A practical guide for the husbandry of cave and surface invertebrates as the first step in establishing new model organisms.

While extensive research on traditional model species has significantly advanced the biological sciences, the ongoing search for new model organisms is essential to tackle contemporary challenges such as human diseases or climate change, and fundamental phenomena including adaptation or speciation. Recent methodological advances such as next-generation sequencing, gene editing, and imaging are widely applicable and have simplified the selection of species with specific traits from the wild. However, a critical milestone in this endeavor remains the successful cultivation of selected species. A historically overlooked but increasingly recognized group of non-model organisms are cave dwellers. These unique animals offer invaluable insights into the genetic basis of human diseases like eye degeneration, metabolic and neurological disorders, and basic evolutionary principles and the origin of adaptive phenotypes. However, to take advantage of the beneficial traits of cave-dwelling animals, laboratory cultures must be established-a practice that remains extremely rare except for the cavefish Astyanax mexicanus. For most cave-dwelling organisms, there are no published culturing protocols. In this study, we present the results of our multi-year effort to establish laboratory cultures for a variety of invertebrate groups. We have developed comprehensive protocols for housing, feeding, and husbandry of cave dwellers and their surface relatives. Our recommendations are versatile and can be applied to a wide range of species. Hopefully our efforts will facilitate the establishment of new laboratory animal facilities for cave-dwelling organisms and encourage their greater use in experimental biology.

Toxicokinetics and bioaccumulation of silver sulfide nanoparticles in benthic invertebrates in an indoor stream mesocosm.

Mesocosms allow the simulation of environmentally relevant conditions and can be used to establish more realistic scenarios of organism exposure to nanoparticles. An indoor mesocosm experiment simulating an aquatic stream ecosystem was conducted to assess the toxicokinetics and bioaccumulation of silver sulfide nanoparticles (Ag2S NPs) and AgNO3 in the freshwater invertebrates Girardia tigrina, Physa acuta and Chironomus riparius, and determine if previous single-species tests can predict bioaccumulation in the mesocosm. Water was daily spiked at 10 µg Ag L-1. Ag concentrations in water and sediment reached values of 13.4 µg Ag L-1 and 0.30 µg Ag g-1 in the Ag2S NP exposure, and 12.8 µg Ag L-1 and 0.20 µg Ag g-1 in the AgNO3. Silver was bioaccumulated by the species from both treatments, but with approximately 1.5, 3 and 11 times higher body Ag concentrations in AgNO3 compared to Ag2S NP exposures in snails, chironomids and planarians, respectively. In the Ag2S NP exposures, the observed uptake was probably of the particulate form. This demonstrates that this more environmentally relevant Ag nanoform may be bioavailable for uptake by benthic organisms. Interspecies interactions likely occurred, namely predation (planarians fed on chironomids and snails), which somehow influenced Ag uptake/bioaccumulation, possibly by altering organisms´ foraging behaviour. Higher Ag uptake rate constants were determined for AgNO3 (0.64, 80.4 and 1.12 Lwater g-1organism day-1) than for Ag2S NPs (0.05, 2.65 and 0.32 Lwater g-1organism day-1) for planarians, snails and chironomids, respectively. Biomagnification under environmentally realistic exposure seemed to be low, although it was likely to occur in the food chain P. acuta to G. tigrina exposed to AgNO3. Single-species tests generally could not reliably predict Ag bioaccumulation in the more complex mesocosm scenario. This study provides methodologies/data to better understand exposure, toxicokinetics and bioaccumulation of Ag in complex systems, reinforcing the need to use mesocosm studies to improve the risk assessment of environmental contaminants, specifically NPs, in aquatic environments.

Metal transfer to sediments, invertebrates and fish following waterborne exposure to silver nitrate or silver sulfide nanoparticles in an indoor stream mesocosm.

