In memory of&#160;Professor Amilcar Roberto.
This work was financially supported by Fundac&#807;a&#771;o para a Cie&#770;ncia e a Tecnologia (FCT, Portugal) under projects UIDB/04567/2020 and UIDP/04567/2020 attributed to CBIOS and PhD grant SFRH/BD/137671/2018 (Vera Isca).

1. Equipment preparation
<ol>
	<li>Acquire commercially available hatching equipment. Select a suitable place to set up the hatching equipment (Figure 2A). Place the funnel-shaped container in the black support (included in the set) and turn the funnel in a suitable direction to see the level mark and the tap.</li>
	<li>To make hand-made migration equipment, cut the top of two 0.5 L (5.8 cm diameter) plastic bottles to obtain a final height of 12 cm. Create a hole of 1.5 cm diameter on one side at 7 cm from the bottom in each bottle and insert a 13 cm rubber tube (1.3 cm outer and 0.9 cm inner diameter) between the two openings. Seal the openings with hot glue (Figure 2B) and leave to dry for 15 min; put the bottles on a flat surface and fill them with water to verify that there is no leakage.</li>
</ol>
2. Preparation of artificial salt solution
<ol>
	<li>In a glass beaker, prepare an artificial salt solution (brine shrimp salt) at a concentration of 35 g/L. To do this, add 28 g of the salt to 800mL of tap water, according to the manufacturer&#39;s instructions. Mix it with a stirring rod until all the salt is thoroughly dissolved. 
	NOTE: Adjust the volume of the prepared saline solution according to the size of the available containers.</li>
</ol>
3. Sample preparation
<ol>
	<li>Prepare all the samples in a microcentrifuge tube by dissolving a suitable amount of extracts (Plectranthus extracts, Pa- P. ambigerus; Pb- P. barbatus; Pc- P. cylindraceus; and Pe- P. ecklonii) or compounds 1-5 (two natural compounds [1 and 2] obtained from Plectranthus spp. and three semi-synthetic derivatives [3, 4, 5]; Figure 3) in dimethyl sulfoxide (DMSO)12, so as to obtain a final concentration of 10 mg/mL (if the sample is water-soluble, use of DMSO is not necessary).</li>
	<li>Dilute 10 &#181;L of each sample (and DMSO for the negative control) in a new microcentrifuge tube using 990 &#181;L of artificial saline solution prepared in step 2.1, to obtain a final concentration of 0.1 mg/mL.</li>
	<li>Under a fume hood, in an Erlenmeyer flask, prepare a solution of potassium dichromate (K2Cr2O7) in distilled water at a concentration of 1 mg/mL13,14,15.</li>
</ol>
4. Brine shrimp lethality bioassay
NOTE: This assay is developed from the works of several authors with modifications1,16,17,18,19.
<ol>
	<li>Fill the hatching vessel with the medium prepared in step 2.1 up to the level mark (500 mL) (Figure 2C).</li>
	<li>Place one spoon (approximately 0.75 g) of brine shrimp cysts in the salt solution, and then close the container. Place a lamp (table lamp, 40 W, 230 V, 50 Hz, with a LED light bulb of 8 W, 4,000 K, 830 lm) pointing directly toward the equipment (Figure 2A) and turn it on.</li>
	<li>Attach the air supplier system (3 W output, 50 Hz, 230 V) to the connector placed on the top of the equipment and turn the pump on.</li>
	<li>Keep the room temperature at 25 &#177; 3 &#176;C. Brine shrimp cysts hatch in the artificial salt solution, under vigorous aeration, continuous lighting, and stable temperature, after 24 h to 48 h. 
	NOTE: Alternatively, a vertical incubator can be used.</li>
	<li>Once the eggs have hatched, turn the air pump off and wait until the nauplii (moving toward the bottom of the funnel) are separated from the empty egg cases (floating at the top).</li>
	<li>In order to separate the unhatched eggs from alive nauplii, open the outlet tap at the bottom and discharge the content of the funnel in one of the containers of the hand-made migration equipment container (described in step 1.2). Ensure that the solution containing the nauplii and the residual unhatched eggs is below the level of the tube. In the second container, add the residual salt solution from step 2.1 above the height of the tube.</li>
	<li>Cover the container with the nauplii and the residual unhatched eggs using aluminum foil. Place the lamp on the second container with just the salt solution. The brine shrimp will be attracted by the light and migrate from one container to the other (harvesting container), leading to efficient separation between eggs (slowly sedimented to the bottom) and alive Artemia.</li>
	<li>Then, place the equipment in the incubator under the same conditions used in step 4.4 for 4 h (Figure 2E). From the harvesting container, collect 900 &#181;L of saline solution containing 10 to 15 nauplii. Place the saline solution with nauplii in each well of a 24-well plate (Figure 2F); all the samples are tested in quadruplicates.</li>
	<li>Add 100 &#181;L each of the negative control (DMSO), the positive control (K2Cr2O7, potassium dichromate), the artificial salt solution, and each of the samples to respective well (Figure 2F)13,14. 
	NOTE: The samples in each well will be at a concentration of 0.01 mg/mL. The final concentration of the positive control in salt solution will be 0.1 mg/mL, to be sure that all the nauplii in the well are exposed to the toxic effect of potassium dichromate and die. The artificial salt solution will act as blank.</li>
	<li>Incubate the plate at 25 &#177; 3 &#176;C under illumination for 24 h (Figure 2G). After 24 h, register the number of dead larvae (non-mobile nauplii for 5 s) in each well under a binocular microscope (12x)20&#160;(Figure 2H). Alternatively, use a hand lens.</li>
	<li>Add 100 &#181;L of potassium dichromate solution, to induce the death of the remaining living larvae, and wait for 6 h. Count the total dead larvae in each well under a microscope. Determine the mortality rate according to the following equation. 
	<img alt="Equation 1" src="/files/ftp_upload/64472/64472eq01.jpg" style="margin: auto;" /></li>
	<li>Perform all the assay in triplicate. Calculate standard deviations (SD), and express the results as the mean of three independent experiments, each with internal quadruplicates (n = 12), &#177; SD. As mentioned by Meyer et al., consider crude extracts and pure compounds with LC50&#60; 1,000 &#181;g/mL as toxic; also, take into account that the mortality rate of brine shrimp is proportional to the concentration of the tested samples21.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64472/64472fig02.jpg" /> 
Figure 2: Artemia salina lethality bioassay method. (A) Commercially available equipment employed for the hatching of brine shrimp cysts; (B) Hand-made migration equipment; (C) Hatching vessel filled with saline solution; (D) Collection of unhatched eggs and nauplii; (E) Hand-made equipment in the incubator during the migration step. The container far from the lamp should be covered with aluminum foil; however, for a better view of the set installation here it was removed; (F) Harvesting of Artemia in wells prior to performing the assay. The compounds should be placed as shown: - refers to the negative control (DMSO), + to the positive control (K2Cr2O7), salt to the artificial salt solution, and 1 to 3 to the samples to test (in this case compounds 1-3); (G) Incubation of the 24-well plate containing Artemia and the selected samples; (H) Artemia count under the binocular microscope. <a href="https://www.jove.com/files/ftp_upload/64472/64472fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64472/64472fig03.jpg" /> 
Figure 3: Structures of selected compounds. Structure of compounds 1-2, extracted from Plectranthus species, and compounds 3-5, obtained by semi-synthesis. <a href="https://www.jove.com/files/ftp_upload/64472/64472fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors declare no conflicts of interest, financial or otherwise.

