Genetic Markers For Species Identification Research Articles

Characterization and Phylogenetic Implication of Complete Mitochondrial Genome of the Medicinal Ant Formica sinae (Hymenoptera: Formicidae): Genomic Comparisons in Formicidae.

Ants (Hymenoptera: Formicidae) are among the largest and most widespread families of terrestrial insects and are valuable to medical and ecological investigations. The mitochondrial genome has been widely used as a reliable genetic marker for species identification and phylogenetic analyses. To further understand the mitogenome-level characteristics of the congeneric Formicidae species, the complete mitogenome of Formica sinae (Hymenoptera: Formicidae) was sequenced, annotated, and compared with other 48 Formicidae species. The results showed that gene composition, content, and codon usage were conserved. The complete mitochondrial genome of F. sinae was 17,432 bp, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and one control region located between rrnS and trnM, which was 1,256 bp long, the longest of all sequenced species. Gene rearrangement was not detected in Formica species (Hymenoptera: Formicidae). All PCGs of F. sinae were initiated with ATN codons and terminated with the TAA codon. The overall nucleotide composition of F. sinae was AT-biased (83.51%), being 80.58% in PCGs, 86.68% in tRNAs, 87.10% in rRNAs, and 88.70% in the control region. Phylogenetic analyses indicated that each subfamily formed a strongly monophyletic group. Furthermore, F. sinae clustered with Formica fusca (Hymenoptera: Formicidae) and Formica selysi (Hymenoptera: Formicidae). This work enhances the genetic data of Formicidae and contributes to our understanding of their phylogenic relationship, evolution, and utilization.

The Identification of Fish Eggs From Four Economically Important Species in Guanghai Bay (China)

By far, very few studies have dealt with the ichthyoplankton diversity in Guanghai Bay (China), which is a potential spawning ground for many important fish species. In this study, fish eggs collected in Guanghai Bay were identified through molecular method combined with visual taxonomic method. We employed two mitochondrial gene regions of cytochromecoxidase subunit I (COI) and 16S ribosomal RNA (16S rRNA) as genetic markers for species identification. Through sequence identification at NCBI, 121 eggs with overlapping size range and easy to be confused were discriminated as four economically important species: seven as silver sillagoSillago sihama, 48 as black-banded sillagoSillago nigrofasciata, 38 as yellow drumNibea albiflora, and 28 as Pacific seabreamAcanthopagrus pacificus. Phylogenetic analyses showed that these 121 eggs clustered in four groups with strong support. To testify the validity of these identification results, species identification through five BarcodingR package methods was also carried out using sequences of 33 fish specimens as a reference library covering four target species. Finally, a highly consensus of species assignment results was achieved across different methods. Morphological characteristics and detailed photographs for eggs from these four species were supplied here. Eggs of each species are pelagic, round, have a smooth chorion and one single oil globule. Embryonic pigment patterns vary as eggs develop and can be used for species distinguishing. Eggs fromS. nigrofasciataandA. pacificuswere described for the first time in this study. One simple and accurate method for identifyingN. albifloraeggs was additionally provided. Moreover, the morphological differences between twoSillagoeggs offered supportive evidence for the recent separation ofS. nigrofasciataas a new species fromS. sihama. All these results would be critical for the discrimination of eggs from these four species and the estimation for their spawning areas. Meanwhile, our study would contribute to the stock assessment and fishery management in Guanghai Bay.

Molecular-genetic analysis markers for identification of <i>Rhodococcus</i> bacteria species

It was established that groEL genes can be used as molecular genetic markers for species identification of Rhodococcus bacteria. A restriction analysis scheme was developed for the PCR products of groEL genes using restriction enzymes BglI, NarI, SfiI, SinI, and RseI to identify the species of natural Rhodococcus bacteria. Fifty-two genome loci determining the resistance to stressful environmental condition were annotated in genome of R. pyridinivorans strain 5Ap (i.e. 49 structural and 4 regulatory genes that determine the synthesis of 23 heat shock proteins, 9 universal stress proteins, 17 P450 cytochromes). The unique nucleotide sequences encoding the synthesis of the heat shock protein DnaK (1 plasmid gene) and P450 cytochromes (2 chromosomal and 1 plasmid) were found among these genes. Thus, they can be used for molecular typing of the biotechnological R. pyridinivorans strain 5Ap.

