Analysis of Genetic Diversity and Structure of Eight Populations of Nerita yoldii along the Coast of China Based on Mitochondrial COI Gene.

Nerita yoldii is a euryhaline species commonly found in the intertidal zone. To investigate the genetic diversity of 233 N. yoldii individuals from eight locations along the coast of China, we utilized the mitochondrial COI gene as a molecular marker. A total of 34 haplotypes were detected, exhibiting a mean haplotype diversity (Hd) of 0.5915 and a mean nucleotide diversity (Pi) of 0.0025, indicating high levels of genetic diversity among all populations. An analysis of molecular variance (AMOVA) indicated that the primary source of genetic variation occurs within populations. In addition, neutral tests and mismatch analyses suggested that N. yoldii populations may have experienced bottleneck events. Moderate genetic differentiation was observed between Xiapu and other populations, excluding the Taizhou population, and may be attributed to the ocean currents. Intensively studying the genetic variation and population structure of N. yoldii populations contributes to understanding the current population genetics of N. yoldii in the coastal regions of China. This not only provides a reference for the study of other organisms in the same region but also lays the foundation for the systematic evolution of the Neritidae family.

Characterization of the complete mitochondrial genome of Desmaulus extinctorium (Littorinimorpha, Calyptraeoidea, Calyptraeidae) and molecular phylogeny of Littorinimorpha.

For the purpose of determining the placement of Calyptraeidae within the Littorinimorpha, we hereby furnish a thorough analysis of the mitochondrial genome (mitogenome) sequence of Desmaulus extinctorium. This mitogenome spans 16,605 base pairs and encompasses the entire set of 37 genes, including 13 PCGs, 22 tRNAs and two rRNAs, with an evident AT bias. Notably, tRNASer1 and tRNASer2 lack dihydrouracil (DHU) arms, resulting in an inability to form a secondary structure. Similarly, tRNAAla lacks a TΨC arm, rendering it incapable of forming a secondary structure. In contrast, the remaining tRNAs demonstrate a characteristic secondary structure reminiscent of a cloverleaf. A comparison with ancestral gastropods reveals distinct differences in three gene clusters (or genes), encompassing 15 tRNAs and eight PCGs. Notably, inversions and translocations represent the major types of rearrangements observed in D. extinctorium. Phylogenetic analysis demonstrates robust support for a monophyletic grouping of all Littorinimorpha species, with D. extinctorium representing a distinct Calyptraeoidea clade. In summary, this investigation provides the first complete mitochondrial dataset for a species of the Calyptraeidae, thus providing novel insights into the phylogenetic relationships within the Littorinimorpha.

Transcriptomic Analysis Provides Insights into Candidate Genes and Molecular Pathways Involved in Growth of Mytilus coruscus Larvae.

Growth is a fundamental aspect of aquaculture breeding programs, pivotal for successful cultivation. Understanding the mechanisms that govern growth and development differences across various stages can significantly boost seedling production of economically valuable species, thereby enhancing aquaculture efficiency and advancing the aquaculture industry. Mytilus coruscus, a commercially vital marine bivalve, underscores this importance. To decipher the intricate molecular mechanisms dictating growth and developmental disparities in marine shellfish, we conducted transcriptome sequencing and meticulously analyzed gene expression variations and molecular pathways linked to growth traits in M. coruscus. This study delved into the molecular and gene expression variations across five larval development stages, with a specific focus on scrutinizing the differential expression patterns of growth-associated genes using RNA sequencing and quantitative real-time PCR analysis. A substantial number of genes-36,044 differentially expressed genes (DEGs)-exhibited significant differential expression between consecutive developmental stages. These DEGs were then categorized into multiple pathways (Q value < 0.05), including crucial pathways such as the spliceosome, vascular smooth muscle contraction, DNA replication, and apoptosis, among others. In addition, we identified two pivotal signaling pathways-the Hedgehog (Hh) signaling pathway and the TGF-beta (TGF-ß) signaling pathway-associated with the growth and development of M. coruscus larvae. Ten key growth-related genes were pinpointed, each playing crucial roles in molecular function and the regulation of growth traits in M. coruscus. These genes and pathways associated with growth provide deep insights into the molecular basis of physiological adaptation, metabolic processes, and growth variability in marine bivalves.

