Response of Propsilocerus akamusi (Diptera: Chironomidae) to the leachates from AMD-contaminated sediments: Implications for metal bioremediation of AMD-polluted areas.

Acid mine water (AMD) is a global environmental problem caused by coal mining with the characteristics of low pH and high concentrations of metals and sulfates. It is a pertinent topic to seek both economical and environmentally friendly approaches to minimize the harmful effects of AMD on the environment. Insect larvae are considered a promising solution for pollution treatment. Chironomidae is the most tolerant family to contaminants in pools and its larvae have a strong capacity for metal accumulation from sediment. This paper aimed to evaluate the larvae of Propsilocerus akamusi, a dominant species in the chironomid community, as a new species for entomoremediation in AMD-polluted areas. We detected the toxic effects of AMD on P. akamusi larvae based on their survival and the trace metals bioaccumulation capabilities of P. akamusi larvae. Moreover, we analyzed the expression patterns of four stress-response genes, HSP70, Eno1, HbV, and Hb VII in P. akamusi larvae. Our results revealed that AMD exposure did not significantly affect the survival of the P. akamusi larvae and individuals exposed to some AMD gradients even exhibited higher survival. We also observed the significantly accumulated concentrations of Fe, Ni, and Zn as well as higher bioaccumulation factors (BAFs) for Ni and Zn in the P. akamusi larvae exposure to AMD. Induced expression of Eno1 and Hb VII may play important roles in the AMD tolerance of P. akamusi larvae. This study indicated the potential application of P. akamusi larvae in the metal bioremediation of AMD-polluted areas. STATEMENT OF ENVIRONMENTAL IMPLICATION: Acid mine drainage (AMD) is a global environmental problem related to coal mining activities. AMD pollution has become a long-term, worldwide issue for its interactive and complex stress factors. Bioremediation is an effective method to remove the metals of AMD from wastewater to prevent downstream pollution. However, the disadvantages of the slow growth rate, susceptibility to seasonal changes, difficult post-harvest management, and small biomass of hyperaccumulating plants greatly limit the usefulness of phytoremediation. Insect larvae may be useful candidate organisms to overcome these shortcomings and have been considered a promising pollution solution. Propsilocerus akamusi is a dominant species in the chironomid community and is distributed widely in many lakes of eastern Asia. This species has extraordinary abilities to resist various stresses. This research is the first time to our knowledge to evaluate the application of P. akamusi as a new species in entomoremediation in AMD-contaminated areas.

Removal of ammonia nitrogen from black and odorous water by macrophytes based on laboratory microcosm experiments.

In recent years, the removal mechanism of ammonia nitrogen in black and odorous water (BOW), especially in the process of phytoremediation, has been a research "hotspot". Here, the migration process of ammonia nitrogen in macrophytes (Acorus calamus, Canna indica and Eichhornia crassipes) was detected by Fourier transform infrared (FT-IR) spectroscopy. Experiments revealed that the concentration of ammonia nitrogen (NH4 +-N) was reduced significantly. Maximum reduction in the NH4 +-N concentration was obtained in 75% BOW: the absorption of NH4 +-N was >90% in A. calamus and C. indica, and >80% in E. crassipes. After two 10 days cultivations, in the culture dishes of A. calamus and C. indica, absorption of NH4 +-N was >90% whereas, in the culture dishes of E. crassipes, absorption of NH4 +-N was ∼50% and ∼60%. FT-IR spectroscopy showed that NH4 +-N, NO2 --N and NO3 --N could be absorbed by the root and migrate to the stem and leaf of macrophytes. NH4 +-N and NO2 --N were transformed, and the direction was NH4 +-N → NO2 --N → NO3 --N. The migration rate of NH4 +-N in C. indica was faster because of its regular and smooth capillaries according to scanning electron microscopy. Our study on the removal and transformation mechanism of ammonia nitrogen in BOW could be an important reference for other bodies of water.

Within-day variation and health risk assessment of trihalomethanes (THMs) in a chlorinated indoor swimming pool in China.

