Morphology and molecular phylogeny of Macrobrachium prachuapense sp. nov. (Decapoda: Palaemonidae) from Southern Thailand.

A new species of small freshwater prawn in the Macrobrachium pilimanus species group was found in the upper southern peninsula of Thailand. The prawns in this group exhibit velvet setae on the telopodites of the second pereiopods. The new species, named M. prachuapense sp. nov., is endemic to Thailand. It has several characteristics that make it standing apart and different from closely related species in the group, e.g. M. naiyanetri, M. forcipatum, M. malayanum, M. dienbienphuense, M. eriocheirum, and M. pilosum. The distinguishing characteristic of the new species is the shape of carpus of the second pereiopod (sub-cylindrical and subequal to palm), similar only to that of M. dienbienphuense. However, a fully-grown male of the new species was less than two-third the size of a fully-grown male M. dienbienphuense. Phylogenetic analysis further enhanced its novel species status with respect to its position in the phylogenetic tree relative to other closely related species.

Tilapia lake virus (TiLV): Genomic epidemiology and its early origin.

Tilapia lake virus (TiLV) is an emerging virus that is rapidly spreading across the world. Over the past 6 years (2014-2020), TiLV outbreaks had been reported in at least 16 countries, spanning three continents, including Asia, Africa, and America. Despite its enormous economic impact, its origin, evolution and epidemiology are still largely poorly characterized. Here, we report eight TiLV whole-genome sequences from Thailand sampled between 2014 and 2019. Together with publicly available sequences from various regions of the world, we estimated the origin of TiLV to be between 2003 and 2009, 5-10 years before the first report of the virus in Israel in 2014. Our analyses consistently showed that TiLV started to spread in 2000s, and reached its peak in 2014-2016, matching well with the timing of its first report. From 2016 onwards, the global TiLV population declined steadily. This could be a result of herd immunity building up in the fish population, and/or a reflection of a better awareness of the virus coupled with a better and more cautious protocol of Tilapia importation. Despite the fact that we included all publicly available sequences, our analyses revealed long unsampled histories of TiLVs in many countries, especially towards its basal diversification. This result highlights the lack and the need for systematic surveillance of TiLV in fish.

Morphology and molecular phylogeny of Macrobrachium saengphani sp. nov. (Decapoda: Palaemonidae) from Northern Thailand.

A small wild prawn of the genus Macrobrachium, found in Chiang Rai Province, Northern Thailand has some morphological features resembling four other closely related species, M. lanchesteri, M. peguense, M. kunjuramani, and M. chainatense. However, it is distinguishable from the above species in terms of distinctive golden colored antennules; number of teeth on the rostrum; number of teeth on the cutting edges of the second pereiopod; and length of carpus relative to that of chela on the second pereiopod. Moreover, DNA analysis places it far apart on the phylogenetic tree from the related species in the genus.

Draft genome sequence of scale drop disease virus (SDDV) retrieved from metagenomic investigation of infected barramundi, Lates calcarifer (Bloch, 1790).

Scale drop disease virus (SDDV) is a novel viral pathogen considered to be distributed in farmed barramundi (Lates calcarifer) in South-East Asia. Despite the severity of the disease, only limited genomic information related to SDDV is available. In this study, samples of SDDV-infected fish collected in 2019 were used. The microbiome of brain tissue was investigated using Illumina HiSeq DNA sequencing. Taxonomic analysis showed that SDDV was the main pathogen contained in the affected barramundi. De novo metagenome assembly recovered the SDDV genome, named isolate TH2019, 131 kb in length, and comprised of 135 ORFs. Comparison between this genome and the Singaporean SDDV reference genome revealed that the nucleotide identity within the aligned region was 99.97%. Missense, frameshift, insertion and deletion mutations were identified in 26 ORFs. Deletion of four deduced amino acid sequence in ORF_030L, identical to the SDDV isolate previously identified in Thailand, would be a potential biomarker for future strain classification. Interestingly, the genome of SDDV TH2019 harboured a unique 7,695-bp-long genomic region containing six hypothetical protein-encoded genes. Collectively, this study demonstrated that the SDDV genome can be sequenced directly, although with limited coverage depth, using metagenomic analysis of barramundi sample with severe infection.