Characterization of Lophiotoma leucotropis Mitochondrial Genome of Family Turridae and Phylogenetic Considerations within the Neogastropoda.

Neogastropoda is a group of marine organisms with an extremely wide distribution that is rich in species and economic and ornamental values, the classification of species in this order has been ongoing for a long time, but there is still a great controversy about whether this order is monophyletic. In this study, we obtained the complete mitogenome of Lophiotoma leucotropis by next-generation sequencing and analyzed the basic structural features of the genome, and we found that the number of genes was consistent with that of most of the Neogastropoda snails, containing 37 genes, including 13 protein-coding genes (PCGs), 2 rRNAs, and 22 tRNAs. Analyzing base content, amino acid content, codon usage preference, and tRNA structure, the mitogenomes of eight species of Turridae were selected for analysis of selection pressures, and it was found that the evolution of species in this family was affected by purifying selection. In addition, by analyzing the rearrangement characteristics, it was found that the sequence of L. leucotropis was consistent with the Conoidea consensus order, and four of the eight species involved in the analysis showed rearrangements. Finally, we constructed a phylogenetic tree by combining PCGs of 60 species within Caenogastropoda and found Neogastropoda to be a monophyletic group, validating the results of morphological classification. The results will provide more references for the classification and species evolution of Neogastropoda, as well as phylogenetic analysis.

Comparison of Mitochondrial Genome Sequences between Two Palaemon Species of the Family Palaemonidae (Decapoda: Caridea): Gene Rearrangement and Phylogenetic Implications.

To further understand the origin and evolution of Palaemonidae (Decapoda: Caridea), we determined the mitochondrial genome sequence of Palaemon macrodactylus and Palaemon tenuidactylus. The entire mitochondrial genome sequences of these two Palaemon species encompassed 37 typical genes, including 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNAs), and 22 transfer RNA genes (tRNAs), and a control region (CR). The lengths of their mitochondrial genomes were 15,744 bp (P. macrodactylus) and 15,735 bp (P. tenuidactylus), respectively. We analyzed their genomic features and structural functions. In comparison with the ancestral Decapoda, these two newly sequenced Palaemon species exhibited a translocation event, where the gene order was trnK-trnD instead of trnD-trnK. Based on phylogenetic analysis constructed from 13 PCGs, the 12 families from Caridea can be divided into four major clades. Furthermore, it was revealed that Alpheidae and Palaemonidae formed sister groups, supporting the monophyly of various families within Caridea. These findings highlight the significant gene rearrangements within Palaemonidae and provide valuable evidence for the phylogenetic relationships within Caridea.

Assessing the Impact of Microplastic Filaments Contaminated with PAHs on Mytilus coruscus Larvae through Surface Contact.

In recent years, microplastics have been of great concern in environmental and health research. In field surgeries and laboratory investigations, research interests were focused on the retention of microplastics inside of animals by ingestion and the series of negative effects after that. However, such large plastic debris and filaments are hardly eaten by small animals, like zooplankton, planktonic larvae, etc. In this study, the surface contact between plastic filaments contaminated with polycyclic aromatic hydrocarbons (PAHs) and mussel pediveliger larvae has been investigated to figure out the effects of "non-digestive tract route of exposure" on subject animals. In a 1600 mL artificial seawater medium, high mortalities of mussel larvae were recorded after being exposed to two PAHs-contaminated (benzo[α]pyrene (BaP) and phenanthrene (Phe)) filaments for 5 days, 68.63% for BaP and 56.45% for Phe on average. We suggest that the surface contact was the dominant pathway to transfer PAHs from contaminated filaments to larvae and that the risk of contaminated plastic ropes transferring hydrophobic organic compounds (HOCs) to larvae in mussel aquaculture should be taken seriously.

Genetic Diversity and Structure Revealed by Genomic Microsatellite Markers of Mytilus unguiculatus in the Coast of China Sea.