Trihalomethanes (THMs) are the most common species of disinfection by-products (DBPs) in swimming pools and have received widespread attention due to their risk to public health. However, studies examining within-day variation and the carcinogenic health risks from exposure to THMs in indoor swimming pools are limited. Our study aimed to detect the within-day variation of four THMs categories and carcinogenic health risk in indoor swimming pool water in Taiyuan, China, and to examine the correlations between THMs and environmental parameters. Our results showed chloroform (TCM) was the most abundant component in THMs with median concentrations from 0.038-0.118 µg/m3. TCM and THMs were significantly positively correlated with FCl and significantly negatively correlated with the cumulative number of swimmers (CNS) in the swimming pool. The concentration of total THMs and TCM, lifetime average daily doses (LADD) of TCM, and the total lifetime cancer risks (ELCR) values of THMs declined with time with the highest level occurring at 8:00 am. ELCR values of THMs were in the range of 1.368 × 10-5-1.968 × 10-5, which exceeded the negligible risk level (10-6) defined by US EPA. Our results suggest that THM occurrence and the carcinogenic health risks in pool water varied temporally. Exposure to pool water THMs may pose a carcinogenic risk to human health, especially at the pool's opening time.

Adiponectin restrains ILC2 activation by AMPK-mediated feedback inhibition of IL-33 signaling.

ILC2s are present in adipose tissue and play a critical role in regulating adipose thermogenesis. However, the mechanisms underlying the activation of adipose-resident ILC2s remain poorly defined. Here, we show that IL-33, a potent ILC2 activator, stimulates phosphorylation of AMPK at Thr172 via TAK1 in primary ILC2s, which provides a feedback mechanism to inhibit IL-33-induced NF-κB activation and IL-13 production. Treating ILC2s with adiponectin or an adiponectin receptor agonist (AdipoRon) activated AMPK and decreased IL-33-NF-κB signaling. AdipoRon also suppressed cold-induced thermogenic gene expression and energy expenditure in vivo. In contrast, adiponectin deficiency increased the ILC2 fraction and activation, leading to up-regulated thermogenic gene expression in adipose tissue of cold-exposed mice. ILC2 deficiency or blocking ILC2 function by neutralization of the IL-33 receptor with anti-ST2 diminished the suppressive effect of adiponectin on cold-induced adipose thermogenesis and energy expenditure. Taken together, our study reveals that adiponectin is a negative regulator of ILC2 function in adipose tissue via AMPK-mediated negative regulation of IL-33 signaling.

Knockdown of α-enolase (Eno1) genes by RNAi does not increase the sensitivity of Propsilocerus akamusi to cadmium stress.

α-enolase (Eno1) is a multifunctional enzyme which can as a stress protein under various environmental stresses. Recent researches also reported that Eno1 appears to have Cd2+ stress-related functions in cadmium tolerant plants. Our previous study inferred that the Eno1 gene might play an important role in the response of Propsilocerus akamusi to exogenous Cd2+. However, reports on the role of the Eno1 gene in coping with cadmium stress are still limited. In this study, we evaluated the roles of PaEno1 in the tolerance of P. akamusi to Cd2+ using RNAi technology and the response of recombinant proteins of PaEno1 in an E. coli expression system under Cd2+ stress. Our results showed that knockdown of PaEno1 did not increase but reduce the sensitivity of P. akamusi larvae to Cd2+ stress. However, bioassays showed the expression of recombinant PaEno1 protein in Rosetta cells enhanced the growth ability of E. coli under Cd2+ stress. These results suggested that overexpression of PaEno1 can significantly enhance the tolerance to heavy metal cadmium stresses in E. coli cells. However, knockdown of PaEno1 genes by RNAi does not increase the sensitivity of P. akamusi to cadmium stress.

Changes in the gut microbiota of honey bees associated with jujube flower disease.

Honeybees are prone to poisoning after collecting jujube nectar during the jujube flowering period ('honeybee's jujube flower disease'). To explore the mechanism of honeybee poisoning, the gut microbiota of honeybees undergoing the disease were characterised based on amplicon sequencing of the 16 S rRNA gene. Our results showed that the composition and diversity of the gut microbiota were significantly altered in diseased honeybees. We observed a decrease in the relative abundance of Proteobacteria and increased abundances of Firmicutes and Actinobacteria in the midgut and hindgut of diseased honeybees. Moreover, linear discriminant analysis (LDA) effect size revealed significantly selected enrichment of Fructobacillus and Snodgrassella in the midguts from diseased honeybees and Lactobacillus, Bifidobacterium, and Snodgrassella in the hindguts from diseased honeybees. Tax4Fun anylasis indicated that the functional potential of the diseased honeybee gut bacterial community was significantly changed relative to the healthy honeybee. Carbohydrate metabolism, nucleotides metabolism, amino acid synthesis metabolism, coenzyme and vitamins metabolism were increased, while energy metabolism and xenobiotic biodegradation and metabolism were decreased in the diseased honeybees. These results provide a new perspective for evaluating the response of honeybees to jujube flower disease based on changes in the intestinal microflora.

iTRAQ-based quantitative proteomic analysis identified Eno1 as a cadmium stress response gene in Propsilocerus akamusi (Tokunaga) hemolymph.