Two-year surveillance of tilapia lake virus (TiLV) reveals its wide circulation in tilapia farms and hatcheries from multiple districts of Bangladesh.

Tilapia lake virus (TiLV) is an emerging pathogen in aquaculture, reportedly affecting farmed tilapia in 16 countries across multiple continents. Following an early warning in 2017 that TiLV might be widespread, we executed a surveillance programme on tilapia grow-out farms and hatcheries from 10 districts of Bangladesh in 2017 and 2019. Among farms experiencing unusual mortality, eight out of 11 farms tested positive for TiLV in 2017, and two out of seven tested positive in 2019. Investigation of asymptomatic broodstock collected from 16 tilapia hatcheries revealed that six hatcheries tested positive for TiLV. Representative samples subjected to histopathology confirmed pathognomonic lesions of syncytial hepatitis. We recovered three complete genomes of TiLV from infected fish, one from 2017 and two from 2019. Phylogenetic analyses based on both the concatenated coding sequences of 10 segments and only segment 1 consistently revealed that Bangladeshi TiLV isolates formed a unique cluster within Thai clade, suggesting a close genetic relation. In summary, this study revealed the circulation of TiLV in 10 farms and six hatcheries located in eight districts of Bangladesh. We recommend continuing TiLV-targeted surveillance efforts to identify contaminated sources to minimize the countrywide spread and severity of TiLV infection.

In experimental challenge with infectious clones of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV), MrNV alone can cause mortality in freshwater prawn (Macrobrachium rosenbergii).

To overcome the lack of immortal shrimp cell lines for shrimp viral research, we constructed and tested DNA infectious clones of Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) often found together in freshwater prawn (M. rosenbergii) exhibiting white tail disease (WTD). Full-length cDNAs of MrNV and XSV genomic RNA were individually inserted into the baculovirus pFastBacDUAL shuttle vector. Individual Sf9 (insect cell line) transfection resulted in production of RNA (RT-PCR) and capsid proteins (immunofluorescence) for both viruses. Presence of respective virions was confirmed by density gradient purification followed by RT-PCR and transmission electron microscopy. Infectivity was by tested in immersion-challenge tests with M. rosenbergii post-larvae (PL) using both semi-purified viruses, individually or combined, and confirmed by histological analysis (morphology and immunofluorescence) and quantitative RT-PCR. Mortality accompanied by WTD lesions occurred with MrNV alone or in combination with XSV but not with XSV alone, despite its replication.

Macrobrachium chainatense sp. nov. (Decapoda: Palaemonidae): a freshwater prawn from Thailand based on morphology and molecular phylogeny.

Many species of freshwater prawns, large and small, inhabit Southeast Asian countries. In Thailand alone, there have been recent reports of new species in the genus Macrobrachium. Morphological differentiation and, recently, DNA sequence differences have been used to distinguish new species from valid ones. Macrobrachium chainatense (Decapoda: Palaemonidae), a small freshwater prawn found in Thailand, is now established as a new species based on both morphological and genetic criteria. Among Thailand Macrobrachium prawns, M. chainatense has smallest body size, largest egg size, and 3-5 teeth on the cutting edges. The most closely related Macrobrachium species to M. chainatense in terms of COI and 18S DNA sequences is M. niphanae. Their COI sequences differ by 7.3% while their 18S sequences are basically identical in all but one site. Observations of some aspects of this prawn in captivity are also utilized to establish its unique species status. This prawn may be useful as a source of cooked food for human consumption.

Morphology and molecular phylogeny of Macrobrachium suphanense sp. nov. (Decapoda: Palaemonidae) from Thailand.

A small freshwater prawn in Thailand has been found to be a new species and is named Macrobrachium suphanense (Decapoda: Palaemonidae). Fully grown male M. suphanense appeared very different from the most closely related male of M. sintangense: size smaller, second pereiopod smaller and less robust, rostrum form different and, in females, fewer eggs. Less closely related, fully grown M. dolatum has sharper distal cutting edge on fixed finger and M. hungi has longer rostrum than M. suphanense. DNA analyses put M. suphanense, M. sintangense and M. nipponense in the same clade with M. nipponense sister to the other two. Two more clades consist of M. dienbienphuense and M. niphanae on the one hand and M. lanchesteri and M. rosenbergii on the other. The relationship among the three clades is not clearly resolved.