The hard-shelled mussel Mytilus unguiculatus plays an important role in mussel aquaculture in China due to its characteristic and nutritive value. In this study, 10 microsatellite loci are used to study the genetic diversity and genetic structure of seven location populations of M. unguiculatus in coastal areas of China. The results of amplification and genotyping show that the observed heterozygosity (Ho) and the expected heterozygosity (He) are 0.61~0.71 and 0.72~0.83, respectively. M. unguiculatus has high genetic diversity. The inbreeding index (FIS) of M. unguiculatus is significantly positive (FIS: 0.14~0.19), indicating that inbreeding might exist within populations. The genetic structure of M. unguiculatus is weak within populations from the East China Sea All results showed that genetic differences existed between the Qingdao population from the Yellow Sea and other populations from the East China Sea. It does not detect a population bottleneck event or expansion event in the populations. The results from this study can be used to provide important insights in genetic management units and sustainable utilization of M. unguiculatus resources and provide a better understand of genetic structure of marine bivalve with similar planktonic larval stage in the China Sea.

The Complete Mitochondrial Genome of Mytilisepta virgata (Mollusca: Bivalvia), Novel Gene Rearrangements, and the Phylogenetic Relationships of Mytilidae.

The circular mitochondrial genome of Mytilisepta virgata spans 14,713 bp, which contains 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, and 22 transfer RNA genes. Analysis of the 13 PCGs reveals that the mitochondrial gene arrangement of Mytilisepta is relatively conserved at the genus level. The location of the atp8 gene in Mytilisepta keenae differs from that of other species. However, compared with the putative molluscan ancestral gene order, M. virgata exhibits a high level of rearrangement. We constructed phylogenetic trees based on concatenated 12 PCGs from Mytilidae. As a result, we found that M. virgata is in the same clade as other Mytilisepta spp. The result of estimated divergence times revealed that M. virgata and M. keenae diverged around the early Paleogene period, although the oldest Mytilisepta fossil was from the late or upper Eocene period. Our results provide robust statistical evidence for a sister-group relationship within Mytilida. The findings not only confirm previous results, but also provide valuable insights into the evolutionary history of Mytilidae.

Insights into the Deep Phylogeny and Novel Gene Rearrangement of Mytiloidea from Complete Mitochondrial Genome.

The extant marine mussels which belong to the Mytiloidea are widespread species inhabiting mostly coastal waters, with some distributed in the deep sea. To clarify the classification systems and phylogenetic relationships range from genus to family level within Mytiloidea, new sequence was used in a phylogenetic analysis including all the available Mytiloidea mitochondrial genomes. In this study, the complete mitochondrial genome of Vignadula atrata is 15,624 bp in length and contains 12 protein-coding genes (PCGs, atp8 is absent), two ribosomal RNA genes, and 22 transfer RNA genes. Phylogenetic analysis based on 12 PCGs showed that it has a close relationship to Bathymodiolus. The analysis of gene rearrangements in the Pteriomorphia showed that the arrangements are highly variable across species, novel gene rearrangements were found within Mytiloidea. The V. atrata mitogenome was provided in detail, with notes on the sequence and a key to the species of Vignadula. This study provides a perspective on the taxonomic histories of the marine mussels and refines the unclear relationship between the origin and evolution of species in Mytiloidea within Bivalvia.

Burrowing invertebrates induce fragmentation of mariculture Styrofoam floats and formation of microplastics.

Secondary microplastics originate from the fragmentation of large plastics, and weathering is supposed to be the main cause of fragmentation. In this study, we investigated burrows and burrowing invertebrates on Styrofoam floats from the mariculture areas of China's coastal waters. Various burrows were found on the submerged surface of Styrofoam floats and could be divided into 'I', 'S', 'J', and 'Y' types based on the burrow entrance number and passage curvature. Different invertebrate species, including 5 isopods, 8 clamworms, and 12 crabs, were found inside the burrows. Micro-foams were found in the bodies of these burrowers, with an average abundance of 4.2 ± 0.3 (isopod), 6.9 ± 2.0 (clamworm), and 3.0 ± 0.5 (crab) micro-foams per individual. In the laboratory, we observed the boring process of crabs in abandoned floats. Field and laboratory evidence suggested that these invertebrates bored various burrows. The total volume of crab burrows on a 3-year-used float was estimated to be 2.6 × 103 cm3, producing 4.1 × 108 microplastics. This study highlights the critical role of bioerosion in destroying man-made substrates and prompting microplastic pollution.

Novel gene re-arrangement in the mitochondrial genome of Pisidiaserratifrons (Anomura, Galatheoidea, Porcellanidae) and phylogenetic associations in Anomura.