Propsilocerus akamusi (Tokunaga) is a common species of midge in Siberia, Japan, and China and an important prey species for fish and aquatic birds. Furthermore, this species has been shown to have an extraordinary capacity to resist cadmium (Cd) toxicity. In this study, isobaric tags for relative and absolute quantitation (iTRAQ) coupled liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze relative changes in the P. akamusi hemolymph proteome following exposure to a sublethal concentration of Cd2+. The results showed that Cd2+ stress affects energy metabolism in P. akamusi. After examining the differentially expressed proteins (DEPs), only one up-regulated protein associated with metabolism, α-enolase (Eno1) was identified and further isolated and characterized. Sequence alignments showed that the deduced P. akamusi Eno1 amino acid sequence is highly conserved, with similarities of 77-95% noted when compared to other Dipteran Eno1 sequences. Furthermore, prolonged Cd2+ exposure impacted Eno1 transcription, protein expression and enzyme activity levels. These results suggest that Eno1 may play a role in the response to Cd2+ stress in P. akamusi.

Cadmium exposure on tissue-specific cadmium accumulation and alteration of hemoglobin expression in the 4th-instar larvae of Propsilocerus akamusi (Tokunaga) under laboratory conditions.

The expression of hemoglobin (Hb) genes has considerable potential as a biomarker for environmental monitoring in Chironomus. However, no sequence information is available regarding Hb genes in Propsilocerus akamusi (Tokunaga), thus the change in Hb mRNA gene expression caused by environmental pollutants remains unknown. In this study, we cloned two Hb gene fragments (PaHbV and PaHbVII) from P. akamusi, analyzed the expression patterns of the PaHbV and PaHbVII transcripts in different tissues using Real-Time quantitative PCR (RT-qPCR), and also measured the Cd levels in different tissues exposed to a sublethal concentration. The results showed significantly increased Cd concentrations and tissue-specific Cd distribution patterns in all of the tissues tested, including the hemolymph, during all time courses. A model describing the roles of specific tissues in Cd uptake and accumulation dynamics was also determined. The Malpighian tubules, gut, and epidermis were the primary sites of Cd accumulation, whereas the hemolymph was the temporary target organ of Cd accumulation, with the Cd being transferred to other internal tissues via the hemolymph. The relative mRNA expression profiles of PaHbV and PaHbVII indicated that their expression levels differed across the different tissues, indicating a tissue-specific response. Our results suggested a reverse effect between Hb expression and Cd accumulation during long-term Cd exposure in comparison with previous studies. The expressions of Hb genes in P. akamusi could be developed as biomarkers for assessing the general health conditions of freshwater ecosystems.

Determination of the protein expression profiles of Propsilocerus akamusi (Tokunaga) Malpighian tubules response to cadmium stress by iTRAQ coupled LC-MS/MS.

Propsilocerus akamusi (Tokunaga) is an important prey species for fish and aquatic birds, which represent an important link in the aquatic food chain as other species of Chironomidae. Malpighian tubules (MT) play an important role in metabolism and detoxification of harmful compounds and metal accumulation. Our previous studies have reported that P. akamusi have an extraordinary resistance to Cd. In this paper, iTRAQ-based proteomics technology was first used to study the differential proteomics of MT of Propsilocerus akamusi under sublethal Cd stress for different time courses. 118 differential expressed proteins (DEPs) were identified through LC-MS/MS and further classified into 26 GO functional groups. Our results showed that metabolic process was the main biological functional categories. Cluster of orthologous groups of proteins (COG) analysis was also performed based on the number of unique proteins identified in each functional category, 21 metabolism-related DEPs were identified. P. akamusi adapted to Cd stress mainly by inducing the processes of lipid metabolism of MT and lipid may play a possible protection role in cadmium resistance in MT. The results provide important information for research on molecular mechanisms of Cd-stress adaption in Malpighian tubules of P. akamusi. BIOLOGICAL SIGNIFICANCE: Propsilocerus akamusi (old species name: Tokunagayusurika akamusi) is an important prey species for fish and aquatic birds, which represent an important link in the aquatic food web as other species of Chironomidae. Malpighian tubules constitute a versatile organ which is able to exert excretory of chemical compounds, acting in the insect metabolism and detoxification of harmful compounds and metal accumulation. Our previous studies have reported that P. akamusi have an extraordinary resistance to Cd. However, up to now, there has been a lack of research on the molecular mechanisms of adaption to Cd stress in the Malpighian tubules of this species. Study on the adaptive mechanisms to Cd stress in Malpighian tubules of Propsilocerus akamusi through comparative proteome analysis is one important way to solve this problem. The aim of the present study was to understand the molecular mechanisms of adaption of the Malpighian tubules of P. akamusi exposed to Cd stress and assess the high expression of proteins involved in cellular adaptation to Cd stress. This will provide information about how this organ is responding to disturbances of contaminated ecosystems.

