Toward invasive mussel genetic biocontrol: Approaches, challenges, and perspectives.

Invasive freshwater mussels, such as the zebra (Dreissena polymorpha), quagga (Dreissena rostriformis bugensis), and golden (Limnoperna fortunei) mussel have spread outside their native ranges throughout many regions of the North American, South American, and European continents in recent decades, damaging infrastructure and the environment. This review describes ongoing efforts by multiple groups to develop genetic biocontrol methods for invasive mussels. First, we provide an overview of genetic biocontrol strategies that have been applied in other invasive or pest species. Next, we summarize physical and chemical methods that are currently in use for invasive mussel control. We then describe the multidisciplinary approaches our groups are employing to develop genetic biocontrol tools for invasive mussels. Finally, we discuss the challenges and limitations of applying genetic biocontrol tools to invasive mussels. Collectively, we aim to openly share information and combine expertise to develop practical tools to enable the management of invasive freshwater mussels.

Cytogenetic characterization of the golden mussel (Limnoperna fortunei) reveals the absence of sex heteromorphic chromosomes.

The golden mussel (Limnoperna fortunei) is an aggressive invasive species in South America, where it endangers native species and freshwater ecosystems, in addition to causing extensive economic losses, mainly to the hydroelectric sector. Currently, there's no efficient control method available and the invasion has progressed across the continent. Its high reproduction rate is one of the key factors of the golden mussel's high invasive potential and, recently, efforts have been done in order to understand the reproduction and the sexual features of this species. However, its cytogenetics characterization is incipient and the possible occurrence of sex-specific cytogenetic features was never investigated. In this study, we aimed to characterize the chromosomal morphometry, the distribution profile of heterochromatin, and to detect possible sex-related epigenetic marks in the golden mussel. Results revealed that the karyotypic structure is similar in both sexes and no chromosome heteromorphism was observed between males and females specimens. The data increment the cytogenetic characterization of Limnoperna fortunei and contribute for future studies that aim to further investigate its reproduction and underlying sex determination processes.

Molecular effects of Microcystin-LA in tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is a freshwater phytoplanktivorous fish species reported to accumulate and tolerate large amounts of cyanotoxins such as microcystins (MCs). The present study aimed to investigate molecular responses to the acute exposure of Nile tilapia to the Microcystin-LA analogue (MC-LA). Thus, the specimens were sublethally exposed to 1000 µg kg-1 of MC-LA for 12, 24, 48, and 96 h. Gene expression of PP1, PP2A, GST, GPX and actin was analyzed by quantitative PCR. The protein abundance profile of PP2A was determined by immunoblotting, while the integrity of its biological function was assessed by a phosphatase enzymatic assay. PP2A activity was significantly and strongly reduced by MC-LA. A resulting feedback mechanism significantly increased PP2A gene expression and protein abundance in all assessed times. However, a recovery of that phosphatase activity was not observed. In this study, the observed increase in GPX gene expression was the only response that could be directly related to the unknown factors associated to the fish survival to such high dose exposure.

Altered substrate specificity of the Pterygoplichthys sp. (Loricariidae) CYP1A enzyme.

Ethoxyresorufin is a classical substrate for vertebrate CYP1A enzymes. In Pterygoplichthys sp. (Loricariidae) this enzyme possesses 48 amino acids substitutions compared to CYP1A sequences from other vertebrate species. These substitutions or a certain subset substitution are responsible for the non-detection of the EROD reaction in this species liver microsomes. In the present study, we investigated the catalytic activity of Pterygoplichthys sp. CYP1A toward 15 potential substrates in order to understand the substrate preferences of this modified CYP1A. The fish gene was expressed in yeast and the accumulation of the protein was confirmed by both the characteristic P450-CO absorbance spectra and by detection with monoclonal antibodies. Catalytic activities were assayed with yeast microsomes and four resorufin ethers, six coumarin derivates, three flavones, resveratrol and ethoxyfluoresceinethylester. Results demonstrated that the initial velocity pattern of this enzyme for the resorufin derivatives is different from the one described for most vertebrate CYP1As. The initial velocity for the activity with the coumarin derivatives is several orders of magnitude higher than with the resorufins, i.e. the turnover number (kcat) for ECOD is 400× higher than for EROD. Nonetheless, the specificity constant (kcat/km) for EROD is only slightly higher than for ECOD. EFEE is degraded at a rate comparable to the resorufins. Pterygoplichthys sp. CYP1A also degrades 7-methoxyflavone and ß-naphthoflavone but not resveratrol and chrysin. These results indicate a divergent substrate preference for Pterygoplichthys sp. CYP1A, which may be involved in the adaptation of Loricariidae fish to their particular environment and feeding habits.

Structural features of cytochrome P450 1A associated with the absence of EROD activity in liver of the loricariid catfish Pterygoplichthys sp.

The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acid substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrate.

Cloning and detection of metallothionein mRNA by RT-PCR in mangrove oysters (Crassostrea rhizophorae).

A semi-quantitative RT-PCR protocol was developed to directly evaluate metallothionein (MT) mRNA expression in different tissues of mangrove oysters (Crassostrea rhizophorae), using beta-Actin (ACT) as a normalizing gene. Clones with high degree of identity from partial coding sequences were obtained for both MT and ACT. Although not statistically significant, high relative accumulation of MT mRNA was observed in the digestive gland (DGG), but not in the gills, from samples collected from both control and contaminated sites. Nevertheless, MT expression was not comparable to the high levels of metal in the contaminated oysters. Results indicate that the variation in relative MT mRNA levels from different samples of the same site could be due to multiple gene copies or different MT isoform induction.

Correlation between bacteria (Klebsiella sp.) and fish (Brachidanio rerio) in wastewater toxicity test results

Because of the practical advantages of thre utilization of microorganisms in screening test for wastewater monitoring, the correlation between results of toxicity tests with bacterium and fish for a chemical mixed effluent was examined. Results showed correlation (r=0,74,n=31,p

Correlation between bacteria (Klebsiella sp.) and fish (Brachidanio rerio) in wastewater toxicity test results

Because of the practical advantages of thre utilization of microorganisms in screening test for wastewater monitoring, the correlation between results of toxicity tests with bacterium and fish for a chemical mixed effluent was examined. Results showed correlation (r=0,74,n=31,p