A Natural Vibrio parahaemolyticus ΔpirA Vp pirB Vp+ Mutant Kills Shrimp but Produces neither Pir Vp Toxins nor Acute Hepatopancreatic Necrosis Disease Lesions.

Acute hepatopancreatic necrosis disease (AHPND) of shrimp is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor a pVA plasmid encoding toxins PirA Vp and PirB Vp These are released from VPAHPND isolates that colonize the shrimp stomach and produce pathognomonic AHPND lesions (massive sloughing of hepatopancreatic tubule epithelial cells). PCR results indicated that V. parahaemolyticus isolate XN87 lacked pirA Vp but carried pirB Vp Unexpectedly, Western blot analysis of proteins from the culture broth of XN87 revealed the absence of both toxins, and the lack of PirB Vp was further confirmed by enzyme-linked immunosorbent assay. However, shrimp immersion challenge with XN87 resulted in 47% mortality without AHPND lesions. Instead, lesions consisted of collapsed hepatopancreatic tubule epithelia. In contrast, control shrimp challenged with typical VPAHPND isolate 5HP gave 90% mortality, accompanied by AHPND lesions. Sequence analysis revealed that the pVA plasmid of XN87 contained a mutated pirA Vp gene interrupted by the out-of-frame insertion of a transposon gene fragment. The upstream region and the beginning of the original pirA Vp gene remained intact, but the insertion caused a 2-base reading frameshift in the remainder of the pirA Vp gene sequence and in the downstream pirB Vp gene sequence. Reverse transcription-PCR and sequencing of 5HP revealed a bicistronic pirAB Vp mRNA transcript that was not produced by XN87, explaining the absence of both toxins in its culture broth. However, the virulence of XN87 revealed that some V. parahaemolyticus isolates carrying mutant pVA plasmids that produce no Pir Vp toxins can cause mortality in shrimp in ponds experiencing an outbreak of early mortality syndrome (EMS) but may not have been previously recognized to be AHPND related because they did not cause pathognomonic AHPND lesions.IMPORTANCE Shrimp acute hepatopancreatic necrosis disease (AHPND) is caused by Vibrio parahaemolyticus isolates (VPAHPND isolates) that harbor the pVA1 plasmid encoding toxins PirA Vp and PirB Vp The toxins are produced in the shrimp stomach but cause death by massive sloughing of hepatopancreatic tubule epithelial cells (pathognomonic AHPND lesions). V. parahaemolyticus isolate XN87 harbors a mutant pVA plasmid that produces no Pir toxins and does not cause AHPND lesions but still causes ∼50% shrimp mortality. Such isolates may cause a portion of the mortality in ponds experiencing an outbreak of EMS that is not ascribed to VPAHPND Thus, they pose to shrimp farmers an additional threat that would be missed by current testing for VPAHPND Moribund shrimp from ponds experiencing an outbreak of EMS that exhibit collapsed hepatopancreatic tubule epithelial cells can serve as indicators for the possible presence of such isolates, which can then be confirmed by additional PCR tests for the presence of a pVA plasmid.

Duplex PCR assay and in situ hybridization for detection of Francisella spp. and Francisella noatunensis subsp. orientalis in red tilapia.

Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.

Knockdown of Litopenaeus vannamei HtrA2, an up-regulated gene in response to WSSV infection, leading to delayed shrimp mortality.

