The gene expression of environmental organisms is useful as a biomarker of environmental pollution. One of its advantages is high sensitivity. We identified the cDNA of a novel cadmium-responsive gene in the soil collembolan Folsomia candida. The deduced protein, designated “metallothionein-like motif containing protein” (MTC), was cysteine-rich and contained a metallothionein-like motif with similarity to metallothionein, but had a much longer sequence than metallothionein and contained repeated sequences of amino acids. Expression of MTC mRNA was sensitively induced by cadmium exposure at 0.3 mg/kg of dry food, a concentration at which toxic effects are not observed, but expression was not affected by γ-ray exposure (an inducer of oxidative stress). These findings suggest that MTC is involved in cadmium-binding processes rather than in oxidative-stress responses. In conclusion, we suggest that gene expression of MTC may be a candidate biomarker for detecting low levels of cadmium contamination in soil.

During the last years, declines in honey bee colonies are being registered worldwide. Cholinergic pesticides and their extensive use have been correlated to the decline of pollinators and there is evidence that pesticides act as neuroendocrine disruptors affecting the metabolism of neuropeptides. However, there is a big absence of studies with quantitative results correlating the effect of pesticide exposure with changes on neuropeptides insects, and most of them are conducted under laboratory conditions, typically with individual active ingredients. In this study, we present an analytical workflow to evaluate pesticide effects on honey bees through the analysis of (neuro)peptides. The workflow consists of a rapid extraction method and liquid chromatography with triple quadrupole for preselected neuropeptides. For non-target analysis, high resolution mass spectrometry, multivariate analysis and automatic identification of discriminated peptides using a specific software and protein sequence databases. The analytical method was applied to the analysis of target and non-target (neuro)peptides in honey bees with low and high content of a wide range of pesticides to which have been exposed in field conditions. Our findings show that the identification frequency of target neuropeptides decreases significantly in honey bees with high concentration of pesticides (pesticide concentrations ≥ 500 μg kg⁻¹) in comparison with the honey bees with low content of pesticides (pesticide concentrations ≤ 20 μg kg⁻¹). Moreover, the principal component analysis in non-target search shows a clear distinction between peptide concentration in honey bees with high level of pesticides and honey bees with low level. The use of high resolution mass spectrometry has allowed the identification of 25 non-redundant peptides responsible for discrimination between the two groups, derived from 18 precursor proteins.

Metallothioneins (MTs) belong to a conserved low-molecular-weight protein family that participates in heavy metal binding and detoxification. EcMT-1 was amplified by PCR from genomic DNA of Exopalaenon carinicauda. It contained a 180 bp open reading frame and encoded 59 amino acids. A total of 18 cysteine (Cys) residues were found in the deduced amino acid sequence, which was consistent with the Cys-rich characteristics of MTs. EcMT-1 was mainly expressed in hepatopancreas, followed by stomach and gill. The expression profiles of EcMT-1 indicated that EcMT-1 was significantly increased at 24, 48 h and 12, 24, and 48 h under the treatment of 2.5 μmol/L CdCl₂ and 50 μmol/L CuSO₄. The expression of EcMT-1 at gastrula stage was very low; it was detectable until nauplius stage, and the highest expression level appeared in the postlarvae stage.

Bivalves use anti-oxidative enzyme systems to defend themselves against excessive reactive oxygen species, which are often catalyzed by environmental pollution. As a key member of anti-oxidative enzyme family, catalase plays a crucial role in scavenging the high level of reactive oxygen species to protect organisms against various oxidative stresses. In this study, a catalase homologue was identified from Mytilus coruscus (named McCAT, KX957929). The open reading frame of McCAT was 1844bp with a 5′ untranslated region of 341bp and a 3′ untranslated region of 927bp. The deduced amino acid sequence was 512 residues in length with theoretical pI/MW 8.02/57.91kDa. BLASTn and phylogenetic analyses strongly suggested that it was a member of catalase, also known as CAT family for its conserved catalytic site motif and proximal heme-ligand signature motif. Real-time fluorescence quantitative PCR showed that constitutive expression of McCAT was occurred, with increasing order in mantle, adductor, gill, hemocyte, gonad and hepatopancreas. It was observed that bacterial infection and heavy metals stimulation up-regulated McCAT mRNA expression in hepatopancreas with time-dependent manners. The maximum expression appeared at 8h after pathogenic bacteria injecting, with 15-fold in Vibrio parahemolyticus and 60-fold in Aeromonas hydrophila than that of 0h. The highest point of McCAT mRNA appeared at different times for exposure to heavy metals with copper at day 5 (0.1mg/L 30-fold, 0.5mg/L 15-fold, 1.5mg/L 6-fold) and plumbum at day 3 (3.0mg/L 20-fold). The enzymatic activity analysis found that McCAT activity in the gill of M. coruscus was affected by heavy metals concentration. The results suggested that McCAT plays a significant role in antioxidation and the expression of McCAT can be used as a biomarker for detection of marine environmental pollution.