Sex-Dependent effects of developmental arsenic exposure on methylation capacity and methylation regulation of the glucocorticoid receptor system in the embryonic mouse brain.

Previously we have shown that prenatal moderate arsenic exposure (50 ppb) disrupts glucocorticoid receptor (GR) programming and that these changes continue into adolescence in males. However, it was not clear what the molecular mechanisms were promoting these GR programming changes or if these changes occurred in arsenic-exposed females. In the present studies, we assessed the effects of arsenic on protein and mRNA of the glucocorticoid receptor (GR) and 11ß-hydroxysteroid dehydrogenase (Hsd) isozymes and compared the levels of methylation within the promoters of the Nr3c1 and Hsd11b1 genes in female fetal brain at embryonic days (E) 14 and 18. Prenatal arsenate exposure produced sex specific effects on the glucocorticoid system. Compared to males, females were resistant to arsenic induced changes in GR, 11ß-Hsd-1 and 11ß-Hsd-2 protein levels despite observed elevations in Nr3c1 and Hsd11b2 mRNA. This sex-specific effect was not due to differences in the methylation of the GR promoter as methylation of the Nr3c1 gene was either unchanged (region containing the egr-1 binding site) or similarly reduced (region containing the SP-1 transcription factor binding site) in both males and females exposed to arsenic. Arsenic did produce sex and age-specific changes in the methylation of Hsd11b1 gene, producing increased methylation in females at E14 and decreased methylation at E18. These changes were not attributed to changes in DNMT levels. Since arsenate metabolism could interfere with the generation of methyl donor groups, we assessed glutathione (GSH), s-adenosylmethionine (SAM) and As 3 methyltransferase (As3MT). Exposed males and females had similar levels of As3MT and SAM; however, females had higher levels of GSH/GSSH. It is possible that this greater anti-oxidative capacity within the females provides protection against low to moderate arsenate. Our data suggest that the GR signaling system in female offspring was not as affected by prenatal arsenic and predicts that female arsenic-exposed mice should have normal GR feedback regulation.

Effect of seabuckthorn (Hippophae rhamnoides ssp. sinensis) leaf extract on the swimming endurance and exhaustive exercise-induced oxidative stress of rats.

BACKGROUND: Seabuckthorn (SBT) leaves have significant antioxidant, immunomodulatory and anti-inflammatory properties. The objective of this study was to assess the anti-fatigue, antioxidant and tissue-protective properties of aqueous lyophilised extracts of SBT dried leaves in the hearts of Wistar male rats undergoing exhaustive physical exercise. Doses of 50, 200 and 800 mg kg⁻¹ body weight (BW) day⁻¹ were given orally for 1 week. A week later the rats were forced to swim in barrels until they were exhausted. The times were noted to establish the effective dose of the extracts in rats. After establishing the effective dose, the rats were then sacrificed and assessed for various biochemical parameters. RESULTS: SBT leaf aqueous extracts (200 and 800 mg kg⁻¹ BW) markedly prolonged the swim time of rats. Supplementation with SBT leaf aqueous extracts helped reduce the exhaustive exercise-induced increase in malondialdehyde level and selenium-dependent glutathione peroxidase activity. Alanine aminotransferase and creatine kinase levels were lowered in the exhaustive exercise with SBT treatment group (E + SBT) compared with the exhaustive exercise group (E). CONCLUSION: The findings suggest that SBT leaf aqueous extract supplements can enhance exercise capacity and protect against oxidative damage caused by exhaustive exercise in rats.

Effects of cadmium exposure on lipid peroxidation and the antioxidant system in fourth-instar larvae of Propsilocerus akamusi (Diptera: Chironomidae) under laboratory conditions.

