Abstract: Background and Objectives: To improve the sustainable management and control of North American, western mosquitofish (Gambusia affinis) in the freshwater of Bangladesh, the most invasive to indigenous species, caused the severe infestation to all carps, genetic diversity of the species were studied using DNA sequences of nucleotides first time in Bangladesh. This study was aimed to develop molecular detection method to confirm the G. affinis in freshwater and construct a genetic baseline to control this species. Materials and Methods: This study consisted of 6 microsatellites nuclear DNA loci that were used for the construction of a genetic baseline used a gene-specific marker (cytb). A total of 6 fish for 6 sites were used for DNA extraction, then genotyped and analyzed to examine genetic diversity to assess the persistence of a species in the environment. Results: Genetic diversity was inferred as some polymorphism and monomorphism with minimal genetic distance, in which a UPGMA dendrogram showed the same cluster. MEGA X [3] computes pairwise distance (0.01) and overall mean distance (0.01) of substitution type is nucleotides between a query and the database sequences were constructed. The optimal tree with the sum of branch length = 0.036 showed a homogenous pattern among lineages. Conclusion: This study confirms the limited genetic diversity of G. affinis, across 6 sampling sites which might be used in the future to control mosquitofish (G. affinis) and the propagation of its parasitic Lernaea sp.
INTRODUCTION
The two species of mosquitofish, Gambusia affinis and Gambusia holbrooki have been introduced to more than 40 countries of the world to control mosquito larvae which in turn negatively affected many other species in each distinct bioregion1-2. No taxonomic record is available from Bangladesh literature about this fish3-4. Mosquitofish is distributed all over Bangladesh but especially highly abundant in Jashore region. It is found in different habitat including the natural ditches, carp rearing pond, brood culture pond, drain and nursery pond. The Mosquitofish or Plague Minnow is serious threat to native fish in Bangladesh that spread pathogens among fish and competes with them for food and habitat also feeds on native fish fry at the water's surface and preys on the eggs. It carries parasites such as Lernaea sp. which is a major threat for the hatchery population also the total amount of annual production. To overcome the issue, a broad study on genetic diversity in hatchery and wild population is obligatory to control the species of G. affinis.
The persistence of a species in the environment and genetic differentiation5 depends upon genetic drift6, overexploitation, habitat destruction and fragmentation and the introduction of alien species7 which affect the demographic dynamics of a population8 and consequently the evolutionary ability and genetic diversity of the population9.
Preservation of genetic diversity and identification of specific genetic units for management are important goals in biodiversity conservation programmes10. Molecular detection of live-bearing fish would also help to control this species and propagation of Lernaea sp. for improved aquaculture production. G. affinis are native to North America and were introduced to many countries for mosquito control in the sixties11-12 control mosquito larvae13. A recent study reported no taxonomic record3,4 in the Jashore aquaculture population, only breeding behavior14 of G. affinis were examined.
This species has been used to examine for a wide variety of conservation and evolutionary questions. Currently, there are only 6 microsatellite loci developed from G. affinis, but one of these is gene-specific15. As a small and fast-growing, live bearing (ovoviviparous) fishes, it has a high reproductive potential and tolerance to a broad range of environmental conditions16-17.
Invasive live-bearing fish in Bangladesh have long been considered to be guppies fish, Poecilia reticulata18-19. Therefore, the possibility that invasive fish is actually G. affinis. To date, this fish is widely distributed in Jashore district (Fig. 1a-b and Table 1) do serious threat to native fish and acts as a vector of crustacean parasite (Lernaea sp.) to cultured fish (Fig. 1a-b). To overcome the issue, a broad study on genetic diversity in hatchery and wild population is obligatory to control the species of G. affinis.