HtrA2 is an apoptosis-activating gene that enhances the apoptotic process by preventing the formation of the IAP-caspase complex, thereby freeing caspase to trigger the apoptosis pathway. In this study, we presented the full-length cDNA sequence of HtrA2 from Litopenaeus vannamei (LvHtrA2). The full-length LvHtrA2 was 1335 bp, encoding 444 amino acids. This deduced amino acid sequence contained five conserved domains: a mitochondrial targeting signal (MTS), a transmembrane (TM) domain, an IAP-binding motif (IBM), a trimerization motif, a serine protease domain, and a PDZ domain normally found in the HtrA2 proteins of other organisms. A phylogenetic analysis revealed that LvHtrA2 clustered with the HtrA2 from other invertebrates and was closely related to Penaeus monodon HtrA2 (PmHtrA2). RT-PCR with RNA extracts from L. vannamei revealed that LvHtrA2 expression was found in several tissues, including the lymphoid organs, the haemocytes, the hepatopancreas, the gill, and the stomach, with different expression levels. When determining the role of LvHtrA2 in WSSV infection, it was found that LvHtrA2 transcription was early up-regulated in the WSSV-infected shrimp at 8h post-infection (p.i.) and expression still remained high at 48 h p.i.. It also demonstrated that dsRNA specific to LvHtrA2 reduced the cumulative mortality in the WSSV-infected shrimp compared with the control group. Additionally, depletion of the LvHtrA2 transcripts reduced expression levels for caspase-3 (Cap-3) gene in shrimp. This result could suggest that LvHtrA2 may involved in apoptosis mediated mortality rather than providing immune protection during WSSV infection.

Mitochondrial COI and nuclear RAG1 DNA sequences and analyses of specimens of the three morphologically established species in the genus Trichopsis (Perciformes: Osphronemidae) reveal new/cryptic species.

Air-breathing fish species of the genus Trichopsis have been reported in Cambodia, Lao PDR, Indonesia, Malaysia, Singapore, Thailand and Vietnam. It is only in Thailand that all three recognized species (Trichopsis vittata, Trichopsis schalleri and Trichopsis pumila), as judged by distinct external features, are found. Cambodia and Lao PDR harbor two species each. The present work involves first-time DNA sequencing and analysis based on mitochondrial (COI) and nuclear (RAG1) DNA of numerous specimens of these species and specimens of a controversial Phetchaburi (Thailand) fish population with a mixed outward appearance. In addition to confirming the morphologically clear-cut taxonomic division of the three fish species, our DNA results show that whereas the T. pumila populations form one single species, there are cryptic species in the T. vittata and T. schalleri populations and possibly a new one in the latter. Members of the putative Phetchaburi fish population have been proven to be hybrids between T. pumila and T. vittata. In addition, a new the phylogenetic tree indicating ancestral relationships is also presented. This study should generate further research to find new/cryptic species of the genus Trichopsis in all countries harboring the fish.

Laminin receptor protein is implicated in hemocyte homeostasis for the whiteleg shrimp Penaeus (Litopenaeus) vannamei.

Here we show that knockdown of laminin receptor (Lamr) with PvLamr dsRNA in the whiteleg shrimp Penaeus (Litopenaeus) vannamei (Pv) caused a dramatic reduction specifically in hyaline hemocytes prior to death. Since apoptosis was not detected in hemocytes or hematopoietic cells, other possible causes of hemocyte loss were investigated. Reports that suppression of crustacean hematopoietic factor (CHF)-like protein or hemocyte homeostasis-associated protein (HHAP) also reduced shrimp hemocyte counts led us to carry out yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays to test for interactions between Lamr and Pv homologues to these proteins (PvCHF-like and PvHHAP). The assays revealed that Lamr bound to both these homologues, but that the homologues did not bind to each other. Subsequent RT-PCR assays confirmed that PvLamr dsRNA injection significantly reduced expression levels for both PvCHF-like and PvHHAP genes. Further work is needed to determine how interaction among these three proteins can regulate shrimp hemocyte homeostasis.

Suppression of shrimp melanization during white spot syndrome virus infection.

The melanization cascade, activated by the prophenoloxidase (proPO) system, plays a key role in the production of cytotoxic intermediates, as well as melanin products for microbial sequestration in invertebrates. Here, we show that the proPO system is an important component of the Penaeus monodon shrimp immune defense toward a major viral pathogen, white spot syndrome virus (WSSV). Gene silencing of PmproPO(s) resulted in increased cumulative shrimp mortality after WSSV infection, whereas incubation of WSSV with an in vitro melanization reaction prior to injection into shrimp significantly increased the shrimp survival rate. The hemolymph phenoloxidase (PO) activity of WSSV-infected shrimp was extremely reduced at days 2 and 3 post-injection compared with uninfected shrimp but was fully restored after the addition of exogenous trypsin, suggesting that WSSV probably inhibits the activity of some proteinases in the proPO cascade. Using yeast two-hybrid screening and co-immunoprecipitation assays, the viral protein WSSV453 was found to interact with the proPO-activating enzyme 2 (PmPPAE2) of P. monodon. Gene silencing of WSSV453 showed a significant increase of PO activity in WSSV-infected shrimp, whereas co-silencing of WSSV453 and PmPPAE2 did not, suggesting that silencing of WSSV453 partially restored the PO activity via PmPPAE2 in WSSV-infected shrimp. Moreover, the activation of PO activity in shrimp plasma by PmPPAE2 was significantly decreased by preincubation with recombinant WSSV453. These results suggest that the inhibition of the shrimp proPO system by WSSV partly occurs via the PmPPAE2-inhibiting activity of WSSV453.