The fate of engineered nanomaterials in ecosystems is unclear. An aquatic stream mesocosm explored the fate and bioaccumulation of silver sulfide nanoparticles (Ag2S NPs) compared to silver nitrate (AgNO3). The aims were to determine the total Ag in water, sediment and biota, and to evaluate the bioavailable fractions of silver in the sediment using a serial extraction method. The total Ag in the water column from a nominal daily dose of 10 µg L-1 of Ag for the AgNO3 or Ag2S NP treatments reached a plateau of around 13 and 12 µg L-1, respectively, by the end of the study. Similarly, the sediment of both Ag-treatments reached ~380 µg Ag kg-1, and with most of it being acid-extractable/labile. The biota accumulated 4-59 µg Ag g-1 dw, depending on the type of Ag-treatment and organism. The oligochaete worm, Lumbriculus variegatus, accumulated Ag from the Ag2S exposure over time, which was similar to the AgNO3 treatment by the end of the experiment. The planarian, Girardia tigrina, and the chironomid larva, Chironomus riparius, showed much higher Ag concentrations than the oligochaete worms; and with a clearer time-dependent statistically significant Ag accumulation relative to the untreated controls. For the pulmonate snail, Physa acuta, bioaccumulation of Ag from AgNO3 and Ag2S NP exposures was observed, but was lower from the nano treatment. The AgNO3 exposure caused appreciable Ag accumulation in the water flea, Daphnia magna, but accumulation was higher in the Ag2S NP treatment (reaching 59 µg g-1 dw). In the rainbow trout, Oncorhynchus mykiss, AgNO3, but not Ag2S NPs, caused total Ag concentrations to increase in the tissues. Overall, the study showed transfer of total Ag from the water column to the sediment, and Ag bioaccumulation in the biota, with Ag from Ag2S NP exposure generally being less bioavailable than that from AgNO3.

Biomarker responses in earthworm coelomocyte extract - Noninvasively collected sample for pesticide effect assessment.

Earthworms are often used as model organisms in ecotoxicological research because of their natural habitat where they can be exposed to many different pollutants, including pesticides. Since a number of them has to be sacrificed for sample collection, it would be useful to develop non-invasive methods and techniques suitable for the analysis of target parameters. The aim of this study is to determine whether the coelomocyte extract, obtained by the non-invasive method, can be used to measure responses of biochemical biomarkers and to establish if it can be used in assessing the effects of pesticides already known to have a negative impact on the earthworms. In the present study Eisenia andrei earthworms were exposed for 48 h to organophosphates dimethoate and pirimiphos-methyl using the filter paper contact test. Following exposure, coelomocyte extracts were prepared and acetylcholinesterase (AChE) and carboxylesterase (CES) activities were measured. The percentage of inhibition of the measured enzymes in the coelomocyte extract was compared with the inhibition of the same enzyme activities in the samples obtained from the whole body homogenate. AChE and CES inhibition was observed at all concentrations for both pesticides in different types of samples. Compared to the coelomocyte extract, the level of AChE inhibition was slightly stronger in the whole body homogenate. Inhibition of CES at the same concentrations in different types of samples did not always coincide, especially in the case of dimethoate, however significant inhibition of CES in coelomocyte extract was recorded. This study indicates the possibility of using the coelomocyte extract for measurement of biochemical biomarkers and assessment of pesticide effects.

Acute toxicity of selenate and selenite and their impacts on oxidative status, efflux pump activity, cellular and genetic parameters in earthworm Eisenia andrei.

Selenium (Se) is an essential element for humans, animals, and certain lower plants, but can be toxic at high concentration. Even though Se is potentially toxic, little information is available about the effects of Se on soil animals. The aim of this study was to assess the impact of different concentrations of two Se forms, selenate and selenite, on earthworm Eisenia andrei. In order to obtain comprehensive overview on the Se effects, different parameters were measured. Namely, acute toxicity, apoptosis, efflux pump activity, different enzymatic and non-enzymatic biomarkers (acetylcholinesterase, carboxylesterase, glutathione S-transferase, catalase, glutathione reductase and superoxide dismutase activities, lipid peroxidation level and GSH/GSSG ratio) and expression of genes involved in oxidative and immune response have been investigated. Additionally, measurement of metallothioneins concentration and concentration of Se in exposed earthworms has been also performed. The assessment of acute toxicity showed a greater sensitivity of E. andrei to selenite exposure, whereas Se concentration measurements in earthworms showed higher accumulation of selenate form. Both Se forms caused inhibition of the efflux pump activity. Decrease in superoxide dismutase activity and increase in lipid peroxidation and glutathione reductase activity indicate that Se has a significant impact on the oxidative status of earthworms. Selenate exposure caused an apoptotic-like cell death in the coelomocytes of exposed earthworms, whereas decreased mRNA levels of stress-related genes and antimicrobial factors were observed upon the exposure to selenite. The obtained data give insight into the effects of two most common forms of Se in soil on the earthworm E. andrei.