| Fig. 1(a-b): | Gambusia affinis act as a vector of parasitic Lernaea sp. to freshwater aquaculture species, (a) Mosquitofish is most susceptible ectoparasites to Lernaea sp. which act more as a vector of infestation to carps in their adult stage than to other freshwater species and (b) In semi-intensive polyculture system, Catla catla is one of the important infested species, so the mosquitofish in this system is completely undesirable with highly reproductive and competitive invasive nature |
The molecular detection, genetic diversity and genetic information could assist to control invasive nature and prevent further expansion of this species in the freshwater aquaculture of Bangladesh. Hence, the aim of this study was to evaluate the SSR DNA and microsatellite markers to facilitate the way of molecular detection, combined morphometric information using20 method (data were not shown) for G. affinis. The sequence variation of the mitochondrial DNA of G. affinis was compared with reference nucleotides of Gene Bank (NCBI, BLAST) to confirm the identity of this species and analyzed the genetic variations of parasite-bearing G. affinis in the freshwater aquaculture of Bangladesh.
MATERIALS AND METHODS
Biological materials of mosquitofish and sites: In the present study, the guppies were searched for in the south-western region of Bangladesh including 26 Upazila of 7 districts from July 2017 to June, 2018 using dip cast nets (2.5 mm mesh size). As an invasive and parasite (anchor worm) carrier, the mosquitofish is a major threat in those areas, especially for carp hatcheries (Fig. 1a-b). In present study mosquitofish was observed in seven (7) district (Shatkhira, Bagerhat, Khulna, Jashore, Narail, Magura and Jhenaida), including 26 Upazila such as Jashore sadar, Chougasa, Jhikorgasa, Monirumpur, Keshoppur, Avainagar, Sharsha, Assasuni, Shyamnagar, Debhata, Rampal, Mollahat, Fakirhat, Mongla, Dumuria, Paikgacha, Phultala, Batiaghata, Kalia, Lohagara, Noril sadar, Mohammadpur, Shalikha, Magura sadar, Khaligong, Kotchandpur and Moheshpur in the south-western region of Bangladesh (Fig. 2a). Initially, the number of respondent fish farmers were 50 for each Upazila. Based on the initial farmer responses from 7 districts, the mosquitofish was found most abundantly in Jashore district including 6 Upazila like Jashore sadar, Chougacha, Jhikorgasa, Monirumpur, Keshoppur, Abhoynagar and Sursha. A total of 52 farmer opinions were compared and analyzed for mosquitofish in those 6 Upazila of Jashore district (Fig. 2b). The major threats obtained from those sites were for carp hatcheries, brood, nursery, grow-out ponds and wild water bodies. A total of one hundred twenty (120) juvenile fish (randomly selected female and male fish/site) of Gambusia affinis (20 for each sampling site) were collected from 6 different Upazilas of Jashore district (Table 1). The geographic locations of the sampling site are also shown in Table 1. Collected fish samples were reared stock in an aquarium, then kept in a zipper bag and stored at -20°C for the extraction of genomic DNA.
Genomic DNA extraction and quantification: The genomic DNA was extracted from fin tissue following methods21-22 of total representative 6 mosquitofish from 6-sampling sites. Approximately 30 mg tissues were cut into small pieces, homogenized by micro-tissue-grinder in a 1.5 mL microfuge tube and digested with proteinase K in extraction buffer [100 mM Tris-HCl, pH 8.0, 10 mM ethylenediaminetetraacetic acid (EDTA), 250 mM NaCl and 1% sodium dodecyl sulphate (SDS)] overnight at 37°C in a hot water bath (JP Selecta). DNA was purified by successive extraction with phenol: chloroform: isoamyl alcohol (25:24:1 v/v/v) and chloroform:isoamyl alcohol (24:1 v/v), respectively. DNA was precipitated first using 0.6 M volume of isopropanol in the presence of 0.3 M sodium acetate, pelleted by centrifugation, then re-suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). DNA was re-precipitated by adding two volumes of absolute ethanol in the presence of 0.3 M sodium acetate and pelleted by centrifugation. The pellets were then washed with 70% ethanol, air-dried and resuspended in an appropriate volume of TE buffer. The DNA quality was checked by on 1% agarose gel electrophoresis using a high-performance ultraviolet trans-illuminator of a gel documentation system BioDoc-ItTM Imaging system, the purity and concentration of eluted DNA were measured using a Nanodrop 2000-Spectrophotometer (Nanodrop Calibri, Titertek Berthold, Germany) in Table 2. DNA was stored at -20°C until required for further analysis.
