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Laguna Lake is an economically important resource in the Philippines, with reports of declining water quality due to fecal pollution. Currently, monitoring methods rely on counting fecal indicator bacteria, which does not supply information on potential sources of contamination. In this study, we predicted sources of Escherichia coli in lake stations and tributaries by establishing a fecal source library composed of rep-PCR DNA fingerprints of human, cattle, swine, poultry, and sewage samples (n = 1,408). We also evaluated three statistical methods for predicting fecal contamination sources in surface waters. Random forest (RF) outperformed k-nearest neighbors and discriminant analysis of principal components in terms of average rates of correct classification in two- (84.85%), three- (82.45%), and five-way (74.77%) categorical splits. Overall, RF exhibited the most balanced prediction, which is crucial for disproportionate libraries. Source tracking of environmental isolates (n = 332) revealed the dominance of sewage (47.59%) followed by human sources (29.22%), poultry (12.65%), swine (7.23%), and cattle (3.31%) using RF. This study demonstrates the promising utility of a library-dependent method in augmenting current monitoring systems for source attribution of fecal contamination in Laguna Lake. This is also the first known report of microbial source tracking using rep-PCR conducted in surface waters of the Laguna Lake watershed. HIGHLIGHTS DNA fingerprinting of E. coli, coupled with machine learning algorithms, can be used to discriminate fecal pollution sources in Laguna Lake, Philippines.; The majority of E. coli isolates can be attributed to sewage contamination, followed by human and agricultural sources.; Source-tracking methods can empower local agencies responsible for water quality management to minimize public health and economic risks.

A genetic assessment was done on the greenblotch parrotfish (Scarus quoyi Valenciennes, 1840) in three bays within southern Mindanao, Philippines. Mitochondrial 16S rRNA recovered 12 haplotypes, one of which was dominant in all sites. Despite the reported phenotypic variation from previous assessments, there was neither evidence of genetic structure (global Φst = 0.012, p = 0.13) nor isolation by distance (r = 0.05, p = 0.50). Genetic diversity was also low (Hglobal = 0.472; πglobal = 0.13%), with ~ 77% of haplotype diversity accounted for with just 69 samples. While connectivity suggests continuous larval exchange within southern Mindanao owing to the species’ high dispersal potential, low genetic diversity implies reduced effective population size, probably due to recent bottlenecks (e.g. overfishing, habitat destruction). Although this study provides baseline genetic information on the local population, inferences are considerably limited by the genetic marker used and the spatial scale under investigation. A thorough understanding of the population will be possible if the entire habitat range of the species is assessed using markers with high resolving power, such as the hypervariable mitochondrial control region, microsatellites, or genome-wide single nucleotide polymorphisms (SNPs).

Laguna Lake is the largest inland freshwater body in the Philippines. Although it is classified to be usable for agricultural and recreational purposes by the country's Department of Environment and Natural Resources (DENR), studies looking at lake ecology revealed severe fecal contamination which contributes to the deterioration of water quality. Determining the sources of fecal contamination is necessary for lake protection and management. This study utilized a library-independent method of microbial source tracking (LIM-MST) to identify sources of fecal contamination in selected Laguna Lake stations and tributaries. Genetic markers of the host-associated Escherichia coli, heat-labile toxin (LTIIA) and heat-stable II (STII), were used to identify cattle and swine fecal contaminations, respectively. Meanwhile, human mitochondrial DNA (mtDNA) was used to identify human fecal contamination. Results identified the presence of agricultural and human fecal contamination in Laguna Lake Stations 1 and 5, Mangangate River, and Alabang River. The selected sites are known to be surrounded by residential and industrial complexes, and most of their discharges find their way into the lake. The identification of the specific sources of fecal contamination will guide management practices that aim to regulate the discharges in order to improve the water quality of Laguna Lake. HIGHLIGHTS The Escherichia coli LTIIa and STII genes, as well as human NADH mtDNA, are useful biomarkers for detecting fecal contamination in Laguna Lake.; Fecal contamination from agricultural and human origins can be detected in some lake stations and rivers near Laguna Lake.; As indicated by the high detection of LTIIa gene, fecal contamination in the sampling sites was mostly of cattle origin.