During the last years, the scientific community has increased its attention toward alternative models for toxicity screenings21. Beside A. salina lethality bioassay, other methodologies are usually performed for the evaluation of sample tolerability and include vertebrate bioassays (such as rodents), invertebrates (such as zebrafish), in vitro methods using yeast strains or cells, and in silico methods22,23,...

Natural products from plants, animals, or microorganisms have been a growing area of interest over the years in the development of new bioactive molecules because of their varied range of biological and pharmacological activities1. However, the associated side effects, drug resistance, or inadequate specificity of the agents, especially when used as anticancer drugs, represent the major factors that can lead to ineffective treatment1,2.
Over the last few decades, several plant-derived cytotoxic agents have been discovered, some of them used as anticancer agents1,2,3. In this context, paclitaxel is reported as one of the best-known and most active chemotherapeutic drugs of natural origin3,4. Currently, it is estimated that more than 35% of all medicines on the market are derived from or are inspired by natural products5. The potential high toxicity of these compounds requires consideration during all of the study phases, since different types of contaminants or even metabolic components of the plant itself can cause toxic effects. For this reason, pharmacological and toxicological profiles should be undertaken in the preliminarily phase, to assess the biological activity and safety of new potential plant-based treatments. To evaluate the toxicity of new bioactive samples, invertebrate animals can be considered as the best models to study. They demand minimal ethical requirements and allow preliminary in vitro assays, to prioritize the most promising products for the next round of testing in vertebrates1,6.
Commonly known as brine shrimp, A. salina is a small halophilic invertebrate belonging to the genus Artemia (family Artemiidae, order Anostraca, subphylum Crustacea; Figure 1). In marine and aquatic saline ecosystems, brine shrimps play an important nutritional role as they feed on microalgae and are constituents of the zooplankton used to feed fish. Moreover, their larvae (known as nauplii) are widely used in the assessment of general toxicity during preliminary studies1,3,7.
Artemia spp. are widely used in lethality studies and are also a convenient starting point for toxicity assessments, by tracking the toxicity of potentially bioactive compounds based on their ability to kill nauplii grown in the laboratory1,8. For this reason, the use of A. salina gained attraction in general toxicity studies, because it is a very efficient and easy-to-use method, compared to other tests on animal models9.
Owing to their simple anatomy, tiny size and short life cycle, a vast number of invertebrates can be studied in a single experiment. As such, they combine genetic amenability and low-cost compatibility with large-scale screenings1. In this context, the use of brine shrimp in a general toxicity assay shows several advantages, such as fast growth (28-72 h is needed from hatching to the first results), cost-effectiveness, and long shelf-life of commercial eggs, that can be used all year round3,10. On the other hand, since invertebrates have a primitive organ system and lack an adaptive immune system, they do not represent a perfect and reliable model for human cells1.
However, it provides a preliminary evaluation method for the general toxicity of selected samples. Since it is widely used as a lethality assay, it can provide provisional indications about the toxic effects of potential anticancer agents. It is often also used to obtain feedback about the general toxicity of compounds endowed with any other biological activities for which it is essential to show the lowest mortality rate possible among the Artemia shrimps.