| Table 1: | Sampling sites and geographical position of the samples in Jashore district |
| Table 2: | Purity and concentration of eluted DNA were measured using a Nanodrop 2000-Spectrophotometer for PCR amplification to get optimal sequencing results |
260/230 (1.0-2.0) ratio is a good indicator of DNA quality, to confirm noise and DNA contamination | |
| Fig. 2(a-b): | (a) Selected questionnaires were observed following FGD (Focus Group Discussion) about mosquitofish from 1300 fish farmers from 26 Upazila in 7 district in the south-western region of Bangladesh and (b) A total of 52 farmers’ opinion were compared and analyzed by SPSS about mosquitofish in the sampling sites (6 in Jashore district) |
37% fish farmers’ response were that mosquitofish is an invasive and parasite carrier, 34% responses were that this is harmful to hatchery and fish culture systems, 29% responded that mosquitofish is an emerging threat to carp aquaculture ponds and hatcheries to require eradication and stopping reproduction |
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| Table 3: | Six primers with corresponding bands sored and their size range, together with polymorphic bands observed in mosquitofish |
*Gene specific DNA marker, a: 23, b-f: 2, #Working annealing temperature | |
Primer selection and PCR amplification: Initially, 18 highly polymorphic microsatellite (SSR) primers were screened on a total of 6 representatives randomly selected mosquitofish from 6 sampling sites (Table 1), to test their character and diversity analysis11,23 that could be accurately scored. Primers were evaluated on the basis of intensity or resolution of bands, repeatability of markers and consistency within individuals and potential to differentiate populations (polymorphism). A final subset of six primers (Table 3) exhibiting good-quality, reproducible banding patterns were used for character and diversity analysis of representative sample sets. DNA profiles of whole sample sets using the 6 primers were repeated and obtained consistent banding patterns. The amplification conditions were performed following the methods described by Bala et al.24, with some modifications. PCR reactions were performed on each DNA sample in a 1 μL reaction mix containing 5 μL of Green Master mix (Go-Taq G2), 2 μL of 10 μM primers, 1 μL of template DNA and 3 μL of nuclease free water. DNA amplification was performed in an oil-free thermal cycler (Professional Standard Gradient, Biometra, Germany). The thermal profile for PCR amplification was set as follows: Preheating at 95°C for 2 min, 35 cycles of 1 min denaturation at 95°C, 1 min annealing at 57.4-58.9°C and elongation or extension at 72°C for 2 min. To ensure the complete extension of all amplified fragments, a final elongation step of 10 min at 72°C was followed.
Agarose gel electrophoresis of PCR-amplified products: The amplified PCR product from each sample was separated electrophoretically on 1% agarose gel (Nacalai tesque, Inc., Kyoto, Japan) containing ethidium bromide in a 1×TAE buffer at 120 V for 90 min (1.4% agarose gel was also tested but could not produce better resolution so 1% gel was used for all samples). TrackIt™ 1 kb plus DNA Ladder (Invitrogen™ by Thermo Fisher Scientific) was used as a molecular weight marker. DNA bands were observed on a high-performance, ultraviolet transilluminator and photographed with a gel documentation system (BioDoc-ItTM Imaging system, Cambridge, UK).
Gene sequencing: The cytochrome b (cytb) gene is one of the most commonly used genetic loci, applied in both taxonomy and forensic science, for the purpose of species identification25-27. In the present study, the sequences of the PCR amplified products using primers cytb_F (ATG GCC AAC CTA CGA AAA AC) and cytb_R (GGG TAG RAC ATA ACC TAC GAA G), in which genomic DNA was isolated from a randomly selected mosquitofish of each of 2 polymorphic populations’ (Jashore sadar and Chougacha) were determined using Big Dye Terminator v3.1 (Life Technologies, Thermo Fisher, Mulgrave, VIC) and resolved using a 3500xL genetic analyzer (Life Technologies). The resolved regions were incorporated into the genome sequence using Sequencer v5.1 (Gene Codes, Ann Arbor). The DNA sequence, alignment-based, phylogenetic analysis was carried out to examine the evolutionary relationships of organisms. Trees are calculated by similarities of sequence and based on the distances. Neighbour-joining methods try to correct the UPGMA method for its assumption that the rate of evolution was the same for all texa. Boots trapping appropriately analyzed the prediction to level of confidence, in which 1.0 is a very high level of confidence in the positioning of the node on the topology of the tree or the relationship of the genomic DNA sequence for all reference and quarry organisms.
