A Simple Method for Estimating the Spatiotemporal Distribution of Phenoloxidase Proteins in Insect Tissues

Materials and reagents

Biological materials

1. Harmonia axyridis laboratory stock established from individuals collected from the field in Aichi Prefecture, Japan, in spring, and maintained under laboratory conditions as described in Nakamura et al. [14]

2. Propylea japonica larvae purchased from Sumika Techno-Service and reared under the same conditions as H. axyridis, following the method described by Nakamura et al. [14]

3. Eurema hecabe pupae provided by Dr. Tatsuro Konagaya, who reared the insects to the pupal stage according to the method described by Konagaya and Maruyama [15]

4. Pea aphid Acyrthosiphon pisum parthenogenetic colony maintained on broad bean plants used as prey for H. axyridis, as described in Nakamura et al. [14]

Reagents

1. Potassium dihydrogen phosphate (KH₂PO₄) (Wako, catalog number: 169-04245)

2. Dipotassium hydrogen phosphate (K₂HPO₄) (Wako, catalog number: 164-04295)

3. 3,4-dihydroxyphenethylamine hydrochloride (dopamine) (Wako, catalog number: 040-15433)

4. Triton X-100 (Sigma-Aldrich, catalog number: X100)

5. 2-Propanol (isopropanol) (Wako, catalog number: 168-21675)

6. Sodium chloride (NaCl) (Wako, catalog number: 195-01663)

Safety note: When handling isopropanol, dopamine, and Triton X-100, appropriate personal protective equipment (e.g., gloves and safety goggles) should be worn. Isopropanol should be used in a well-ventilated area, avoiding inhalation of vapors, and should be kept away from sources of ignition.

Solutions

1. 0.5 M KH₂PO₄/K₂HPO₄ (see Recipes)

2. K-PO₄ buffer (see Recipes)

3. K-PO₄ T buffer (see Recipes)

4. Dopamine solution (see Recipes)

5. Staining solution (see Recipes)

Recipes

1. 0.5 M KH₂PO₄/K₂HPO₄

Add 0.5 M K₂HPO₄ dropwise to 100 mL of 0.5 M KH₂PO₄ until the pH reaches 6.3 (approximately 88 mL of 0.5 M K₂HPO₄). Sterilize the buffer by autoclaving at 121 °C and 1.5 atm for 20 min. The solution is stable at room temperature, and fresh preparation prior to the assay is unnecessary.

2. K-PO₄ buffer

The solution is stable at room temperature, and fresh preparation prior to the assay is unnecessary.

3. K-PO₄ T buffer

The solution is stable at room temperature, and fresh preparation prior to the assay is unnecessary.

4. Dopamine solution

Because dopamine is required in a very small amount, weigh dopamine within the range of 4–15 mg. Calculate the volume of K-PO₄ buffer to be added according to the measured mass of dopamine. The solution should be freshly prepared prior to each assay.

5. Staining solution

The solution should be freshly prepared prior to each assay.

Laboratory supplies

1. 200 and 1,000 μL pipette tips (Watson, catalog numbers: 123R-757CS and 110-705C)

2. Slide glass (Matsunami, catalog number: S1111)

3. Cover glass (Matsunami, catalog number: C018181)

4. 1.5 mL microtube flat-bottom slight-stiff-touch cap (Watson, catalog number: 131-415C)

5. 24-well and 48-well microplates (Corning, catalog numbers: 258201 and 258301)

6. Paper wipe (Daio Paper, catalog number: DSI20703128)

7. Artificial diet for ladybird beetles [16]

Equipment

1. Dissection/stereomicroscope (Leica or equivalent)

2. Pipetman [GILSON, catalog numbers: F123602 (P1000) and F123601 (P200)]

3. Analytical balance (Shimadzu, model: AUW120D)

4. pH meter (METTLER TOLEDO, model: S220)

5. Vortex mixer (LMS, model: VTX-3000L)

6. E-Centrifuge (Wealtec, catalog number: 1090003)

7. Petri dish (MonotaRO, catalog number: 34680572)

8. Forceps (Fine Science Tools, catalog number: 11252-00)

9. Time-lapse camera (SANWA SUPPLY, model: 400-CAM109)

10. Stir bar (AS ONE, catalog number: 9-870-07)

11. Magnet stirrer (AS ONE, model: CT -1AT)

Procedure

Here, we describe the protocol using the pupal forewing primordia of H. axyridis as an example; we validated this protocol with the other two species, P. japonica and E. hecabe.

A. Determination of pupal duration

Note: Lac2, a PO protein involved in melanin synthesis and cuticle sclerotization, is expressed during the late pupal stage in many insect species [6,17–19]. Therefore, it is important to determine the pupal duration in advance to ensure reproducible staining patterns. Here, we describe a method to determine pupal duration, using H. axyridis as an example.