Taxonomic classification of strain PO100/5 shows a broader geographic distribution and genetic markers of the recently described Corynebacterium silvaticum.

The bacterial strain PO100/5 was isolated from a skin abscess taken from a pig (Sus scrofa domesticus) in the Alentejo region of southern Portugal. It was identified as Corynebacterium pseudotuberculosis using biochemical tests, multiplex PCR and Pulsed Field Gel Electrophoresis. After genome sequencing and rpoB phylogeny, the strain was classified as C. ulcerans. To better understand the taxonomy of this strain and improve identification methods, we compared strain PO100/5 to other publicly available genomes from C. diphtheriae group. Taxonomic analysis reclassified it and three others strains as the recently described C. silvaticum, which have been isolated from wild boar and roe deer in Germany and Austria. The results showed that PO100/5 is the first sequenced genome of a C. silvaticum strain from livestock and a different geographical region, has the unique sequence type ST709, and could be could produce the diphtheriae toxin, along with strain 05–13. Genomic analysis of PO100/5 showed four prophages, and eight conserved genomic islands in comparison to C. ulcerans. Pangenome analysis of 38 C. silvaticum and 76 C. ulcerans genomes suggested that C. silvaticum is a genetically homogeneous species, with 73.6% of its genes conserved and a pangenome near to be closed (α > 0.952). There are 172 genes that are unique to C. silvaticum in comparison to C. ulcerans. Most of these conserved genes are related to nutrient uptake and metabolism, prophages or immunity against them, and could be genetic markers for species identification. Strains PO100/5 (livestock) and KL0182T (wild boar) were predicted to be potential human pathogens. This information may be useful for identification and surveillance of this pathogen.

Application of a Faecalibacterium 16S rDNA genetic marker for species identification of dog fecal waste.

A dog-associated 16S rDNA genetic marker (ED-1) was designed to detect dog fecal contamination in water through a comparative bioinformatics analysis of Faecalibacterium sequences. For the dog fecal samples, ED-1 had 100% specificity, a high positive rate (89% in dog feces and 92.3% in dog fecal-contaminated water samples), and a low detection limit (107 copies/100mL) in dog-contaminated water samples. Detection of water samples from seven provinces or cities of China showed that ED-1 was stable enough to be applied in practice. Furthermore, the abundance and diversity of dog gut microbiota from two private house pets (PHP) and Third Military Medical University (TMMU) dogs were estimated by using operational taxonomic units, and the significant differences of dog feces were found, as the PHP dogs have a more diverse diet and closer contact with human than dogs in TMMU. However, ED-1 could detect the feces from the two regions, indicating that ED-1 has good reliability.

Five new mitogenomes of Phylloscopus (Passeriformes, Phylloscopidae): Sequence, structure, and phylogenetic analyses

The genus Phylloscopus belongs to the order Passeriformes and subfamily Phylloscopinae within the family Sylviidae. Phylloscopus, a small insectivore widely distributed in the old world, includes 66 species and 112 subspecies. We identified five new Phylloscopus mitogenomes: P. tenellipes (16,904 base pairs (bp), MK390475), P. coronatus (16,905 bp, MK533705), P. borealis (16,881 bp, MK390476), P. schwarzi (16,920 bp, MK411584), and P. borealoides (16,904 bp, MN125373). All contained 37 genes, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and two control regions. Seven Phylloscopus species were compared to the five new mitogenomes and the published complete P. inornatus and P. proregulus mitochondrial sequences. The genetic distance of 13 single protein-coding genes exhibited low variation within all seven Phylloscopus mitogenomes. Based on the Ka/Ks ratios, the molecular evolution patterns of single protein-coding genes were relatively consistent among Phylloscopus bird species. Phylogenetic analysis verified that subspecies of the Pale-legged Leaf Warbler P. tenellipes borealoides could be promoted to the Sakhalin Leaf Warbler P. borealoides. Gene order and genome organization information is useful to understand evolutionary relationships among Phylloscopus species. The complete mitogenomes of these five Phylloscopus species provide genetic markers for species identification, population genetics, and phylogeographic studies of birds.