Anti-lipopolysaccharide factor isoform 3 from Penaeus monodon (ALFPm3) exhibits antiviral activity by interacting with WSSV structural proteins.

In innate immunity, antimicrobial peptides (AMPs) play a vital role in combating microbial pathogens. Among the AMPs identified in Penaeus monodon, only anti-lipopolysaccharide factor isoform 3 (ALFPm3) has been reported to exhibit activity against white spot syndrome virus (WSSV). However, the mechanism(s) involved are still not clear. In the present study, ALFPm3-interacting proteins were screened for from a WSSV library using the yeast two-hybrid screening system, revealing the five potential ALFPm3-interacting proteins of WSSV186, WSSV189, WSSV395, WSSV458 and WSSV471. Temporal transcriptional analysis in WSSV-infected P. monodon revealed that all five of these WSSV gene transcripts were expressed in the late phase of infection (24h and 48h post-infection). Of these, WSSV189 that was previously identified as a structural protein, was selected for further analysis and was shown to be an enveloped protein by Western blot and immunoelectron microscopy analyses. The in vitro pull-down assay using recombinant WSSV189 (rWSSV189) protein as bait confirmed the interaction between ALFPm3 and WSSV189 proteins. Moreover, pre-incubation of rWSSV189 protein with rALFPm3 protein interfered with the latter's neutralization effect on WSSV in vivo, as shown by the increased cumulative mortality of shrimp injected with WSSV following prior treatment with pre-incubated rWSSV189 and rALFPm3 proteins compared to that in shrimp pre-treated with rALFPm3 protein. Thus, ALFPm3 likely performs its anti-WSSV action by binding to the envelope protein WSSV189 and possibly other WSSV structural proteins.

Two new anti-apoptotic proteins of white spot syndrome virus that bind to an effector caspase (PmCasp) of the giant tiger shrimp Penaeus (Penaeus) monodon.

White spot syndrome virus proteins WSSV134 and WSSV322 have been shown to bind with the p20 domain (residues 55-214) of Penaeus monodon caspase (PmCasp) protein through yeast two-hybrid screening. Binding was confirmed for the p20 domain and the full-length caspase by co-immunoprecipitation. WSSV134 is also known as the WSSV structural protein VP36A, but no function or conserved domains have been ascribed to WSSV322. Discovery of the caspase binding activity of these two proteins led to an investigation of their possible anti-apoptotic roles. Full-length PmCasp was confirmed to be an effector caspase by inducing apoptosis in transfected Sf-9 cells as assessed by DAPI staining. Using the same cell model, comparison of cells co-transfected with PmCasp and either WSSV134 or WSSV322 revealed that both of the binding proteins had anti-apoptotic activity. However, using the same Sf-9 protocol with anti-apoptosis protein-1 (AAP-1; also called WSSV449) previously shown to bind and inactivate a different effector caspase from P. monodon (Pm caspase) did not block apoptosis induced by PmCasp. The results revealed diversity in effector caspases and their viral protein inhibitors in P. monodon.

Knockdown of a novel G-protein pathway suppressor 2 (GPS2) leads to shrimp mortality by exuvial entrapment during ecdysis.

A novel G-protein pathway suppressor 2 (GPS2) has been identified from hemocytes of the whiteleg shrimp Penaeus vannamei (Pv) and appears to play a role in ecdysis. The full-length of PvGPS2 cDNA consisted of a 1230-bp open reading frame, encoding 409 deduced amino acids with significant sequence homology to GPS2 sequences of crustaceans and insects. RT-PCR revealed that PvGPS2 was expressed in all P. vannamei tissues examined, but that expression was molt stage specific in eyestalk tissue. Relative expression was higher in the period before molting (i.e., intermolt and pre-molt stages) than in the post-molt stage. When double-stranded RNA (dsRNA)-mediated RNA interference was employed to inhibit PvGPS2 formation in shrimp, it led to significant mortality due to unsuccessful separation of new cuticle from old cuticle (exuvial entrapment) during ecdysis.