Fecal contamination is one of the factors causing deterioration of Laguna Lake. Although total coliform levels are constantly monitored, no protocol is in place to identify their origin. This can be addressed using the library-dependent microbial source tracking (MST) method, repetitive element sequence-based polymerase chain reaction (rep-PCR) fingerprinting. Serving as a prerequisite in developing the host-origin library, we assessed the discriminatory power of three fingerprinting primers, namely BOX-A1R, (GTG)5, and REP1R-1/2-1. Fingerprint profiles were obtained from 290 thermotolerant Escherichia coli isolated from sewage waters and fecal samples of cows, chickens, and pigs from regions surrounding the lake. Band patterns were converted into binary profiles and were classified using the discriminant analysis of principal components. Results show that: (1) REP1R-1/2-1 has a low genotyping success rate and information content; (2) increasing the library size led to more precise estimates of library accuracy; and (3) combining fingerprint profiles from BOX-A1R and (GTG)5 revealed the best discrimination (average rate of correct classification (ARCC) = 0.82 ± 0.06) in a two-way categorical split; while (4) no significant difference was found between the combined profiles (0.74 ± 0.15) and using solely BOX-A1R (0.76 ± 0.09) in a four-way split. Testing the library by identifying known isolates from a separate dataset has shown that a two-way classification performed better (ARCC = 0.66) than a four-way split (ARCC = 0.29). The library can be developed further by adding more representative isolates per host source. Nevertheless, our results have shown that combining profiles from BOX-A1R and (GTG)5 is recommended in developing the MST library for Laguna Lake.

In lieu of the importance of species authentication of processed fishery products, no reports have been made on sardine products in the Philippines. This is partly because of the challenges in dealing with biological material that has been subjected to extreme conditions during the process. Although molecular tools are now within reach, they are often reliant on proprietary kits which make the process expensive. In this paper, we used hot sodium hydroxide and tris (HotSHOT) DNA extraction protocol and cytochrome oxidase I (COI) minibarcode amplification to genotype and identify selected brands of locally-produced canned and dried sardines. To validate, fresh representatives based on advertised and suspected identities of the processed samples were genotyped as well. Success rate of amplification has gone from 0% to 64% when the minibarcode was amplified, as opposed to using the full-length COI barcoding region. Phylogenetic analysis allowed for the successful identification of processed Sardinella lemuru. Species substitution was detected on samples marketed as dried tawilis; however, the resolving power of minibarcode was not enough to discriminate the samples up to the species level. Regardless, coupling HotSHOT extraction with minibarcode amplification proved to be effective with processed sardines. Using this method to authenticate other processed fishery products that have underwent extreme postharvest processing warrants further exploration.

DNA barcoding was done to identify the dominant species of sardines that comprised the landed catch of the sardine fishery in the Northern Mindanao Region (NMR), and juveniles or "lupoy" in the Northern Zamboanga Peninsula (NZP). Cytochrome oxidase I (COI) minibarcodes were sequenced for all samples, while 16S ribosomal subunit sequences were obtained from a subset of samples. In reference to a previously reported land catch survey, sequence data confirmed the identity of the most dominant sardines in NMR and lupoy from NZP as Sardinella lemuru. Meanwhile, those reported as S. gibbosa were identified as Herklotsichthys quadrimaculatus, while those reported as S. pacifica were identified as the recently described S. goni. Results highlight: 1) the limitation in identifying sardines during land catch surveys without further taxonomic verification, and 2) the utility of DNA barcoding as a tool for species identification. Moreover, genetic data revealed that S. goni occurs in the fisheries of NMR, thereby expanding its known habitat range. Since this study was limited only in using DNA barcodes for identification, a detailed morphological examination of the dominant sardines is recommended.

Population genetic structure of Sardinella lemuru in Philippine waters was examined using mitochondrial DNA markers, control region (CR) and cytochrome oxidase I (COI). The sampling effort was focused along the northern coast of Mindanao where the species was previously reported to have regionally structured stocks. Sequences from online databases were included to expand geographic coverage of the analyses. Phylogeographic and population genetic inferences revealed the presence of genetically structured sympatric haplogroups across the archipelago (CR: n = 493, K = 3, between-group genetic distance = 0.038 – 0.054, global Φ ST = 0 . 41 p = 159, K = 2, between-group genetic distance = 0.012, global Φ ST = 0 . 77 , p 0.001). In contrast, little to no genetic differentiation was inferred when samples were assigned based on geographic location (CR global Φ ST = 0 . 01 , p Φ ST = 0 . 001 , p > 0.05). These results suggest that (1) gene flow occurs across space, but is limited among sympatric haplogroups, (2) these sympatric haplogroups do not correspond to cryptic species, and (3) differences in mutation rates between the two markers may have led to a different number of haplogroups being recovered. High haplotype diversity (H CR = 0 . 99 ; H COI = 0 .798), low nucleotide diversity ( π CR = 0 . 041 ; π COI = 0 . 005 ), and significantly negative Fu’s F S values implied demographic expansion for all haplogroups. The mismatch distribution and Bayesian Skyline plot indicated that expansion occurred during the late Pleistocene. Based on our results, we hypothesize that the Philippines is a secondary contact zone for S. lemuru, and testing this requires that samples be obtained from the entire habitat range of the species along the Indo-West Pacific Region.