Statistical analysis: Farmer’s responses obtained using different questionnaire forms were analyzed with IBM SPSS statistic 19. Allelic frequency, gene diversity, heterozygosity and polymorphism in 6 different populations of mosquitofish were analyzed using power marker software. Genomic DNA sequences were analyzed using BLAST, which computes a pairwise significant alignment between a query based on partial DNA nucleotides sequences with the database sequences from BLAST. The evolutionary relationships of the database, genomic DNA sequences with quarry sequence of mosquitofish were inferred using the neighbour-joining method. Evolutionary analysis were conducted in MEGA X [3].
RESULTS
Farmers’ opinion and status of mosquitofish: Mosquitofish is highly abundant in pond aquaculture systems and susceptible to ectoparasite Lernaea sp. (anchor worm) that infested carps (Catla catla) as shown in Fig. 1a-b. Jashore district is the most potential area for producing carp hatchling in Bangladesh. The most of the carp hatcheries in 7 districts in the south-western region of Bangladesh highly abound with mosquitofish, mosquitofish carries the parasitic Lernaea sp. (Fig. 2a). Among the 52 farmers, 37% said mosquitofish is an invasive and parasite carrier, 34% said this is harmful to hatchery and fish culture system and the rest of 29% according to fish farmers said mosquitofish is an emerging threat that needs to eradication and stop their reproduction in those six Upazila of Jashore district (Fig. 2b).
Allelic and loci variation within the genotypes: The microsatellite enriched DNA fingerprints were constructed using the standard procedures. In this study, 6 genotypes of mosquitofish were analyzed. Six SSR primers were used in this study. Amplified microsatellite loci were analyzed for polymorphism using 1% agarose gel electrophoresis (AGE). The result revealed that all the primer pairs (Table 3) detected monomorphism among the fish genotypes analyzed except cytb (Fig. 3a-e).
Size of alleles, genetic diversity and similarity indices (SI): The cytb primer only resulted in a polymorphic band as shown in Table 3. Primers (cytb, Gaaf10 and Gaaf14) generated the highest number of bands (1-2) whereas Gaaf15, Gaaf22 and Gaaf23 produced the least number of bands (1). The 6 primers yielded a total of 50 distinct bands of which 2 (4%) were polymorphic (either occurring in or absent in <95% of all individuals) and therefore used in the analysis (Fig. 3a-e. Among the 6 primers, all primers resulted in the maximum number of monomorphic bands and thus a high level of monomorphism in where gene diversity, heterozygosity and PIC (Polymorphism Information Content) was zero (0.0) analyzed by PowerMarker V3.25. DNA profiles of different primers are shown in Fig. 3. Despite some polymorphism, overall genetic diversity across all populations for all loci studied was 0.00. The Jashore Sadar and Chougasa population showed higher genetic diversity compared with the Jhikorgasha, Monirumpur, Keshoppur and Avainagar populations (Table 4). The genetic diversity values for the Jhikorgasha, Monirumpur, Keshoppur and Avainagar population are the lowest. However, the 95% confidence intervals showed no significant differences in genetic diversity between the populations. Intrapopulation SI (similarity indices) for the Jhikorgasha population was the highest (100%) followed by that of the Monirumpur, Keshoppur and Avainagar populations, respectively. The Jashore sadar was closer to the Chougasa population with a genetic similarity of 99% when compared with the 6 populations together.