Enzymatic antioxidants such as selenium-dependent glutathione peroxidase (GPx), glutathione transferase (GST), glutathione reductase (GR), and superoxide dismutases (SOD), as well as the concentration of hydrogen peroxide (H2O2) and malondialdehyde (MDA, an indicator of lipid peroxidation) were determined to identify which antioxidant enzymes participate in the efficient scavenging of ROS generated upon exposure to high doses of Cd2+ in fourth-instar Propsilocerus akamusi (Tokuna) (Diptera: Chironomidae) larvae after 72-h exposure. A significant increase in MDA levels and a change in GR and GPx activities in the Cd(2+)-treated P. akamusi were observed. The MDA in 25.0 and 50.0 mmol/liter treatments was significantly higher than that of the control dose after 72 h exposure. GPx activity was significantly induced by Cd2+ exposure only in the 50.0-mmol/liter treatment with a 0.59-fold increase in the control. All doses of Cd2+ significantly suppressed GR activity compared with the findings for the control dose, with an inhibited rate up to 0.55-fold in the 25.0 mmol/liter Cd2+ treatment. SOD and GST activities were not altered. The results indicate that Cd2+ can induce oxidative stress as indicated by the changes in lipid peroxidation and antioxidant status. For P. akamusi, an increase in the dose that the threshold needed for defense (namely, MDA level and GPx activity) activation was achieved. From this, organisms can be hypothesized to enable cells to avoid oxidant stress up to a certain extent where damage is again measurable (higher Cd2+ concentration).

Genetic variation and population structure of oriental migratory locust, Locusta migratoria manilensis, in China by allozyme, SSRP-PCR, and AFLP markers.

Allozyme analysis, microsatellite primer PCR (SSRP-PCR), and amplified fragment length polymorphism (AFLP) techniques were used to assess genetic diversity and population structure of the Chinese oriental migratory locust, Locusta migratoria manilensis. A total of 299 PCR markers (67 SSRPs and 232 AFLPs) were detected in eight populations, of which 98.7% were polymorphic markers. The proportion of polymorphic loci (95.5-98.8%) by SSRP+AFLP markers indicated no significant differences between populations, and all populations exhibited a similar level of variability; results of the allozyme analysis demonstrated that 19 loci gave rise to a lower level of polymorphism (55.6-66.7%). The genetic distances between the populations were relatively low. Shannon's index and Nei's gene diversity showed low differentiation among the populations. Allozyme analysis, however, reflected greater similarity and smaller differentiation between the populations than those shown by SSRP and AFLP markers. Neighbor-joining dendrograms derived from both the allozyme and SSRP+AFLP markers showed that the genetic distances among Chinese oriental migratory locust populations were not greatly influenced by geographic distance and breeding habitats.

Genetic differentiation of different populations of four locust species in China.

The genetic structure of eight populations of four locust species in three families (Catantopidae, Pamphagidae and Pyrgomorphidae) was examined by means of horizontal starch gel electrophoresis, the locusts were collected from Shanxi, Jiangsu and Hebei Province in China. The allele frequency and allozyme polymorphism of 12 enzymes (18 loci) were analyzed. Low variability with a few alleles was observed in Shirakiacris shirakii and Atractomorpha sinensis, however, Oxya Chinensis and Haplotropis brunneriana showed high polymorphism. In the eight populations of the four locust species, high percentage of polymorphic loci was observed (53.3%-100.0%). Owing to heterozygote deficiency of some species, all the four species demonstrated low overall heterozygosity (Ho = 0.034-0.139), which lead to the genotype frequency deviated significantly from Hardy-Weinberg equilibrium. However, many of the examined loci were fit for or close to H-W equilibrium in Atractomorpha sinensis. A certain differentiation in mean heterozygosity was found among the four species. Due to the difference of migratory capability, reproductive manner and living bound, mean heterozygosity (Ho) is higher in Haplotropis brunneriana (Ho = 0.089-0.139) and Oxya Chinensis (Ho = 0.073-0.090) than in Atractomorpha sinensis (Ho = 0.034-0.050) and Shirakiacris shirakii (Ho = 0.048-0.068). Genetic differentiation from F-statistics was also different at population level among four locust species. It was high in Haplotropis brunneriana (Fst = 0.32) and Atractomorpha sinensis (Fst = 0.31), and low genetic differentiations were observed in Oxya Chinensis (Fst = 0.20) and Shirakiacris shirakii (Fst = 0.18). It was confirmed that migratory capability, adaptability and surrounding factors had an important influence on the genetic differentiation of locust populations. The taxon relationships based on Nei's genetic identity (I) and genetic distance (D) were consistent with results obtained from karyotypic analyses. Oxya Chinensis and Shirakiacris shirakii in the same family have a higher genetic identity (I = 0.576) and a smaller genetic distance (D = 0.559); the species examined in Pampagidae displayed somewhat closer relationship to those in Pyrgomorphidae (D = 0.776, I = 0.464) than to those in Catantopidae (D = 0.908, I = 0.406). It is suggested that the allozyme analysis is an useful molecular marker for phylogenetic reconstruction of locust at both species and population levels.