To improve the taxonomy and systematics of Porcellanidae within the evolution of Anomura, we describe the complete mitochondrial genomes (mitogenomes) sequence of Pisidiaserratifrons, which is 15,344 bp in size, contains the entire set of 37 genes and has an AT-rich region. Compared with the pancrustacean ground pattern, at least five gene clusters (or genes) are significantly different with the typical genes, involving eleven tRNA genes and four PCGs and the tandem duplication/random loss and recombination models were used to explain the observed large-scale gene re-arrangements. The phylogenetic results showed that all Porcellanidae species clustered together as a group with well nodal support. Most Anomura superfamilies were found to be monophyletic, except Paguroidea. Divergence time estimation implies that the age of Anomura is over 225 MYA, dating back to at least the late Triassic. Most of the extant superfamilies and families arose during the late Cretaceous to early Tertiary. In general, the results obtained in this study will contribute to a better understanding of gene re-arrangements in Porcellanidae mitogenomes and provide new insights into the phylogeny of Anomura.

The Complete Mitochondrial Genome of Pilumnopeus Makianus (Brachyura: Pilumnidae), Novel Gene Rearrangements, and Phylogenetic Relationships of Brachyura.

Pilumnopeus makianus is a crab that belongs to Pilumnidae, Brachyura. Although many recent studies have focused on the phylogeny of Brachyura, the internal relationships in this clade are far from settled. In this study, the complete mitogenome of P. makianus was sequenced and annotated for the first time. The length of the mitogenome is 15,863 bp, and includes 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNA), and 2 ribosomal RNA genes (rRNA). The mitogenome exhibits a high AT content (72.26%), with a negative AT-skew (-0.01) and a GC-skew (-0.256). In the mitogenome of P. makianus, all the tRNA genes are folded into the typical cloverleaf secondary structure, except trnS1 (TCT). A comparison with the ancestors of Brachyura reveals that gene rearrangement occurred in P. makianus. In addition, phylogenetic analyses based on thirteen PCGs indicated that P. makianus, Pilumnus vespertilio, and Echinoecus nipponicus clustered into a well-supported clade that supports the monophyly of the family Pilumnidae. These findings enabled a better understanding of phylogenetic relationships within Brachyura.

Insights into the Deep Phylogeny and Novel Divergence Time Estimation of Patellogastropoda from Complete Mitogenomes.

To further understand the origin and evolution of Patellogastropoda, we determined the mitochondrial genome sequence of Cellana toreuma, and compared its mitogenome characteristics with the other four limpets of Nacellidae. The ratio of Ka and Ks indicated that these Nacellidae species were suffering a purifying selection, with exception of the atp6 gene. The gene sequence is basically consistent among families, while there are great differences among Lottidae species. According to the mitogenome sequences of selected gastropod species, we reconstructed a new phylogenetic tree with two methods. The data complement the mitogenome database of limpets and is a favorable research tool for the phylogenetic analysis of Gastropoda. It is found that there is a long-branch attraction (LBA) artefact in the family Lottiidae of Patellogastropoda. Therefore, the Patellogastropoda was separated by Heterobranchia, and Lottiidae is located at the root of the whole phylogenetic tree. Furthermore, we constructed the divergence time tree according to the Bayesian method and discussed the internal historical dynamics, and divergence differences among the main lineages of 12 Patellogastropoda under an uncorrelated relaxed molecular clock. In turn, we made a more comprehensive discussion on the divergence time of limpets at the molecular level.

Sequence comparison of the mitochondrial genomes of five brackish water species of the family Neritidae: Phylogenetic implications and divergence time estimation.

Neritids are ancient gastropod species which can live in marine, brackish water, and freshwater environments. In this study, we sequenced and annotated the mitochondrial genomes of five brackish water neritids (i.e., Clithon corona, Clithon lentiginosum, Clithon squarrosum, Neritina iris, and Septaria lineata). The mitogenomes ranged from 15,618 to 15,975 bp, and all contain 13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes, with a closed ring structure. We calculated the Ka/Ks values of all 13 PCGs of Neritidae species, all ratios are less than 1, under purification selection. Phylogenetic analysis of the 13 PCGs showed that Neritimorpha is a sister group with Vetigastropoda and Caenogastopoda, genus Clithon is a sister group with Neritina and Septaria. Estimation of divergence time for all species of Neritidae showed that the main differentiation of Neritidae occurred in Cenozoic period (65 Mya), C. corona and C. lentiginosum were differentiated in the Cenozoic Neogene, the other three species diverged in the Cenozoic Paleogene. These results will help to better understand the evolutionary position of Neritidae and provide reference for further phylogenetic research on Neritidae species.