In an ongoing study from our group, different extracts from Plectranthus species showed antioxidant and antimicrobial activities (unpublished results). In parallel, isolated compounds were obtained by purification of the extracts and were then chemically modified. The extracts, pure compounds, and semisynthetic derivatives were then tested in terms of general toxicity. In this context, the present work aims to give an overview of the use of the Artemia lethality bioassay for the evaluation of general toxicity and potential cytotoxic activity of bioactive extracts and isolated compounds from different plants of the genus Plectranthus11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64472/64472fig01.jpg" /> 
Figure 1: Artemia salina under the microscope. Newly hatched nauplii of A. salina as seen under the microscope (magnification 12x). <a href="https://www.jove.com/files/ftp_upload/64472/64472fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>24-well plates</td><td>Thermo Fisher Scientific, Denmark</td><td>174899</td><td>Thermo Scientific Nunc Up Cell 24 multidish</td></tr><tr><td>Aluminium foil</td><td>Albal</td><td>-</td><td>Can be purchased in supermarket</td></tr><tr><td>Artemio Set</td><td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td><td>61066000</td><td>Can be purchased in pet shops</td></tr><tr><td>Binocular microscope</td><td>Ceti, Belgium</td><td>&amp;nbsp;1700.0000</td><td>Flexum-24AED, 220-240 V, 50 Hz</td></tr><tr><td>Bottles</td><td>-</td><td>-</td><td>0.5 L Diameter: 5.8 cm; Height: 12 cm</td></tr><tr><td>Brine shrimp cysts</td><td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td><td>3090700</td><td>Can be purchased in pet shops</td></tr><tr><td>Brine shrimp salt</td><td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td><td>3090600</td><td>Can be purchased in pet shops</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>VWR chemicals</td><td>CAS: 67-68-5</td><td>&amp;nbsp;99% purity</td></tr><tr><td>Discartable tips</td><td>Diamond</td><td>F171500</td><td>Volume range: 100 - 1000 &amp;micro;L</td></tr><tr><td>Eppendorf microtubes</td><td>BRAND</td><td>7,80,546</td><td>Microtubes, PP, 2 mL, BIO-CERT&amp;nbsp;PCR QUALITY</td></tr><tr><td>Erlenmeyer flask</td><td>VWR chemicals</td><td>4,47,109</td><td>volume: 100 mL</td></tr><tr><td>Glass beaker</td><td>Normax</td><td>3.2111654N</td><td>Volume: 1000 mL</td></tr><tr><td>Gloves</td><td>Guantes Luna</td><td>GLSP3</td><td>-</td></tr><tr><td>GraphPad Prism</td><td>GraphPad Software, San Diego, CA, USA</td><td>-</td><td>GraphPad Prism version 5.00 for Windows, www.graphpad.com, accessed on 5 February 2021; commercial statistical analysis software</td></tr><tr><td>Home-made A. salina Grower</td><td>&amp;nbsp;-</td><td>&amp;nbsp;-</td><td>Home made: two plastic bottles connected by a hose</td></tr><tr><td>Hot glue</td><td>Parkside</td><td>PHP500E3</td><td>230 V, 50 Hz, 25 W</td></tr><tr><td>Incubator</td><td>Heidolph Instruments, Denmark&amp;nbsp;</td><td>&amp;nbsp;-</td><td>One Heidolph Unimax 1010 equipment and one Heidolph Inkubator 1006</td></tr><tr><td>Light</td><td>Roblan</td><td>SKYC3008FE14</td><td>LED light bulb</td></tr><tr><td>Micropipettes</td><td>VWR chemicals</td><td>613-5265</td><td>Volume range: 100 - 1000 &amp;micro;L</td></tr><tr><td>Potassium dichromate (K&lt;sub&gt;2&lt;/sub&gt;Cr&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;7&lt;/sub&gt;)</td><td>VWR chemicals</td><td>CAS: 7778-50-9</td><td>&amp;nbsp;99% purity</td></tr><tr><td>Pump ProAir a50</td><td>JBL GmbH and Co. KG, D-67141, Neuhofen Germany</td><td>&amp;nbsp;-</td><td>Included in the Artemio Set+1 kit</td></tr><tr><td>Rubber tube</td><td>-</td><td>-</td><td>1.3 cm outer and 0.9 cm inner diameter</td></tr><tr><td>Stirring rod</td><td>VWR chemicals</td><td>441-0147</td><td>&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/64472/64472eq02.jpg&quot; style=&quot;margin: auto;&quot;/&gt; 6 mm, 250 mm</td></tr><tr><td>Termometer</td><td>VWR chemicals</td><td>620-0821</td><td>0 - 100 &amp;deg;C</td></tr></tbody></table>