Genetic studies on eight populations of eight locust species from Shanxi Province, China.

The genetic structure of eight locust species in three families (Catantopidae, Oedipodidae and Arcypteridae) from Shanxi Province in China was compared using allozyme analysis with horizontal starch gel electrophoresis. Among 17 loci identified in zymograms, Ao-1, Est-3, G3pd-1, Idh-2 and Mdh-2 had low variability with a few alleles. High polymorphism was observed at Ldh-1, Me-1 and Gpi-1. Each of the eight species demonstrated high percentage of polymorphic loci (P = 64.7%-94.1%) but low observed heterozygosity (H0 = 0.024-0.087) due to heterozygote deficiency. It was noted that the migratory locusts usually had higher percentage of polymorphic loci (P = 88.2%-94.1%) than non-migratory species (P = 64.7%-94.1%). The only exception is Oxya chinensis(P = 94.1%). It is reasoned that the higher polymorphism is necessary for migratory species to cope with the environments that might be drastically different from the habitats before migration. The taxon relationships using cluster analysis based on Nei's genetic identity (I) and Roger's genetic distance (D) were the same at species and genus levels. The differences were found at family level, possibly due to the alternative algorithms. The cladogram using Roger's genetic distance (D) overlapped the relationship obtained from karyotypic analyses, which demonstrated that the species examined in Catantopidae displayed somewhat closer relationship to those in Oedipodidae than to those in Arcypteridae. It is suggested that the allozyme analysis is useful as molecular marker for locusts in phylogenetic reconstruction at the species and genus level, while additional data from other studies are necessary when used for higher taxa.

Comparative allozyme analysis of Oriental migratory locust Locusta migratoria manilensis from two breeding areas in north China.

The allozyme analysis using horizontal starch gel electrophoresis was employed to compare the genetic structure in the population of oriental migratory locust Locusta migratoria manilensis from two breeding areas, Beidagang(Tianjin) and Huanghua(Hebei). The two areas are adjacent but with distinct ecological features, with the recorded locust outbreaks and migration. The zymograms showed that among nineteen loci four (Mdh-1, Pgm, Adk and G3pd) showed extremely low variability level with the frequency of the most common allele higher than 0.95 in the populations from both sites. The rest loci had 2 to 4 alleles but the allele frequencies between the two populations were all similar except Fbp and Got-2 loci. In the 27 chi 2-tests for the genotypes at polymorphic loci only two (Pgi and Got-1) of beidagang population did fit the Hardy-Weinberg's expectations. This is due to high frequencies of the most common homozygotes and the corresponding heterozygote deficiency. The allozyme data demonstrated that the locusts had remarkable genetic variability within each population, but little divergence between the populations. The genetic variability measurements were found similar: Percentage of polymorphic loci (P) was between 73.7% and 78.9%; The mean number of alleles per locus (A) was from 2.9 to 3.1; and the mean heterozygosity (Ho) was nearly identical (about 0.138). The F-statistics (FST = 0.053) also showed the genetic uniformity of the populations, corresponding to the high Nei's genetic identity (I = 0.938). These results of the allozyme analysis suggested that the two populations appeared to be a part of a large population. It is reasoned that the genetic polymorphism and differentiation at certain loci between the two populations may depend on at least two agnostic factors that are all related to migration. First, the unusual dispersal capability of L. m. manilensis tends to make a continuous genetic structure distribution. Second, the frequent migration also results in the individuals to be exposed to drastically various environments. Since the broad adaptability is crucial to survive the changing environments, the genetic variation at population level is necessarily required to offer the population resilience for successful survival and reproduction under those ecologically divergent abiotic and biotic conditions. Thus, the migration contributes to the maintenance of dynamic equilibrium of genetic polymorphism in this highly specialized subspecies.