Shifting national surveillance of Shigella infections toward geno‐serotyping by the development of a tailored Luminex assay and NGS workflow

The phylogenetically closely related Shigella species and enteroinvasive Escherichia coli (EIEC) are responsible for millions of episodes of bacterial dysenteriae worldwide. Given its distinct epidemiology and public health relevance, only Shigellae are subject to mandatory reporting and follow‐up by public health authorities. However, many clinical laboratories struggle to differentiate non‐EIEC, EIEC, and Shigella in their current workflows, leading to inaccuracies in surveillance and rising numbers of misidentified E. coli samples at the National Reference Centre (NRC). In this paper, we describe two novel tools to enhance Shigella surveillance. First, we developed a low‐cost Luminex‐based multiplex assay combining five genetic markers for species identification with 11 markers for serotype prediction for S. sonnei and S. flexneri isolates. Using a test panel of 254 clinical samples, this assay has a sensitivity of 100% in differentiation of EIEC/Shigella pathotype from non‐EIEC strains, and 68.7% success rate in distinction of Shigella and EIEC. A novel, and particularly successful marker was a Shigella‐specific deletion in the spermidine acetyltransferase gene speG, reflecting its metabolic decay. For Shigella serotype prediction, the multiplex assay scored a sensitivity and specificity of 96.6% and 98.4%, respectively. All discrepancies were analyzed with whole‐genome sequencing and shown to be related to causative mutations (stop codons, indels, and promoter mutations) in glycosyltransferase genes. This observation spurred the development of an in silico workflow which extracts the Shigella serotype from Next‐Generation Sequencing (NGS) data, taking into account gene functionality. Both tools will be implemented in the workflow of the NRC, and will play a major role in the shift from phenotypic to genotyping‐based surveillance of shigellosis in Belgium.

Comparative Mitogenomic Analysis of Forensically Important Sarcophagid Flies (Diptera: Sarcophagidae) and Implications of Species Identification

The flesh flies (Diptera: Sarcophagidae) are significant in forensic investigations. The mitochondrial genome (mitogeome) has been widely used as genetic markers for phylogenetic analysis and species identification. To further understand the mitogenome-level features in Sarcophagidae, the complete mitogenome of Sarcophaga formosensis (Kirneret Lopes, 1961) (Diptera: Sarcophagidae) and Sarcophaga misera (Walker, 1849) (Diptera: Sarcophagidae) was firstly sequenced, annotated, and compared with other 13 Sarcophagidae species. The result indicated that the gene arrangement, gene content, base composition, and codon usage were conserved in the ancestral arthropod. Evolutionary rate of the mitogenome fragments revealed that the nonsynonymous and synonymous substitution rates (Ka and Ks) ratio was less than 1.00, indicating these variable sites under strong purifying selection. Almost all transfer RNA genes (tRNAs) have typical clover-leaf structures within these sarcophagid mitogenomes, except tRNA-Ser (AGN) is lack of the dihydrouridine arm. This comparative mitogenomic analysis sheds light on the architecture and evolution of mitogenomes in the Sarcophagidae. Phylogenetic analyses containing the interspecific distances from different regions in these species provided us new insights into the application of these effective genetic markers for species identification of flesh flies.

Specific primer design of mitochondrial 12S rRNA for species identification in raw meats

Polymerase chain reaction (PCR) is a molecular technique that widely used in agriculture area including species identification in animal-based products for halalness and food safety reasons. Amplification of DNA using PCR needs a primer pair (forward and reverse primers) to isolate specific DNA fragment in the genome. This objective of this study was to design specific primer from mitochondrial 12S rRNA region for species identification in raw beef, pork and chicken meat. Three published sequences, HQ184045, JN601075, and KT626857, were downloaded from National Center for Biotechnology Information (NCBI) website. Furthermore, those reference sequences were used to design specific primer for bovine, pig, and chicken species using primer3 v.0.4.0. A total of 15 primer pairs were picked up from primer3 software. Of these, an universal forward primer and three reverse primers which are specific for bovine, pig, and chicken species were selected to be optimized using multiplex-PCR technique. The selected primers were namely UNI_F (5’-ACC GCG GTC ATA CGA TTA AC-3’), SP_R (5’-AGT GCG TCG GCT ATT GTA GG-3’), BB_R (5’-GAA TTG GCA AGG GTT GGT AA-3’), and A_R (5’-CGG TAT GTA CGT GCC TCA GA-3’). In addition, the PCR products were visualized using 2% agarose gels under the UV light and sequenced to be aligned with reference sequences using Clustal Omega. The result showed that those primers were specifically amplified mitochondrial 12S rRNA regions from bovine, pig, and chicken using PCR. It was indicated by the existence of 155, 357, and 611 bp of DNA bands for bovine, pig, and chicken species, respectively. Moreover, sequence analysis revealed that our sequences were identically similar with reference sequences. It can be concluded that mitochondrial 12S rRNA may be used as a genetic marker for species identification in meat products.