lethality bioassay using artemia salina l.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural sources and used against a plethora of diseases. Unfortunately, most of these compounds often display systemic toxicity and adverse effects. In order to better evaluate the tolerability of selected potentially bioactive samples, brine shrimp (Artemia salina) is generally used as a model in lethality studies. The A. salina test is based on the ability of the studied bioactive compounds to kill the microcrustaceans in their larval stage (nauplii). This method represents a convenient starting point for cytotoxicity studies, as well as for the general toxicity screening of synthetic, semisynthetic, and natural products. It can be considered a simple, quick, and low-cost assay, compared to many other assays (in vitro cells or yeast strains, zebrafish, rodents) generally suitable for the aforementioned purposes; moreover, it can be easily performed even without any specific training. Overall, A. salina assay represents a useful tool for the preliminary toxicity evaluation of selected compounds and the bio-guided fractionation of natural product extracts.

The general toxicity of some natural products recently studied by our group was evaluated through the brine shrimp lethality bioassay. Four extracts (Pa- P. ambigerus; Pb- P. barbatus; Pc- P. cylindraceus; and Pe- P. ecklonii) from Plectranthus genus, known for their antioxidant activity (unpublished results), were tested. Additionally, two natural compounds (1 and 2) obtained from Plectranthus spp., and three semi-synthetic derivatives (3, 4, 5; ...

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Lethality Bioassay Using Artemia salina L.

This work aims to evaluate and review the Artemia salina lethality bioassay procedure, also identified as brine shrimp lethality assay. This simple and cheap method gives information about the general toxicity (considered as a preliminary toxicity evaluation) of samples, namely, natural products.

Lethality Bioassay Using Artemia salina L.

Natural products have been used since ancient times to produce medicines. Nowadays, there are plenty of chemotherapeutic drugs obtained from natural ...

lethality-bioassay-using-artemia-salina-l

Research

JoVE Journal

Chemistry

9.7K Views.  Universidade Lusófona de Humanidades e Tecnologias. Dit protocol is een zeer nuttig hulpmiddel voor een voorlopige algemene toxiciteitsevaluatie van verschillende soorten monsters, zoals extracten, fracties en verbindingen uit verschillende natuurlijke of synthetische bronnen. Het belangrijkste voordeel van ons protocol is dat het heel gemakkelijk en goedkoop is en op veel monsters tegelijkertijd kan worden gebruikt. Bovendien verbetert de handgemaakte apparatuur de betrouwbaarheid van de site.In deze techniekfase doen we een screening ...