A novel molluscan TLR molecule engaged in inflammatory response through MyD88 adapter recruitment.

Toll-like receptors (TLRs) mediated signaling plays a vital role in activating innate and adaptive immunity. Although TLR mediated signaling has been comprehensively investigated in mammalian species, the mechanisms underlying TLR signaling in molluscs remain obscure. In the present study, a novel TLR isoform namely McTLR-like1 was identified in the thick shell mussel Mytilus coruscus. McTLR-like1 was highly expressed in molluscan immune-related tissues, and its transcriptional levels in hemocytes were significantly increased when challenged by V. alginolyticus. McTLR-like1 activated nuclear factor κB (NF-κB) and strengthened the transcription and phosphorylation of NF-κB subunit P65 in mammalian cells. Upon the silencing of McTLR-like1, the mRNA expression levels of pro-inflammatory cytokines were down-regulated, and the animals exhibited higher levels of resistance when challenged with V. alginolyticus. McMyD88a mRNA expression was also downregulated alongside McTLR-like1. Furthermore, GST-pull down assays revealed a visible affinity between McTLR-like1 and McMyD88a. Collectively, these results demonstrated that the newly identified gene affiliated to the molluscan TLR family and plays a role in the TLR-mediated activation of inflammatory response via its affinity with MyD88. The present study enhances our knowledge of TLR signaling mechanisms in molluscs and provides new insights into the evolution of TLRs.

The mitochondrial genome of Grapsus albolineatus (Decapoda: Brachyura: Grapsidae) and phylogenetic associations in Brachyura.

Complete mitochondrial genomes (mitogenomes) can provide useful information for phylogenetic relationships, gene rearrangement, and evolutionary traits. In this study, we determined the complete mitochondrial DNA sequence of the herbivorous crab Grapsus albolineatus. It is a typical metazoan mitochondrial genome. The total size is 15,583 bp, contains the entire set of 37 genes, and has an AT-rich region. Then, 23 of the 37 genes were encoded by the heavy (+) strand while 14 are encoded by the light (-) strand. Compared with the pan-crustacean ground pattern, two tRNA genes (tRNA-His and tRNA-Gln) were rearranged and the tandem duplication/random loss model was used to explain the observed gene rearrangements. The phylogenetic results showed that all Grapsidae crabs clustered together as a group. Furthermore, the monophyly of each family was well supported, with the exception of Menippidae. In general, the results obtained in this study will contribute to the better understanding of gene rearrangements in Grapsidae crab mitogenomes and provide new insights into the phylogeny of Brachyura.

Particles rather than released Zn2+ from ZnO nanoparticles aggravate microplastics toxicity in early stages of exposed zebrafish and their unexposed offspring.

Knowledge on the interaction between microplastics (MPs) and zinc oxide nanoparticles (ZnO NPs) is limited. Here, we investigated effects of embryo-larvae exposure to 500 µg/L polystyrene MPs (5 µm), 1200 µg/L ZnO NPs (< 100 nm), 500 µg/L dissolved Zn2+ from ZnSO4, and the mixtures of MPs and ZnO NPs or ZnSO4 on exposed F0 larvae and unexposed F1 larvae. Consequently, ZnO particles adhered to MPs surfaces rather than Zn2+, and increased Zn transport into larvae. Growth inhibition, oxidative stress, apoptosis, and disturbance of growth hormone and insulin-like growth factor (GH/IGF) axis were induced by MPs and ZnO NPs alone, which were further aggravated by their co-exposure in F0 larvae. MPs + ZnO increased apoptotic cells in the gill and esophagus compared with MPs and ZnO NPs alone. Reduced growth and antioxidant capacity and down-regulated GH/IGF axis were merely observed in F1 larvae from F0 parents exposed to MPs + ZnO. Contrary to ZnO NPs, dissolved Zn2+ reversed MPs toxicity, suggesting the protective role of Zn2+ may be not enough to ameliorate thfie negative effects of ZnO particles. To summarize, we found that particles rather than released Zn2+ from ZnO nanoparticles amplified MPs toxicity in early stages of exposed zebrafish and their unexposed offspring.