UPGMA on genetic distance: The genetic distance value for the Jashore Sadar-Avoinagar population pair was the highest (1.00), whereas the value for the Jashore Sadar-Chougasa-Jikorgacha-Monirumpur-Kesobpur population was the lowest (0.00) (Fig. 4). The UPGMA dendrogram based on genetic distance resulted in the segregation of four populations of mosquitofish into 2 clusters: the Avoinagar alone belonged to one cluster, whereas the Jashore Sadar-Chougasa-Jikorgacha-Monirumpur-Kesobpur populations made another cluster. There was no second cluster that further separated subgroups.
Genome DNA sequences and evolutionary distance tree analysis: The DNA sequences of mosquitofish have been checked in NCBI using standard nucleotides BLAST (FASTA sequences).
| Table 4: | Estimates of genetic variation number and proportion of polymorphic bands and gene diversity obtained in different populations of mosquitofish |
| Fig. 3(a-e): | DNA profiles of 6 representative mosquitofish collected from 6 sampling sites generated using primers (a) cytb, (b) Gaaf10, (c) Gaaf14, (d) Gaaf15, (e) Gaaf22 and (f) Gaaf23 |
TrackIt™ 1 Kb plus DNA ladder (Invitrogen™ by Thermo Fisher Scientific) was used as molecular weight marker |
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The genome sequences of mosquitofish produced significant alignment with query cover (98%), E-value (0.0) and identities (99%) between G. affinis sequences and other published templet genome sequences of host species (Table 5). Comparisons between genome sequences are summarized in Table 5. The rooted phylogenetic tree with a common ancestor of cytb as a primer for genomic DNA sequences of mosquitofish in which neighbor genetic distance is 0.001 nucleotides for all sites that compared with database sequences. The optimal tree with the sum of branch length = 0.036 is shown (next to the branches) (Fig. 5). Despite the several clusters computed pairwise distance (0.01) and overall mean distance (0.01) substitution type is nucleotides’ using Kimura 2-parameter methods showed a homogenous pattern among lineages.
| Fig. 4: | Clustering of different population of Gambusia affinis using the unweighted pair group method of arithmetic mean (UPGMA) algorithm, genetic distance constructed and summarizing differentiation according to microsatellite profiles |
| Fig. 5: | Evolutionary relationships of database genomic DNA sequences (EF017514.1, HM443905.1, U18107.1, AP004422.1, AF412123.1, DQ075683.1, DQ075686.1, DQ075861.1, KF013227.1, HM443911.1) with quarry sequence cytb of Gambusia affinis was examined using the neighbor-joining method |
| Table 5: | Summary of metadata for genome sequences using BLAST nucleotides sequence in NCBI producing significant alignments |
DISCUSSION
In this study, the genomic DNA of Gambusia affinis were tested for selected SSR DNA and microsatellite markers, amplified by polymerase chain reaction (PCR), alleles were separated on 1% agarose gel electrophoresis and species detection was confirmed by genome DNA sequences. The software analysis and graphical presentations of all genotypic data revealed that all the primer pairs detected monomorphism among the fish genotypes except cytb (polymorphic), in where no significant differences in genetic diversity between the populations, heterozygosity and PIC (Polymorphism Information Content) was zero (0.0), the genetic distance was highest (1.00) and lowest (0.00) resulted into 2 cluster. Whereas, the genome sequences of mosquitofish produced significant alignment with reference nucleotides sequences in NCBI (BLAST).
In previous studies, presented the genetic differentiation and phylogeography of blue shark from the Mediterranean Sea and North-Eastern Atlantic Ocean and examined 2 mitochondrial genes (cytb and control region) amplified from 207 and 170 individuals respectively, with no obvious pattern of geographical differentiation28. In the present study, 6 SSR DNA and microsatellite markers (cytb, Gaaf10, Gaaf14, Gaaf15, Gaaf22, Gaaf23) generated difference genetic structure of in 6 different regions of G. affinis in the Jashore district. The population structure was often changed may with time due to either environmental effect or evolution. Previously, some researchers revealed, the genetic structure and diversity of Gambusia holbrooki1, genetic variation in wild and hatchery populations of (Macrobrachium rosenbergii) by DNA markers2,24 and genetic characterization and invasiveness of G. affinis were analyzed by Sanz et al.9.
