Qualitative Detection of Lipid Peroxidation in Mosquito Larvae Using Schiff’s Reaction: A Simple Histochemical Tool for In Situ Assessment of Oxidative Damage

Materials and reagents

Biological materials

1. Mosquito larvae at the stage of interest; in our study, second-stage larvae (L2) (Figure 1)

Reagents

1. Distilled water

2. Schiff’s reagent (Biopack, catalog number: 2000938600- 100 mL)

3. Hydrochloric acid (HCl) (Biopack, catalog number: 9632.08)

4. Hydrogen peroxide (H₂O₂) (Sigma-Aldrich, catalog number: H1009)

5. Sodium metabisulfite (Na₂S₂O₅) (Sigma-Aldrich, catalog number: S9000)

6. Sodium hydroxide (NaOH) (Anedra, catalog number: 71690)

7. Potassium hydroxide (KOH) (Mallickrodt, catalog number: 6984)

8. Disodium phosphate (Na₂HPO₄) (Fluka, catalog number: 71645)

9. Monopotassium phosphate (KH₂PO₄) (Timper, catalog number: 6504)

Laboratory supplies

1. Standard 24-well flat-bottom microplate made of optically clear polystyrene

2. Microscope slides (Sail Brand, catalog number: 7102)

3. Parafilm^TM M plastic wrap

4. Aluminum foil

Solutions

1. Phosphate-buffered saline (PBS) (see Recipes)

2. Permeabilization solution (see Recipes)

3. Schiff’s reagent staining solution [6] (see Recipes)

4. Sulfite solution 0.5% (w/v) (see Recipes)

5. BG11 medium (see Recipes)

Recipes

1. PBS

2. Permeabilization solution

Add 0.1% Triton X-100 to PBS (Recipe 1).

This solution increases cell membrane permeability, allowing reagents to penetrate into cells or tissues.

3. Schiff’s reagent staining solution [6]

Prepare a 10% (v/v) Schiff’s reagent solution in distilled water and mix well. Prepare fresh.

4. Sulfite solution 0.5% (w/v)

Na₂S₂O₅ in 0.05 M HCl.

5. BG11 medium

* Trace metal mix A5 + Co contains (in g/L) H₃BO₃ 2.86, MnCl₂·4H₂O 1.81, ZnSO₄·7H₂O 0.222, Na₂MoO₄·2H₂O 0.390, CuSO₄·5H₂O 0.079, and Co(NO₃)₂·6H₂O 0.0494

Equipment

1. Pipettes (Gilson PIPETMAN, Finnpipette^TM, 2-20, 20-200 and 100-1,000 μL)

2. pH meter (pH Tutor, Eutech Instruments)

3. Weighing balance (Sartorius, 0.1 mg to 220 g)

4. Vacuum pump and vacuum desiccator (SIGMA, model: Z119008)

5. Stereomicroscope (Nikon, model: SMZ800 stereoscope)

6. Digital camera (Olympus, model: DP72 with CellSens Entry imaging software)

7. Orbital shaker (Haimen Kylin-Bell Lab Instruments Co., Ltd., China, model: TS-8)

8. Brush (Artística Dibu, Marta 100 series, size 5/0, Argentina)

9. Insulin syringe needle (Micro-Fine, catalog number: 328418)

Procedure

A. Larval treatments

1. Select larvae of a known stage (see Figure 1) and pre-treat them according to experimental conditions (e.g., exposure to treatments, like ferroptotic Synechocystis) [5]. Include a positive control (larvae treated with 100 mM H₂O₂ for 20 min) and a negative control (larvae reared in BG11 medium) [7]. BG11 medium is used as a control because it is the Synechocystis growing medium (see Figure 2).

Figure 2. Histochemical detection of lipid peroxidation in Culex quinquefasciatus larvae using Schiff’s reagent under different experimental conditions. Representative images of larvae showing magenta staining indicative of lipid peroxidation. (A) Positive control: larvae treated with 100 mM H₂O₂ for 20 min. (B) Negative control: larvae reared in water. (C) Larvae reared on BG11 medium. (D) Larvae exposed to live Synechocystis. (E) Larvae exposed to ferroptotic Synechocystis (heat-treated at 50 °C). Larvae were fed with commercial fish food (Shulet Carassius). Each staining was performed in triplicate, and the entire procedure was independently repeated three times. Scale bars, 1 mm.

2. Prepare PBS (Recipe 1) and permeabilization solution (Recipe 2).

3. Transfer 10 larvae per well in a 24-well plate for each treatment and its corresponding controls.

Note: The experimental layout in the 24-well plate was organized as follows: Row A contained the negative control larvae; wells A1–A3 included larvae processed with Schiff’s reagent (stained negative controls), whereas wells A4–A6 contained larvae processed without Schiff’s reagent (unstained negative controls). Row B contained the positive control larvae exposed to 100 mM H₂O₂; wells B1–B3 were processed with Schiff’s reagent (stained positive controls), and wells B4–B6 were processed without Schiff’s reagent (unstained positive controls). Row C contained larvae exposed to live Synechocystis, and row D contained larvae exposed to ferroptotic Synechocystis; in both rows, wells 1–3 were processed with Schiff’s reagent (stained treatments), and wells 4–6 were processed without Schiff’s reagent (unstained treatments).

1. Larval preparation: Wash all larvae in 2 mL of PBS.

Caution: Larvae should be handled gently to prevent tissue damage. All liquid removal steps are performed using a pipette.