Video: Lethality Bioassay Using Artemia salina L.

Salinity-dependent Toxicity Assay of Silver Nanocolloids Using Medaka Eggs

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and seawater. Tests of the toxicity of chemicals and assessments of their ecological risks to aquatic organisms are frequently performed in freshwater, but the toxicity of chemicals to aquatic organisms depends on pH, temperature, and salinity. There is no method, however, for testing the salinity dependence of toxicity to aquatic organisms. Here, we used medaka (Oryzias latipes) because they can adapt to freshwater, brackish water, and seawater. Different concentrations of embryo-rearing medium (ERM) (1x, 5x, 10x, 15x, 20x, and 30x) were employed to test the toxicity of silver nanocolloidal particles (SNCs) to medaka eggs (1x ERM and 30x ERM have osmotic pressures equivalent to freshwater and seawater, respectively). In six-well plastic plates, 15 medaka eggs in triplicate were exposed to SNCs at 10 mg/L&#8722;1 in different concentrations of ERM at pH 7 and 25 &#176;C in the dark.
We used a dissecting microscope and a micrometer to measure heart rate per 15 sec and eye diameter on day 6 and full body length of the larvae on hatching day (section 4). The embryos were observed until hatching or day 14; we then counted the hatching rate every day for 14 days (section 4). To see silver accumulation in embryos, we used inductively coupled plasma mass spectrometry to measure the silver concentration of test solutions (section 5) and dechorionated embryos (section 6).The toxicity of the SNCs to medaka embryos obviously increased with increasing salinity. This new method allows us to test the toxicity of chemicals in different salinities.

Embryonic stages are the most susceptible to xenobiotics. Although chemical toxicity depends on salinity, no method exists to test the salinity dependence of toxicity to aquatic organisms. Here, we describe a new and high-throughput method for determining the salinity dependence of toxicity to aquatic embryos.

Salinity is an important characteristic of the aquatic environment. For aquatic organisms it defines the habitats of freshwater, brackish water, and ...

Developmental Biology

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food webs. Toxicity testing based on microorganisms are widely used because they are relatively quick, reproducible, cheap, and are not associated with ethical issues. Here, we describe an ecotoxicological method to evaluate the biological response of the marine bacterium Vibrio anguillarum. This method assesses the acute toxicity of chemical compounds, including new contaminants such as nanoparticles, as well as environmental samples. The endpoint is the reduction of bacterial culturability (i.e., the capability to replicate and form colonies) due to exposure to a toxicant. This reduction can be generally referred to as mortality. The test allows for the determination of the LC50, the concentration that causes a 50% decrease of bacteria actively replicating and forming colonies, after a 6 h exposure. The culturable bacteria are counted in terms of colony forming units (CFU), and the &#34;mortality&#34; is evaluated and compared to the control. In this work, the toxicity of copper sulphate (CuSO4) was evaluated. A clear dose-response relationship was observed, with a mean LC50 of 1.13 mg/L, after three independent tests. This protocol, compared to existing methods with microorganisms, is applicable in a wider range of salinity and has no limitations for colored/turbid samples. It uses saline solution as the exposure medium, avoiding any possible interferences of growth medium with the investigated contaminants. The LC50 calculation facilitates comparisons with other bioassays commonly applied to ecotoxicological assessments of the marine environment.

Ecotoxicological Method with Marine Bacteria Vibrio anguillarum to Evaluate the Acute Toxicity of Environmental Contaminants

This work describes a new protocol to assess the ecotoxicity of pollutants, including emerging contaminants such as nanomaterials, using the marine bacterium Vibrio anguillarum. This method allows for the determination of the LC50, or mortality, the concentration that causes a 50% decrease in bacteria culturability, after a 6 h exposure.

Bacteria are an important component of the ecosystem, and microbial community alterations can have a significant effect on biogeochemical cycling and food ...

