The EHP-infected L. vannamei and the EHP-infected M. rosenbergii were collected from the same farm in Fengxian District, Shanghai Province, China (N30°53’18.6”, E121°35’32.3”), in March 2020. The farm suffered severe EHP infection in 2019. The infected L. vannamei were 2.0 ~ 3.0 cm in length. The unnormal M. rosenbergii were 1.0 ~ 1.8 cm in length. Both healthy L. vannamei and healthy M. rosenbergii were collected from the normal ponds on another farm in Fengxian District. Samples were transported to the laboratory with oxygen and then fixed in 95% ethanol for PCR analysis. The handling of shrimps followed the guidelines for the Ethical Committee of Experimental Animal Care at the Shanghai Ocean University of China.

For EHP detection, 10 individual samples of each shrimp and prawn were dissected. The hepatopancreas DNA was extracted using an animal organization DNA Extraction Kit (Tiangen Biotechnology, China) according to the manufacturer’s instructions, and stored at −20°C for PCR assays.

The target genes were amplified using the nested SWP-PCR method. Briefly: the PCR mixtures (25 μL) for both steps contained 0.625 units of Ex Taq DNA polymerase (Takara Bio) and 0.2 μM of each primer. For the first PCR reaction, outer primers SWP1F and SWP1R ( Table 1 ) were used to amplify a 514 bp fragment. The PCR cycling conditions include an initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 68°C for 45 s, and a final extension at 68°C for 5 min. The PCR products were checked using 1% agarose gel with DNA ladder.

The DNA fragments of the SWP1 gene from both EHP isolates were purified and cloned into the pMD18-T vector. The resulting plasmids were transferred into competent cells DH5α and cultured in Luria-Bertani (LB) medium. Recombinant colonies were selected by the Blue-White screening. The transformants were identified by colony PCR and then cultured in a shaking incubator for 4 hours. Finally, the positive colonies were sequenced using M13 sequencing primers by the ABI 3730xl DNA Analyzer.

For sequence homology analysis, the full-length SWP1 gene (EhSWP1, GenBank accession nos. MG015710, GenPept accession nos. AVQ09707) previously published by Jaroenlak et al. (2018) was used as the reference sequence, which was amplified from EHP strain isolated from P. vannamei. After cloning and sequencing, the nucleotide sequences obtained were edited and alignments performed using Clustal W and compared with other nucleotide sequences in the GenBank using the BLAST program at National Center for Biotechnology Information (NCBI).

For the SWP1 gene phylogeny, multiple nucleotide sequence alignment was carried out using Clustal W. The “find best DNA/Protein models” program was run to determine the best-fit model with the lowest Bayesian information criterion (BIC). Phylogenetic tree was constructed using Neighbor-Joining method based on the Tamura 3-parameter model in MEGA-X. Bootstrap with 1000 replications was set to assess branch support.

To solve the problem that the primers SWP2F and SWP2R could not amplify the SWP1 gene of EHP _Mr , the new inner primer pairs were designed with the aid of Primer Premier 6.0 software. Based on multiple sequence alignments results, primer positions were derived from the conserved regions of SWP1 gene from all EHP isolates. Specificity of the primers was initially checked using primer blast (www.ncbi.nlm.nih.gov/tools/primer-blast/). The new inner primer pairs, forward primer SWP2F′ (5’-GCAGAGTGTTGTTAAGGGTTTAAG-3’) and reverse primer SWP2R′ (5’-GCTGTTTGTCWCCAACTGTATT-3’), were designed to target 182 bp internally to the first PCR product ( Table 1 ).

To compare the validity of newly designed inner primers and the original inner primers, the external PCR products of three SWP-PCR positive L. vannamei (EHP _Lv ) and three SWP-PCR positive M. rosenbergii (EHP _Mr ) were selected as DNA template respectively.

For the original SWP-PCR method, the inner primers SWP2F and SWP2R ( Table 1 ) were used to generate a 147 bp fragment. The thermal cycling conditions include an initial denaturation at 95°C for 5 min, followed by 20 cycles of 95°C for 20 s, 64°C for 30 s, and 68°C for 20 s, and a final extension at 68°C for 5 min (Jaroenlak et al., 2016).

For the modified SWP-PCR method, 2nd-step (nested) PCR was carried out with the inner primers SWP2F′ and SWP2R′ ( Table 1 ) to amplify a 182 bp product. PCR cycling conditions were initiation denaturation at 95°C for 5 min, followed by 20 cycles of 95°C for 30 s, 55°C for 30 s, and 68°C for 20 s, and a final extension at 68°C for 5 min. All secondary PCR products were analyzed by electrophoresis on a 1% agarose gel.

The plasmid containing the SWP1 gene of the EHP _Lv (named pGEM-SWP1) was extracted from the positive colonies as described above. The series of 10-fold dilutions of pGEM-SWP1 were used as positive templates. The single PCR with the two primers sets SWP2F/2R and SWP2F′/2R′ were carried out respectively, for testing the comparative sensitivity of the modified nested PCR and original nested PCR.

