KEGG enrichment analyses. Specifically, the metabolic processes, including the metabolism of carbohydrates, tyrosine, glycerolipids, tryptophan, methane, purine nucleobases, and amino sugars, were significantly altered following enrofloxacin exposure. Moreover, the functions of the mitochondria and lysosomes were also affected by enrofloxacin. Significant changes in the enrichment results relative to the biological processes (the response to oxidative stress, tricarboxylic acid cycle, and protein import into the mitochondrial matrix) and molecular functions of the GO analysis (the ATPase activity, NADH dehydrogenase activity, and oxidoreductase activity) and KEGG analysis (the lysosome metabolism, oxidative phosphorylation, pantothenate and CoA biosynthesis, and the fatty acid metabolism) were all involved in the resultant damage of the mitochondrial and lysosomal functions following enrofloxacin exposure.

The metabolic pathways are well recognized as important regulators of immune differentiation and activation [41–43]. Chen et al. reported that Ophiopogon japonicus increased the immune response in *E. sinensis*, inhibited the proliferation of the white spot syndrome virus (WSSV), and improved the survival of WSSV-challenged crabs [44]. Abamectin insecticides and anthelmintics inflict oxidative damage on aquatic animals and impair the immune defenses, which may further cause a sharp drop in the hemocyte counts in *E. sinensis* [29]. Therefore, immune homeostasis is crucial for *E. sinensis* survival. We determined that enrofloxacin exposure affected the biological processes, including the response to oxidative stress, regulation of autophagy, and viral release from the host cells,

all of which were related to the homeostasis of the immune system [45–49]. Moreover, some DEGs related to the immune system showed significant changes in expression, including those of AKP, dual oxidase 1, and NF-κB inhibitor alpha, in the hepatopancreases of the *E. sinensis* specimens.

The widely used indicators ACP and AKP are potential indicators for evaluating the impact of pollutants on the immune defense of biological organisms [28]. Oxidative stress markers, GSH-ST, and AchE are important indicators that affect the metabolic processes of the liver and hepatopancreas in animals [50]. Overall, the ACP, AKP, GSH-ST, and AchE levels were clearly decreased following enrofloxacin exposure in the hepatopancreases of the *E. sinensis* specimens, confirming that enrofloxacin exposure affects the metabolism and immune response in the hepatopancreas of *E. sinensis*. However, the mechanisms by which enrofloxacin affects the metabolism and the immune system remain unclear and require further investigation.

#### **5. Conclusions**

In conclusion, this study not only provided novel evidence for the toxicological effects exerted on *E. sinensis* following enrofloxacin exposure but also helped elucidate the possible mechanisms underlying this toxicity and the corresponding cellular pathways that were activated, which notably involved the metabolic processes and immune responses.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/ijerph20031836/s1, Table S1: Statistics of gene annotation success rate.

**Author Contributions:** Conceptualization, S.L. and F.Z.; methodology, S.L., F.Z. and Q.W.; software, Q.W. and Z.X.; validation, Z.X. and Y.W.; formal analysis, Q.W., Z.X. and Y.W.; investigation, F.Z.; resources, C.H.; data curation, S.L.; writing—original draft preparation, S.L. and Q.W.; writing—review and editing, G.H., X.Z., J.L. and F.Z.; visualization, Z.X. and Y.W.; supervision, F.Z. and C.H.; project administration, S.L.; funding acquisition, S.L., C.H. and F.Z. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Key Research and Development Project (No. 2019YFC1605800), Key Subject of Ecology of the Jiangsu Province (SUJIAOYANHAN (2022) No. 2), Excellent Scientific and Technological Innovation Team of the Colleges and Universities of the Jiangsu Province (SUJIAOKE (2021) No. 1), and Key Subject of Biology of Nanjing (NINGJIAO-GAOSHI (2021) No. 16).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data sets used and analyzed during the current study are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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