2. Permeabilization: Incubate larvae in 2 mL of permeabilization solution using a pipette for 15–30 min at room temperature with gentle agitation. Rinse three times in 2 mL of PBS for 5 min each to remove detergent.

Notes:

1. In this particular experiment, larvae are treated while alive and die later during the staining process. For larvae treated with ferroptotic Synechocystis and in the death process, staining is performed at 24 h post-treatment to assess lipid oxidation prior to death, which typically occurs between 48 and 72 h.

2. Gentle agitation is achieved with an orbital shaker. This should be performed occasionally, approximately every 2–3 min, throughout the 15–30 min incubation period.

3. Staining

a. Prepare fresh 10% (v/v) Schiff’s reagent solution. Protect from light.

Caution: Keep the reagent and the plate covered with aluminum foil during incubation in the dye to prevent light interference. It is important to emphasize that the primary components and reaction products of Schiff’s reagent are considered potentially toxic and carcinogenic. For this reason, Schiff’s reagent must be handled with appropriate personal protective equipment, including gloves, and in accordance with standard laboratory safety guidelines.

b. Add 2 mL of reagent, ensuring full immersion of larvae.

c. After immersion of larvae, cover the entire 24-well plate with Parafilm M, perforated with approximately 15 small needle holes per well to allow controlled air exchange while maintaining vacuum conditions. Place the covered plate inside a vacuum desiccator and apply vacuum infiltration for 2 h in darkness. To prevent light-induced degradation of the dye, cover the desiccator with aluminum foil for the entire duration of the vacuum incubation. The vacuum strength applied corresponds to standard laboratory desiccator conditions (approximately -70 to -80 kPa; 200–250 mmHg), which ensures effective reagent infiltration without damaging the larvae.

Caution: During vacuum infiltration, some larvae may adhere to the parafilm. If this occurs, gently detach them using a fine brush under PBS to avoid tissue damage.

4. Rinsing and mounting

a. Gently replace the reagent with 2 mL of sulfite solution (Recipe 4) using a pipette and incubate for 15 min. Wash the larvae by performing three consecutive rinses with distilled water. For each rinse, add ~2 mL of distilled water (or enough to fully immerse the larvae), gently agitate, and carefully aspirate the liquid with a pipette to avoid damaging the specimens.

b. Carefully mount the larvae on glass slides using a fine needle and a soft brush. Distilled water is used as the mounting medium. Coverslips are not required and are intentionally avoided to prevent compression of the larvae and to preserve their structural integrity. Acquire representative images under brightfield illumination using a light background (preferably white) to maximize contrast and allow reliable comparison across samples. Keep illumination conditions consistent among samples. Capture images using a stereomicroscope equipped with a 4× objective lens (see Figure 2).

Note: It is recommended to photograph the stained samples immediately after staining to ensure optimal image quality. If immediate imaging is not possible, samples may be stored at 4 °C and photographed within 24 h. Beyond this period, staining intensity may fade, and tissue integrity may deteriorate, which can compromise image quality.

Data analysis

For potential larvicidal evaluation for mosquito larval control, Schiff’s staining was applied to larvae exposed to the cyanobacterium Synechocystis sp. PCC 6803 subjected to ferroptosis (by thermal treatment). The staining revealed a widespread accumulation of oxidized lipids in larvae filtering ferroptotic Synechocystis, whereas those exposed to live cyanobacterial cells or BG11 medium exhibited coloration primarily restricted to the gastric caecum, with markedly lower intensity. These findings indicate that ferroptotic cyanobacteria induce elevated lipid peroxidation, correlating with increased larval mortality [5].

As this is a qualitative assay, results were documented by acquiring representative photographs under standardized imaging conditions, and interpretation was based on the presence, localization, and relative intensity of the staining signal, as described in Cuniolo et al. [5].

Validation of protocol

This protocol (or parts of it) has been used and validated in the following research article:

1. Cuniolo et al. [5]. Ferroptotic cyanobacteria as biocontrol agent of the southern house mosquito. Culex quinquefasciatus. J Invert Pathol.

General notes and troubleshooting

General notes

1. Light protection during incubation: Keep the plate covered with aluminum foil during incubation in the dye to prevent light interference.

2. Vacuum handling: When applying vacuum, wrap the uncovered plate with plastic wrap. Create small holes in the wrap to allow air entry. Larvae may adhere to the plastic wrap.

3. Light control: Cover the vacuum desiccator with aluminum foil to maintain darkness.

4. Photography tip: Use a light-colored background for clearer images. Ensure consistent lighting conditions during observation to achieve reliable results.

Acknowledgments

This study was partially supported by Grants of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), PUE 2017-0101, Agencia Nacional de Promoción de la Investigación, el Desarrollo Tecnológico y la Innovación (ANPCyT), PICT-2020-SERIE A-02641 and PICT-2021-GRF-TII-00106, and Universidad Nacional de Mar del Plata (800 202405 00109 MP).

Antonella Cuniolo: Writing—original draft, Writing—review & editing, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization.

Corina M. Berón: Writing—original draft, Writing—review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition.

María Victoria Martin: Writing—original draft, Writing—review & editing, Visualization, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Competing interests

The authors declare no conflicts of interest.

Ethical considerations

The protocol for Cx. quinquefasciatus laboratory rearing was reviewed and approved by the CONICET Safe Procedures Policy (NPS: NBTC017-version 1.3). All experimental procedures should comply with institutional biosafety and environmental handling standards.

References

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