The genomic DNAs of five different aquatic microsporidians were selected to evaluate the specificity of the designed inner primers. Enterospora epinepheli isolated from Epinephelus spp.; Nucleaspora hippocampi isolated from Hippocampus erectus (Wang et al., 2022); Enterocytospora artemiae isolated from Palaemonetes sinensis; Ameson portunus isolated from Portunus trituberculatus; Potaspora sp. (unidentified) isolated from Exopalaemon carinicauda. Enterospora and Nucleaspora were the closely related genus in the Enterocytozoon group Microsporidia (EGM) that mainly infect gastrointestinal tracts of their hosts (Stentiford et al., 2019). E. artemiae infected the hepatopancreas and gut of crustacean hosts (Rode et al., 2013). Ameson and Potaspora were selected as the representative species of microsporidia infecting the skeletal muscles of hosts (Ding et al., 2016; Wang et al., 2017). The DNA template extracted from EHP _Mr -infected M. rosenbergii was used as a positive control. The nested PCR conditions referred to the above.

To validate the two protocols, the hepatopancreas DNAs, isolated from naturally EHP _Lv -infected L. vannamei samples and naturally EHP _Mr -infected M. rosenbergii samples, were subjected to the first round of amplification. The outer primers SWP1F/1R successfully amplified a 514 bp DNA fragment from both infected L. vannamei and infected M. rosenbergii ( Figure 4 , top).

In the second round, the inner primers SWP2F/2R amplified the expected 147 bp fragment from all EHP _Lv -infected L. vannamei but were negative for any of the EHP _Mr -infected M. rosenbergii ( Figure 4 , middle), it means the existing SWP-PCR method can only detect the EHP _Lv . Whereas the novel inner primers SWP2F′/2R′ produced the predicted 182 bp fragment for both infected shrimp and infected prawn ( Figure 4 , bottom), indicating that the modified SWP-PCR method can detect not only EHP _Lv but also EHP _Mr .

In addition, comparing the second-round PCR products by the two methods, the typical products of the existing SWP-PCR method contained an unexpected DNA fragment which migrated very closely together with the target band of 147 bp ( Figure 4 , middle), similar to previous reports (Jaroenlak et al., 2016; Munkongwongsiri et al., 2022). While only one prominent band of 182 bp was formed by the novel primers, indicating that the modified method improved the specificity of PCR amplification ( Figure 4 , bottom).

The sensitivity of the two nested PCR was tested using the 10-fold dilution series of pGEM-SWP1 plasmid DNA. The result was shown in Figure 5 . In the single PCR, the modified method displayed a high sensitivity identical to the original SWP-PCR method, which could detect as low as 10³ copies of pGEM-SWP1 per reaction mix.

In cross-amplification assays, none of the other microsporidian showed any amplification product in the nested PCR ( Figure 6 ). This confirmed the specificity of the designed primers for EHP detection.

Microsporidia are known as the obligate intracellular parasite. To adapt to the host cell life, its genome has been extremely compressed (Corradi and Slamovits, 2011). The natural genetic variation of pathogenic microorganisms can determine the success of infecting the host and favorable mutations may help to expand its host range (Bonneaud and Longdon, 2020). Variants have been found in a variety of microsporidia, such as Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis and Enterocytozoon bieneusi (Duzlu et al., 2019; Li et al., 2019). According to the results of this study, a mutant strain of EHP has been detected in M. rosenbergii for the first time, and the difference of its SWP1 gene suggested that EHP is also quietly changing itself to infect other crustacean hosts.

The nested PCR method has become the common method for disease surveillance because of its high specificity and sensitivity. However, due to the strain differentiation of EHP, the single specific primers cannot recognize different strains of EHP. To solve this problem, in this study, we designed a pair of degenerate primers to meet the demand for the EHP mutants’ detection in disease control and prevention.

Furthermore, the combined use of SWP2F′/2R′ primer pairs and SWP2F/2R primer pairs will help us to identify EHP _Lv and EHP _Mr strains. In a batch of shrimp infected with EHP, if SWP2F′/2R′ is positive and SWP2F/2R test is negative, it suggests that this batch of samples is infected with EHP _Mr strain; if SWP2F′/2R′ is positive and SWP2F/2R test is also positive, it shows that this batch of samples is infected with EHP _Lv strain.

Most researches on genotyping of microsporidia mutant strains use ITS site as diagnostic target and seldom use SWP gene. However, in recent years, more and more studies demonstrated that SWP gene was a promising target for genotyping. Xiao et al. (2001) found that the SWP1 gene of E. cuniculi had genetic diversity; Ou et al. (2021) confirmed the canine-adapted genotypes (Group 11) of E. bieneusi are one unique group of genotypes, and genetically divergent from other genotype groups by the sequence difference in SWP1 gene. Polonais et al. (2010) found that the EhSWP1 C-terminal of four strains of human microsporidia E. hellem showed significant interspecific and intraspecific polymorphisms, suggesting that SWP gene is more suitable for genotyping than internal transcribed spacer (ITS) or small subunit ribosomal DNA (SSU-rDNA) sequences.

To investigate the spread of EHP in global shrimp aquaculture, genotyping will become an important problem that needs to be solved in epidemiology. The polymorphism of SWP1 gene in different isolates will make it a good marker for studying EHP genotyping. In addition, the mutation of SWP1 gene may help to increase our understanding of the adhesion of EHP spore wall proteins to different host cell surface receptors.