1. Rear H. axyridis at 25 °C under 16L:8D and feed with aphids and an artificial diet (Niimi et al. [16]), as described previously [14].

2. Position a time-lapse camera approximately 40 cm above the rearing container. Set the camera to a resolution of 1,280 × 960 pixels, with the LED flash turned on (allowing image acquisition under dark conditions), and programmed to automatically capture images at 3-min intervals to obtain time-lapse images (Figure 1A, B).

Note: To facilitate accurate tracking of pupal position in the images, the angle of light is adjusted to minimize reflected light, and obstructions within the field of view are removed as much as possible (Figure 1A, B).

3. Define the time point of pupation as 0 h after pupation (0 h AP) and, in H. axyridis, collect pupae at approximately 80% of the total pupal duration.

Note: In a previous study applying this protocol to the wing primordia of H. axyridis, weak staining across the entire wing primordium was observed at 70 h after pupation (approximately 65% of the total pupal duration), whereas reproducible staining patterns corresponding to adult melanin-forming regions were obtained at 80 h after pupation (approximately 75% of the total pupal duration) and later stages [1]. Thus, in this section, the protocol is demonstrated using pupae at approximately 80% of the total pupal duration.

Figure 1. Experimental setup and procedure. (A) Time-lapse imaging using a time-lapse camera. (B) Representative images captured by the time-lapse camera (red circles indicate the position of H. axyridis pupa). The bottom-left image shows the pupa. (C) Instruments used for dissection and staining. (D) Dissection procedure. (E) Well plate containing wing primordia during phenoloxidase (PO) activity staining.

B. PO activity staining

Here, we describe the dissection and staining protocol for pupal forewing primordia of H. axyridis.

1. Place a pupa on a paper wipe and, under a stereomicroscope, grasp the proximal region of the pupal forewing primordia with forceps. Dissect the forewing primordia from the pupa together with the pupal cuticle (Video 1, Figure 1D).

Notes:

1. Using a piece of paper as a support allows us to easily position the pupa within the microscope field of view without direct manual handling.

2. Depending on the species and tissue, use forceps or scissors as appropriate.

2. Hold the proximal region of the dissected forewing primordia with forceps and transfer it into the K-PO₄ buffer in a Petri dish. Carefully isolate the wing primordia from the pupal cuticle (Video 1).

3. Transfer the isolated wing primordia into a 48-well plate containing 500 μL of staining solution per well or a 24-well plate containing 1 mL of staining solution per well (Figure 1E).

Notes:

1. The volume of staining solution and well size are adjusted according to tissue size. For H. axyridis and P. japonica, we used a 48-well plate with 500 μL of staining solution. For E. hecabe, which has larger wing primordia, we used a 24-well plate with 1 mL of staining solution.

2. Staining can also be performed in microcentrifuge tubes; however, visual monitoring of staining progression is difficult.

3. ProPOs are artificially activated by isopropanol in the staining solution [10]; therefore, the observed activity may not fully reflect endogenous PO activity under physiological conditions.

4. Put the lid on the plate and incubate at room temperature (25–27 °C), checking the staining progression every 20 min (Figure 1E).

Notes:

1. Because the timing of PO activity staining may vary depending on pupal stage and species, staining should be monitored at 20-min intervals to determine the optimal time point at which to terminate the staining reaction. In H. axyridis, staining beyond 180 min leads to nonspecific darkening of regions unrelated to future adult melanization (Figure 2), which could be caused by the presence of PO proteins involved in cuticle sclerotization outside the adult melanin-forming regions, as well as by spontaneous oxidation of dopamine.

2. In H. axyridis, staining post-80 h pupal stages for 120 min consistently produced staining patterns corresponding to adult melanized regions, and the patterns became clearer as pupal development progressed [1].

Figure 2. Time-lapse images of the phenoloxidase (PO) activity staining process in pupal forewing primordia of H. axyridis. Images were captured at 20-min intervals up to 120 min, at 1-h intervals thereafter, and again after overnight incubation.

5. Once staining is observed exclusively in regions corresponding to future melanized regions in adults (Figure 2), terminate the reaction by washing the tissue three times with K-PO₄ T buffer.

Notes:

1. Although the reaction is terminated by washing, the tissue may continue to darken gradually over time; images should be acquired immediately after washing.