A CYP4 cDNA was cloned and characterized to identify the relationship between persistent organic pollutants and stress response in marine polychaete Perinereis aibuhitensis. The full length of PaCYP4 cDNA is 1857bp and encodes 481 amino acids. The deduced amino acid sequence showed 73% identity with CYP4BB1 from polychaete Nereis virens and shared high homology to other known CYP4 sequences. The expression level of PaCYP4 under petroleum hydrocarbon (PH) and deltamethrin (DM) exposure was detected using Real-time PCR. PH and combined toxicity treatments elevated the mRNA level of PaCYP4 in a dose- and time-dependent manner. The mRNA transcripts of PaCYP4 increased at the beginning of DM exposure and then eventually decreased, and the expression level of PaCYP4 down-regulated with increasing concentration of DM. CYP4 in P. aibuhitensis plays an important role in the metabolism of petroleum hydrocarbon and organochlorine pesticide.

Toll-like receptors (TLRs), type I transmembrane pattern recognition receptors (PRRs), are composed of the extracellular domain that is implicated in the recognition of microbial products and initiates the innate and adaptive immune response. Previous reports on TLRs in birds showed significant levels of inter- and intraspecific genetic variation. Little is known about the structure and function of the avian immune system, especially waterfowl species. This work aimed to identify and clone Anas platyrhynchos (mallard duck) TLR-3 (dTLR-3) and its expression level following challenge with velogenic Newcastle disease virus (NDV) as a model for waterfowl species. The mallard duck TLR-3 full-length cDNA sequence had been cloned, which consisted of 2457 nucleotides. The translated amino acid sequence showed identity degree as 97% with Muscovy duck, 95% with geese, 89% with helmeted guineafowls, 88% with the chickens TLR-3 gene, 82% with turkey TLR-3, and 79% with zebra finch, while it showed 54% with human one; the analysis data suggested that the new sequence is probably homologous to vertebrates’ TLR-3. The predicted protein encoded by the duck dTLR-3 mRNA sequence is composed of 819 amino acids. Analysis of the deduced amino acid sequence indicated that dTLR-3 has typical structural features and contains the main components of proteins in the TLR family. The dTLR-3 expressed in almost all examined tissues of mallard duck following quantitative real-time polymerase chain reaction (qPCR) analysis and using B-actin as a housekeeping gene. To check the functionality of the receptor and its role in viral infection, we evaluate the expression level in different tissues and its changes following NDV infection. The results showed significant (P < 0.05) upregulated in the brain at 24 h (1.84-fold), reached a peak at 48 h (4.82-fold), and recovered to normal levels at 72 h post-infection. These results indicate a complete and functional dTLR-3 that is orthologous to other vertebrate receptors with its potential role in early response against viral infection in mallard duck species.

Currently, there are five known types of mercury transporters in bacteria: MerC, MerE, MerF, MerH, and MerT. Their general function is to mediate mercuric ion uptake into the cell in preparation for reduction to Hg°. They are present in several bacterial phyla and comprise five distinct families. We have utilized standard statistical bioinformatic tools and the superfamily principle to show that they are related by common descent. After using programs such as Global Alignment Program and SSearch to establish homology, we aligned and analyzed their amino acid sequences to find a single well conserved motif. Although these proteins exhibit 2, 3, or 4 transmembrane helical segments (TMSs), TMSs 1 and 2 are common to all superfamily members. An ancestral sequence was determined, and reliable phylogenetic trees were constructed. The results support the conclusion of homology, establishing that these proteins belong to a single superfamily. This important discovery allows extrapolation of information about structure, function, and mechanism from one protein to all superfamily members to degrees inversely proportional to their phylogenetic distances.

The emissions of CO₂ into the atmosphere have been constantly rising due to anthropogenic activities, which have led to global warming and climate change. Among various methods proposed for mitigating CO₂ levels in the atmosphere, carbonic anhydrase (CA)-mediated carbon sequestration represents a greener and safer approach to capture and convert it into stable mineral carbonates. Despite the fact that CA is an extremely efficient metalloenzyme that catalyzes the hydration of CO₂ (CO₂ + H₂O ↔ HCO₃ ⁻ + H⁺) with a kcat of ∼10⁶ s⁻¹, a thermostable, and alkalistable CA is desirable for the process to take place efficiently. The purified CA from alkaliphilic, moderately thermophilic, and halotolerant Bacillus halodurans TSLV1 (BhCA) is a homodimeric enzyme with a subunit molecular mass of ~37 kDa with stability in a broad pH range between 6.0 and 11.0. It has a moderate thermostability with a T₁/₂ of 24.0 ± 1.0 min at 60 °C. Based on the sensitivity of CA to specific inhibitors, BhCA is an α-CA; this has been confirmed by nucleotide/amino acid sequence analysis. This has a unique property of stimulation by SO₄ ²⁻, and it remains unaffected by SO₃ ²⁻, NOx, and most other components present in the flue gas. BhCA is highly efficient in accelerating the mineralization of CO₂ as compared to commercial bovine carbonic anhydrase (BCA) and is also efficient in the sequestration of CO₂ from the exhaust of petrol driven car, thus, a useful biocatalyst for sequestering CO₂ from flue gas.