Construction and application of a protein interaction map for white spot syndrome virus (WSSV).

White spot syndrome virus (WSSV) is currently the most serious global threat for cultured shrimp production. Although its large, double-stranded DNA genome has been completely characterized, most putative protein functions remain obscure. To provide more informative knowledge about this virus, a proteomic-scale network of WSSV-WSSV protein interactions was carried out using a comprehensive yeast two-hybrid analysis. An array of yeast transformants containing each WSSV open reading frame fused with GAL4 DNA binding domain and GAL4 activation domain was constructed yielding 187 bait and 182 prey constructs, respectively. On screening of ∼28,000 pairwise combinations, 710 interactions were obtained from 143 baits. An independent coimmunoprecipitation assay (co-IP) was performed to validate the selected protein interaction pairs identified from the yeast two-hybrid approach. The program Cytoscape was employed to create a WSSV protein-protein interaction (PPI) network. The topology of the WSSV PPI network was based on the Barabási-Albert model and consisted of a scale-free network that resembled other established viral protein interaction networks. Using the RNA interference approach, knocking down either of two candidate hub proteins gave shrimp more protection against WSSV than knocking down a nonhub gene. The WSSV protein interaction map established in this study provides novel guidance for further studies on shrimp viral pathogenesis, host-viral protein interaction and potential targets for therapeutic and preventative antiviral strategies in shrimp aquaculture.

Biodiversity of the Betta smaragdina (Teleostei: Perciformes) in the northeast region of Thailand as determined by mitochondrial COI and nuclear ITS1 gene sequences.

In Thailand, there are currently five recognized species members of the bubble-nesting Betta genus, namely Betta splendens, B. smaragdina, B. imbellis, B. mahachaiensis and B. siamorientalis. In 2010, we indicated the possibility, based on COI barcoding evidence, that there might be two additional species, albeit cryptic, related to the type-locality B. smaragdina in some provinces in the northeast of Thailand. In the present study, after a more extensive survey of the northeast, and phylogenetic analyses based on COI and ITS1 sequences, the B. smaragdina group may be composed of at least 3 cryptic species members. The phylogenetic positions of these B. smaragdina group members in the bubble-nesting bettas' tree together with those of their congeners have been consolidated by better DNA sequence quality and phylogenetic analyses. With a better supported tree, the species statuses of B. siamorientalis and the Cambodian B. smaragdina-like fish, B. stiktos, are also confirmed.

Southeast Asian mouth-brooding Betta fighting fish (Teleostei: Perciformes) species and their phylogenetic relationships based on mitochondrial COI and nuclear ITS1 DNA sequences and analyses.

Fighting fish species in the genus Betta are found in several Southeast Asian countries. Depending on the mode of paternal care for fertilized eggs and hatchlings, various species of the betta fish are classified as mouth brooders or nest builders whose members in turn have been grouped according to their similarities mainly in morphology. The mouth brooders as well as some nest builders involved in the present study include fishes discovered and identified subsequent to previous reports on species groupings and their positions on phylogenetic trees based on DNA sequences that differ from those used by us in this study. From the mitochondrial COI gene and nuclear ITS1 gene sequences and more accurate analyses we conclude that the following members of the mouth-brooding pairs, named differently previously, are virtually identical, viz the Betta prima-Betta pallida pair and Betta ferox-Betta apollon pair. The Betta simplex, hitherto believed to be one species, could possibly be genetically split into 2 distinct species. In addition, several other established type-locality fishes could harbor cryptic species as judged by genetic differences. Assignments of fish species to groups reported earlier may have to be altered somewhat by the present genetic findings. We propose here a new Betta fish phylogenetic tree which, albeit being similar to the previous ones, is clearly different from them. Our gene-based evidence also leads to assignments of some fishes to new species groups and alters the positions of some species on the new phylogenetic tree, thus implying different ancestral relationships.