Micro-polyethylene particles reduce the toxicity of nano zinc oxide in marine microalgae by adsorption.

Contaminant adsorption by microplastics (MPs) allows them to act as contaminant vehicles or vectors, complicating eco-toxicological study of MPs. The contaminants adsorbed are mainly organic contaminants, especially hydrophobic organic contaminants (HOCs), although heavy-metal adsorption has also been reported. Compared to the mechanisms of HOC adsorption, those for metals are not fully understood. In the present study, combined-exposure assays revealed that polyethylene microplastics (PEMPs, 150 µm) alleviate the toxic effect of nano zinc oxide (nZnO, 20-30 nm) on marine microalgal growth by 14.4%. Thus, we hypothesized that nZnO adsorption onto PEMP surfaces ameliorates its toxicity to microorganisms. To test this hypothesis, PEMP samples isolated from nZnO suspensions were characterized. Their surfaces were observed by SEM, their Zn levels were measured by ICP-MS, and the compound form of Zn on the PEMP surface was determined by XRD analysis. The results indicated that 5.53%-7.16% of the Zn in the suspension is adsorbed during the first 24 h of exposure and that the Zn remains as the ZnO form upon adsorption. The findings in the present study provide important information on the role of MPs as metal oxide vehicles.

Characterization of four mitochondrial genomes of family Neritidae (Gastropoda: Neritimorpha) and insight into its phylogenetic relationships.

Neritidae is one of the most diverse families of Neritimorpha and possesses euryhaline properties. Members of this family usually live on tropical and subtropical coasts and are mainly gregarious. The phylogenetic relationships between several subclasses of Gastropoda have been controversial for many years. With an increase in the number of described species of Neritidae, the knowledge of the evolutionary relationships in this family has improved. In the present study, we sequenced four complete mitochondrial genomes from two genera (Clithon and Nerita) and compared them with available complete mitochondrial genomes of Neritidae. Gene order exhibited a highly conserved pattern among three genera in the Neritidae family. Our results improved the phylogenetic resolution within Neritidae, and more comprehensive taxonomic sampling of subclass Neritimorpha was proposed. Furthermore, we reconstructed the divergence among the main lineages of 19 Neritimorpha taxa under an uncorrelated relaxed molecular clock.

Effects of continuous and intermittent cadmium exposure on HPGL axis, GH/IGF axis and circadian rhythm signaling and their consequences on reproduction in female zebrafish: Biomarkers independent of exposure regimes.

Typical biomarkers of cadmium (Cd) pollution have well been confirmed in fish from continuous exposure pattern. However, in a natural environment, fish may be exposed to Cd intermittently. In this study, juvenile female zebrafish were exposed for 48 days to 10 µg/L Cd continuously, 20 µg/L for 1 day in every 2 days or 30 µg/L for 1 day in every 3 days. The toxic effects were evaluated using 8 various physiological and biochemical endpoints like specific growth rate (SGR), 17ß-estradiol (E2) and vitellogenin (VTG) concentrations in plasma, reproductive parameters (gonadosomatic index (GSI), egg-laying amount, spawning percentage, and hatching and mortality rate of embryos). Transcription of 59 genes related to hypothalamic-pituitary-gonadal-liver (HPGL) axis, circadian rhythm signaling and insulin-like growth factor (IGF) system was examined. SGR, spawning percentage, E2 and VTG levels declined in fish exposed to 10 and 20 µg/L Cd but remained relatively stable in fish exposed to 30 µg/L Cd. Exposure to 10, 20 and 30 µg/L Cd significantly reduced GSI, hatching rate and mortality rate. Similarly, mRNA expression of 27 genes were sensitive to both continuous and intermittent Cd exposure. Among these genes, expression levels of 10 genes had more than 5-fold increase or decrease, including mRNA levels of vtg1, vtg2, vtg3, esr1, igf2a, igf2b, igfbp5b, nr1d1, gnrh3 and gnrhr4. The most sensitive molecular biomarker was vtg3 expression with 1500-3100 fold increase in the liver. The present study, for the first time, provides effective candidate biomarkers for Cd, which are independent of exposure regimes.