2. Extraction of RNAs should be performed after washing.

C. Observation of staining results

To validate this protocol, we performed staining in three insect species (Figure 3). As described in the previous section, we performed staining at approximately 80% of the total pupal duration for all three examined species. Specifically, pupae were sampled at 4 days after pupation for H. axyridis (total pupal duration: 4.5 days [20]), 3 days after pupation for P. japonica (total pupal duration: 3.76 ± 0.04 days [21]), and 5 days after pupation for E. hecabe (total pupal duration: 6.5 ± 0.4 days [22]). We obtained PO activity staining patterns corresponding to adult melanin-forming regions in all three species by incubating wing primordia for 120 min (Figure 3G–I). Based on these results, we propose that performing staining at approximately 80% of the total pupal duration serves as a valuable baseline when applying this protocol to other insect species, although incubation times may need optimization.

Conducting stage-specific analyses in other species could allow for the precise identification of the timing of color pattern determination, as previously demonstrated in H. axyridis [1], in which we obtained a transition in the staining pattern from weak staining across the entire wing primordium to a pattern corresponding to adult melanin-forming regions between 70 and 80 h after pupation [1]. Applying this approach to define critical developmental time points in other insects would facilitate further analyses, such as RNA-seq, aiming to identify genes involved in color pattern formation.

The staining patterns obtained at this stage are considered to reflect the spatial distribution of proPOs, rather than endogenous active POs. This is supported by the observation that staining without isopropanol-mediated artificial activation yielded no patterns corresponding to adult melanin-forming regions (data not shown). Consequently, while this method effectively estimates proPO distribution, it does not provide insight into the precise timing of in vivo proPO conversion to active POs.

Figure 3. Phenoloxidase (PO) activity staining in pupal wing primordia of different insect species. (A–C) Adult right forewings. (D–F) Right pupal forewing primordia before staining. (G–I) Right pupal forewing primordia after staining for 120 min. Scale bars: 1 mm.

Validation of protocol

This protocol has been used and validated in the following research article:

• Ando et al. [1]. Repeated inversions within a pannier intron drive diversification of intraspecific colour patterns of ladybird beetles. Nat Commun. (Figure 2a and Supplementary Figures 1 and 9)

General notes and troubleshooting

General notes

We observed that the pupal stage, suitable for estimating the spatial distribution of PO proteins, may be associated with external morphological features. In all three species examined, wing primordia were easily dissected at approximately 80% of the total pupal duration, and staining for 120 min yielded clear patterns corresponding to adult melanin-forming regions. Based on external morphological features, H. axyridis pupae at this stage (later than 80% development) exhibit both black compound eyes and black hindwing primordia, whereas earlier stages (>60%) show dark brown eyes with no wing pigmentation (Figure 4). Similar pigmentation changes in the compound eyes and hindwings were observed in P. japonica at comparable stages (data not shown). In E. hecabe, while eye pigmentation was distinct, yellow pigmentation of the wings became apparent at this stage (data not shown). These external markers may thus serve as practical indicators for identifying the optimal timing for staining.

Figure 4. Developmental stages of H. axyridis pupae. Immediately after pupation, the pupa is entirely white; after 1 h, black melanin pigmentation appears on the dorsal side. The compound eye primordia progressively darken during development and become fully black by day 4. In addition, melanization of the hindwing primordia located beneath the forewing primordia becomes apparent by day 4. The numbers above the images indicate the percentage (%) of the total pupal duration (4.5 days), and the numbers below indicate the time after pupation for each image.

Troubleshooting

We caution that staining patterns corresponding to adult melanin-forming regions cannot be reliably obtained at earlier developmental stages. In our previous paper, in H. axyridis wing primordia at 70 h after pupation (~65% of the total pupal duration), weak staining was observed across the entire wing primordium rather than patterns corresponding to adult melanin-forming regions [1]. Therefore, we recommend performing staining at later developmental stages.

Acknowledgments

T.N. conceived and developed the protocol. Y.M., S.M., and T.N. supervised the study. Y.N. performed experiments to validate the protocol. Y.N. wrote the manuscript with feedback from all authors. All authors reviewed and approved the final version of the manuscript. We are grateful to Tatsuro Konagaya for providing E. hecabe pupae and photographs of adults, and to Toshiya Ando for helpful advice. This protocol was adapted from True et al. [11] and the master’s thesis of Kumiko Goto (2009). This work was supported by JST SPRING, Japan Grant Number JPMJSP2104 (to Y.N.), and JSPS KAKENHI grant numbers 19H01004 and 23H02227 (to T.N.). We thank the Model Plant Research Facility/NIBB BioResource Center and the Emerging Model Organisms Facility of NIBB Trans-Scale Biology Center for technical assistance. This protocol was used in [1].

Competing interests

No conflict of interest is declared regarding this article.

Ethical considerations

No ethical requirements are needed regarding this article.

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