One of the major objectives of our study was to see if populations locked genetic variation for G. affinis. All studies specimens from Jashore region yielded the exact same sequences of the six primer cytochrome b fragment. Based on the low degree of genetic variability, all mosquitofish in 6 Upazilas of Jashore district may have been introduced from the same source population, but future analysis using more markers (including fast-evolving nuclear markers), coupled with powerful, population genetic approaches are needed to shed additional light on this question, that’s investigated already by Prodöhl et al.29 and Sanz et al.9 using European G. holbrooki; Ayres et al.1 using Australian G. holbrooki; Purcell and Stockwell30 using G. affinis in New Zealand.
The results of the research indicated that an incomplete understanding of the farmers in the introduction of G. affinis (i.e., man-made dispersal) in freshwater aquaculture due to active disposal along watersheds causes invasive nature to native species as well as parasite carriers (such as: anchor worm-like Larneae sp.) that were the limitations for research. In previous work, the mosquitofish introduction continues to impose a massive threat not only to the conservation of indigenous biodiversity but also to fisheries31. Where mosquitofish become invasive, they cause in population reductions of native aquatic species, including different detritivores, zooplankton and microplankton (rotifers, crustaceans, backswimmers, etc.), amphibians and fishes, all of which have the potential to affect vital components of ecosystem functioning32-35. Even the introduction of only a few mosquitofish individuals risks of local population establishment and further range expansion, as mosquitofish have a high reproductive potential1,33. It remains to be studied how aquatic faunas in freshwater ecosystems will change that relative to the roles of genetic and environmental factors36 throughout Jashore district, as the further spread of mosquitofish is likely hampered only by colder climates towards higher latitudes37.
In this study, it is clear that the SSR DNA and microsatellite markers showed genetic diversity was inferred as some polymorphism and monomorphism with minimal genetic distance, in which a UPGMA dendrogram showed the same cluster into the revealed genotypes. As a recommendation, the information about molecular detection and genetic diversity might be utilized in future to control treatment programs for eradication of G. affinis. In the current study, combined molecular information (based on phylogenetic analyses of sequence variation of the mitochondrial cytochrome b gene) used to confirm the presence of only one species, G. affinis, across 6 sampling sites in greater Jashore district of Bangladesh.
CONCLUSION
This study will help to broaden our understanding of the population genetic structure and genetic diversity of G. affinis. It was clearly shown that the genotypic data using all molecular techniques and software analysis confirmed the G. affinis in freshwater aquaculture systems as invasive nature with minimal genetic distance. It can be concluded that this information’s on molecular detection and genetic variability might be of interest as a starting point for a control program to eradicate the G. affinis along with Larneae that parasitic to fish in hatchery, wild and pond aquaculture system of Bangladesh.
SIGNIFICANCE STATEMENT
This study discovers the understanding of population genetic structure and genetic diversity of G. affinis that can be beneficial for fisheries community (hatchery, wild and pond aquaculture) in Bangladesh. The information about molecular detection and genetic diversity might be utilized in future to control treatment programs for eradication of invasive species (G. affinis). The size of the alleles, allelic variation within the genotypes, genetic variability, distance matrix and sequence data of mitochondrial DNA of G. affinis revealed the significant effects on control measures. This study will help the researcher to uncover the critical areas of molecular detection and genetic analysis that many researchers were not able to explore in Bangladesh. This new approach can be applied to data in several science fields and which can help reduce the drug use by farmers to control G. affinis and also complete eradication using all genetic information’s may have arrived,
ACKNOWLEDGMENTS
The authors wish to thank Jashore University of Science and Technology for fundings (Serial Number: 1). We gave special thanks to the Chief Scientific Officer of the Bangladesh Institute of Nuclear Agriculture (BINA), for providing the facilities to undertake this work. We thank Ph.D. candidate (Md. Monjur Hossain) of BINA and my research student (Md. Ariful Haque Rupom, Jannatul Ferdoush) for their contributions in research and preparing this manuscript. We thank the Chairman of the Department of Fisheries and Marine Bioscience. We are grateful to the carp hatchery owners in the greater Jashore district for fish samples.