Multiple Resistance to Glyphosate and Acetolactate Synthase Inhibitors in Palmer Amaranth (Amaranthus palmeri) Identified in Brazil

Palmer amaranth is native to the United States, but was discovered in 2015 in Brazil. Palmer amaranth populations in Brazil were very difficult to control using glyphosate, which resulted in many changes to standard weed management practices. A genotyping assay was used to confirm that the population detected in Mato Grosso State, Brazil, was correctly identified as Palmer amaranth and that it was not tall waterhemp. Greenhouse dose–response curves and shikimate accumulation assays showed that the Brazilian population was highly resistant to glyphosate, with an LD50 value (3,982 g glyphosate ha−1) more than twice the typical use rates and very little shikimate accumulation at 1 mM glyphosate concentrations in a leaf-disk assay. The Brazilian population was also resistant to sulfonylurea and imidazolinone acetolactate synthase (ALS) inhibitor herbicides. The resistance mechanisms in the Brazilian population were identified as increasedEPSPSgene copy number for glyphosate resistance (between 50- and 179-fold relativeEPSPSgene copy number increase) and two different alleles for target-site mutations in theALSgene (W574L and S653N). These results confirm the introduction of Palmer amaranth to Brazil using a genetic marker for species identification, as well as resistance to glyphosate and ALS inhibitors.

The complete mitochondrial genome of Dixella aestivalis (Diptera: Nematocera: Dixidae)

Dixidae, meniscus midges, belong to the suborder Nematocera of the order Diptera. The family includes 197 known species classified in nine genera. The complete mitochondrial genome of the Dixella aestivalis (Meigen) from the United Kingdom is reported here, along with its annotation and comparison with the genome of an unidentified species of Dixella from China. The circular genome consists of 16 465 bp and has a gene content consisting of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and a non-coding, A + T-rich, control region. The mitochondrial genome of D. aestivalis can be used to identify genetic markers for species identification, and will be valuable for resolving phylogenetic relationships within the genus, family Dixidae and suborder Nematocera.

Genetic approach for species identification: Forensic application of DNA barcoding

Species identification is an essential issue in forensic casework. The introduction of DNA barcoding highlighted the expanding use of Cytochrome c Oxidase I mitochondrial gene (COI) as a genetic marker for species identification. This study aims to look at the current method for species identification and how the COI methodology could be applied in a forensic environment. In particular, to test the effective application of COI analysis in forensics, we created experimentally critical conditions such as environmental and mixtures, commonly found in forensic cases, when stain morphology is often compromised.

Genetic diversity of medically important and emerging Candida species causing invasive infection.