Environment

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity testing has been demonstrated. Overall, the sensitivity of the species to toxic compounds is in the range with, or higher than, that of other model species.
This work describes protocols for acute, chronic, and multigenerational bioassays of single and combined stressor effects on N. furzeri. Due to its short maturation time and life-cycle, this vertebrate model allows the study of endpoints such as maturation time and fecundity within four months. Transgenerational full life-cycle exposure trials can be performed in as little as 8 months. Since this species produces eggs that are drought-resistant and remain viable for years, the on-site culture of the species is not needed but individuals can be recruited when required. The protocols are designed to measure life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

Protocol for Acute and Chronic Ecotoxicity Testing of the Turquoise Killifish Nothobranchius furzeri

In this work, we describe an acute, chronic and multigenerational bioassay to study the effects of single and combined stressors on the Turquoise killifish Nothobranchius furzeri. This protocol is designed to study life-history traits (mortality, growth, fecundity, weight) and critical thermal maximum.

The killifish Nothobranchius furzeri is an emerging model organism in the field of ecotoxicology and its applicability in acute and chronic ecotoxicity ...

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult forms has revealed the genetic basis of some of these differences. Less is known about how the populations differ at post-larval stages (at the onset of feeding). Such studies may provide insight into how cavefish survive through adulthood in their natural environment. Methods for comparing post-larval development in the laboratory require standardized aquaculture and feeding regimes. Here we describe how to raise fish on a diet of nutrient-rich rotifers in non-recirculating water for up to two-weeks post fertilization. We demonstrate how to collect post-larval fish from this nursery system and perform whole-mount immunostaining. Immunostaining is an attractive alternative to transgene expression analysis for investigating development and gene function in A. mexicanus. The nursery method can also be used as a standard protocol for establishing density-matched populations for growth into adults.

Raising the Mexican Tetra Astyanax mexicanus for Analysis of Post-larval Phenotypes and Whole-mount Immunohistochemistry

In this protocol, we demonstrate how to breed Astyanax mexicanus adults, raise the larvae, and perform whole-mount immunohistochemistry on post-larval fish to compare the phenotypes of surface and cave morphotypes.

River and cave-adapted populations of Astyanax mexicanus show differences in morphology, physiology, and behavior. Research focused on comparing adult ...

Genetics

Protocols for Oral Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

In this video, we demonstrate oral infection techniques of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be ...

Biology

Protocols for Microapplicator-assisted Infection of Lepidopteran Larvae with Baculovirus

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. . This video shows how lepidopteran larvae can be infected with microapplicator techniques in the gut with baculovirus polyhedra and in the hemolymph with budded virus. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents.  Formulation and application of baculoviruses for pest control purposes are described elsewhere.

In this video, we demonstrate two microapplicator techniques used to infect of lepidopteran larvae with baculovirus in order to determine insecticidal efficiency.

Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. . This video shows how lepidopteran larvae can be ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...

Immunology and Infection

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

A Toxicological and Ecotoxicological Assay Based on Mussel (Mytilus galloprovincialis) Hemocytes Motility

Hemocytes are the circulating immune-competent cells in bivalve mollusks and play a key role in several important functions of cell-mediated innate immunity. During the early stages of the immune response, hemocytes actively migrate to the site of infection. This inherent motility is a fundamental characteristic of these cells. It represents a key cellular function that integrates multiple processes, such as cell adhesion, cell signaling, cytoskeletal dynamics, and changes in cell volume. Therefore, alterations in cell motility following exposure to drugs or pollutants can serve as a useful toxicological endpoint. Despite the fundamental role of cell motility in cellular physiology, it has been poorly investigated from a toxicological perspective. This work proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on evaluating the hemocyte motility of Mytilus galloprovincialis. We developed a cell motility assay on hemocytes adhering to the bottom of a 96-well polystyrene microplate. Following exposure to increasing concentrations of drugs, cell trajectories, and velocities were quantified by cell tracking under time-lapse microscopy, allowing us to measure the effects on hemocyte motility. Due to the ease of hemocyte collection from the animals in a relatively non-invasive manner, the proposed method offers an alternative test for screening the effects and mechanisms of action of pollutants and drugs. It aligns with the 3Rs (Replacement, Reduction, and Refinement) criteria, addressing ethical concerns and contributing to the reduction of vertebrate in vivo animal testing.

The article proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on the motility of Mytilus galloprovincialis hemocytes. The method aims to contribute to the development of more ethical and sensitive toxicological and ecotoxicological exposure tests.

Hemocytes are the circulating immune-competent cells in bivalve mollusks and play a key role in several important functions of cell-mediated innate ...

Lethality Bioassay Using Artemia salina L.

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