BackgroundGenetic variation in the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region has been studied among fungi. However, the numbers of ITS sequence polymorphisms in the various Candida species and their associations with sources of invasive fungal infections remain poorly investigated. Here, we characterized the intraspecific and interspecific ITS diversity of Candida spp. strains collected from patients with bloodstream or oroesophageal candidiasis.MethodsWe selected cultures of representative medically important species of Candida as well as some rare and emerging pathogens. Identification was performed by micromorphology and by biochemical testing using an ID32C® system, as well as by the sequencing of rDNA ITS. The presence of intraspecific ITS polymorphisms was characterized based on haplotype networks, and interspecific diversity was characterized based on Bayesian phylogenetic analysis.ResultsAmong 300 Candida strains, we identified 76 C. albicans, 14 C. dubliniensis, 40 C. tropicalis, 47 C. glabrata, 34 C. parapsilosis (sensu stricto), 31 C. orthopsilosis, 3 C. metapsilosis, 21 Meyerozyma guilliermondii (C. guilliermondii), 12 Pichia kudriavzevii (C. krusei), 6 Clavispora lusitaniae (C. lusitaniae), 3 C. intermedia, 6 Wickerhamomyces anomalus (C. pelliculosa), and 2 C. haemulonii strains, and 1 C. duobushaemulonii, 1 Kluyveromyces marxianus (C. kefyr), 1 Meyerozyma caribbica (C. fermentati), 1 Pichia norvegensis (C. norvegensis), and 1 Lodderomyces elongisporus strain. Out of a total of seven isolates with inconsistent ID32C® profiles, ITS sequencing identified one C. lusitaniae strain, three C. intermedia strains, two C. haemulonii strains and one C. duobushaemulonii strain. Analysis of ITS variability revealed a greater number of haplotypes among C. albicans, C. tropicalis, C. glabrata and C. lusitaniae, which are predominantly related to endogenous sources of acquisition. Bayesian analysis confirmed the major phylogenetic relationships among the isolates and the molecular identification of the different Candida spp.ConclusionsMolecular studies based on ITS sequencing are necessary to identify closely related and emerging species. Polymorphism analysis of the ITS rDNA region demonstrated its utility as a genetic marker for species identification and phylogenetic relationships as well as for drawing inferences concerning the natural history of hematogenous infections caused by medically important and emerging Candida species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-015-0793-3) contains supplementary material, which is available to authorized users.

Novel SNP Genotyping Assays Facilitate Species Identification of Salvelinus Collected in a Recreational Fishery

AbstractMembers of the genus Salvelinus often occur sympatrically, exhibit a high degree of phenotypic plasticity, and commonly hybridize. These factors often complicate species identification and hybrid detection based solely on morphology. Many species and populations of Salvelinus are threatened or endangered and accurate species identification and hybrid detection are important for conservation. Single nucleotide polymorphism (SNP) markers offer several advantages over other classes of genetic markers for species identification. We used DNA sequences from six species of Salvelinus to identify 16 SNPs that exhibited fixed differences among species and 16 additional SNPs that were variable within different species. These markers were used for species identification of putative Bull Trout Salvelinus confluentus that were misidentified as Lake Trout S. namaycush in a recreational fishery in Lake Pend Oreille, Idaho. The markers developed in this study will be useful for Salvelinus species identification and provide important data for conservation efforts.

Analysis of three leafminers' complete mitochondrial genomes

Liriomyza trifolii (Burgess), Liriomyza huidobrensis (Blanchard), and Liriomyza bryoniae (Kaltenbach), are three closely related and economically important leafminer pests in the world. This study examined the complete mitochondrial genomes of L. trifolii, L. huidobrensis and L. bryoniae, which were 16141bp, 16236bp and 16183bp in length, respectively. All of them displayed 37 typical animal mitochondrial genes and an A+T-rich region. The genomes were highly compact with only 60–68bp of non-coding intergenic spacer. However, considerable differences in the A+T-rich region were detected among the three species. Results of this study also showed the two ribosomal RNA genes of the three species had very limited variable sites and thus should not provide much information in the study of population genetics of these species. Data generated from three leafminers' complete mitochondrial genomes should provide valuable information in studying phylogeny of Diptera, and developing genetic markers for species identification in leafminers.

Variations in BM224 microsatellite in green frogs of genus Rana

The variable microsatellite repeat BM224 has been revealed in the genomes of eight species of green frogs (Rana ridibunda, R. cf. bedriagae, R. cretensis, R. esculenta, R. lessonae, R. shquiperica, R. saharica, R. nigromaculata). Previously, this repeat was observed in members of the genus Bufo. In this paper, the possibility of using this genetic marker for species identification is discussed.

Validation of the barcoding gene COI for use in forensic genetic species identification

The application of forensics to wildlife crime investigation routinely involves genetic species identification based on DNA sequence similarity. This work can be hindered by a lack of authenticated reference DNA sequence data resulting in weak matches between evidence and reference samples. The introduction of DNA barcoding has highlighted the expanding use of the mtDNA gene, cytochrome c oxidase I (COI), as a genetic marker for species identification. Here, we assess the COI gene for use in forensic analysis following published human validation guidelines. Validation experiments investigated reproducibility, heteroplasmy, mixed DNA, DNA template concentration, chemical treatments, substrate variation, environmental conditions and thermocycling parameters. Sequence similarity searches using both GenBank BLASTn and BOLD search engines indicated that the COI gene consistently identifies species where authenticated reference sequence data exists. Where misidentification occurred the cause was attributable to either erroneous reference sequences from published data, or lack of primer specificity. Although amplification failure was observed under certain sample treatments, there was no evidence of environmentally induced sequence mutation in those sequences that were generated. A simulated case study compared the performance of COI and cytochrome b mtDNA genes. Findings are discussed in relation to the utility of the COI gene in forensic species identification.

ITS2 variability of Biomphalaria (Mollusca, Planorbidae) species from the Paranapanema Valley (São Paulo State, Brazil): diversity patterns, population structure, and phylogenetic relationships

The ribosomal DNA internal transcribed spacer 2 (ITS2) has been shown to be a useful genetic marker for species identification and phylogenetic reconstruction in the genus of freshwater snails Biomphalaria (Preston 1910). Additionally, ITS2 studies in Biomphalaria have uncovered significant intra-specific genetic variability suggesting the presence of cryptic species complexes. We obtained ITS2 sequences for the Biomphalaria species B. glabrata, B. tenagophila, B. occidentalis and B. peregrina from the Paranapanema Valley (Sao Paulo State, Brazil) and compared them with a comprehensive set of published Biomphalaria ITS2 sequences using Bayesian inference of phylogeny. Analysis of the resulting trees showed that the newly obtained B. glabrata sequences did not cluster with those from other Brazilian localities and that sub-structuring occurred among Brazilian B. tenagophila populations. Moreover, although ITS2 sequences seem to indicate clear genetic differentiation within both B. glabrata and B. tenagophila, evidence in support of the occurrence of cryptic species is more compelling for the latter. We discuss the significance and implications of the detected patterns of ITS2 variability for taxonomic studies in Biomphalaria.

Arbitrary Primer Based RAPD — A Useful Genetic Marker for Species Identification in Morels

Internal transcribed spacer (ITS) regions of 5.8S ribosomal DNA gene of 46 monospore cultures representing different species of family Morchellaceae were sequenced. Eight putative species namely Morchella esculenta, M. crassipes, M. angusticeps, M. conice, Mitrophora semilibera, Morchella spongiola, M. vulgaris and Verpa conica were designated to the sequenced cultures analyzed based on similarity with the best aligned sequence of the BLAST search. We recorded interspecific polymorphism in ITS regions of the 5.8S r-DNA gene amongst putative species of morels. However no intraspecific ITS polymorphism could be visualized amongst monospores from single and or different ascocarps collected from various geographical regions. PCR RAPDamplified profiles of different monospores were identical at intraspecific levels and could not be distinguished by eight arbitrary primers tested. RAPD profiles exhibited significant interspecific polymorphism distinguishing all the eight putative species from each other by primer OPP-6 (5’- GTG GGT TGA C- 3’) and a custom primer (5’-CGC ACC GCA G-3’). RAPDprofiles generated using aforesaid arbitrary primers can serve as useful genetic markers for species identification and to improve morel systematics. Present study is the first report on exploitation of random primers in species differentiation in morels.

Characterization of lactate dehydrogenase isozyme pattern and morphology of three marine fish cell lines

Three continuous marine fish cell lines of FG (i.e., Flounder Gill) from flounder (Paralichthys olivaceus) gill, SPH (i.e., Sea Perch Heart) from sea perch (Lateolabrax japonicus) heart and RSBF (i.e., Red Sea Bream Fin) from red sea bream (Pagrosomus major) fin, were characterized by lactate dehydrogenase (LDH) isozyme and morphological analysis. The LDH isozyme patterns of these three cell lines and their corresponding tissues of origin were investigated and compared. The results showed: (1) No difference was found in the LDH isozyme patterns of FG and flounder gill tissue. However, the LDH isozyme patterns of SPH and RSBF were significantly different from their corresponding tissues of origin; (2) LDH isozyme patterns of FG, SPH and RSBF were markedly different from each other and could serve as genetic markers for species identification and detection of cross contamination. Morphological change analysis of these three cell lines in comparison to their original tissues indicated that FG cells still appeared epithelioid without morphological transformation. However, morphological changes were found in SPH and RSBF compared to their original tissues. Therefore, the cellular morphology was still plastic in the relatively stable culture conditions, and it was possible that change of LDH patterns was related to morphological changes of fish cellsin vitro.