**1. Introduction**

Tuberculosis (TB) is a disease, which requires more than just biomedical treatment. WHO-recommended standard TB treatment requires a minimum duration of 6 months. The patients have to regularly take treatment without interruption to get a cure. However, discontinuation of treatment because of loss to follow-up (LTFU) is a significant problem, especially among patients suffering from multidrug-resistant tuberculosis (MDR-TB), requiring urgent attention. The proportions of LTFU and its associated factors differ among various countries. A clear understanding of these underlying causes is essential for the success and effectiveness of the National Tuberculosis Program (NTP) of every nation. Hence, appropriate measures targeting LTFU are needed to achieve the goals of the NTP.


**1. Introduction**

The increase in joining dissimilar metals has been prompted by the increasing demand for light weights and fuel efficiency in modern industries in recent years [1]. The emerging trend towards lightweight, high performance and emissions reduction is leading to the increasing use of multi-material hybrid structures in electric vehicles [2]. The hybrid structure of aluminum and steel are superior to conventional materials because of the high mechanical properties of steel and the excellent corrosion resistance of aluminum [3]. Naturally, the combination of aluminum alloy and steel shows huge research value and future potential, and has become the subject of many investigators [4].

However, it remains an unconquerable problem to get stable brazed joints between aluminum/metals dissimilar to steel because of the formation of brittle and hard intermetallic compounds, like FexAly [5]. The complexity and randomness of the brazing process makes the design and development of brazing materials more complicated and time-consuming than ordinary materials [6]. Therefore, it is absolutely imperative to control the formation of Fe-Al intermetallic compounds. EI-Sayed and Naka [7] found that the maximum bond shear strength of 127 MPa was obtained for an Al-steel joint brazed at 663 K with a 3 s ultrasound application time using a Zn-14Al alloy. It was reported that the shear strength of brazed joints reached the peak at 131 MPa when the Zn–15Al filler metal was added in the joining of lap joints of 6061 aluminum alloy to the 304 stainless steel via a flame brazing process, with the Zn-xAl filler metals matching the CsF-0.5 wt.% RbF-AlF3 flux [8]. The interfacial layer in the weld made with the Zn-15Al filler metal was comprised of (FeAl3)Znx and (Fe2Al5)Znx [9].

Nanomaterials have been widely applied due to their unique properties. Joining technology at the nanometer scale has gradually developed with the popularity of nanomaterials, which have broad application prospects in the fields of electronics, aero-space, biology and health care [10]. Previous studies have indicated that [11] trace amounts of Ga2O3 addition in the CsF-0.5 wt.% RbF-AlF3 flux could obviously strengthen the Zn-2Al filler metal in its wetting and spreading on the surface of 5052 aluminum alloy and Q235 low-carbon steel. One previous study discussed how the addition of GaF3 and Ga2O3 nanoparticles influenced the wettability and spreadability of the CsF-AlF3 flux under the same conditions [12].

The influence that adding heavy metal fluoride, ZnF2, SnF2, CdF2, PbF2 and KBF4 into the KF-AlF3 flux has on the spreadability of brazing aluminium was investigated, and adding ZnF2 could greatly improve the brazing area [13]. Therefore, the fourth component, ZnF2, has been considered for doping into CsF-0.5 wt.% RbF-AlF3 flux in order to reduce the price in this paper. ZnCl2 and SnCl2 were added into the CsF-AlF3 flux for connecting aluminium alloys [14]. The joints were bonded soundly when the mass fractions of ZnCl2 and SnCl2 are about 4%.

The effect of KBF4 addition on the microstructure of the Mg-6Zn-1Si alloy has been investigated [15], and the morphology of the Mg2Si phase changed with the addition of 1.5 wt.% KBF4. To compare the activity of BF4− and its unified positive ions in reducing the variables, Zn(BF4)2 has been chosen.

In this paper, brazing AA6061 to Q235 steel using flame brazing has been performed with improved CsF-0.5 wt.% RbF-AlF3 fluxes doped with a GaF3, ZnF2, Zn(BF4)2 and Ga2O3 nanoparticles-matched Zn-15Al filler metal, and the spreadability of the filler metal and the mechanical properties of the brazed joints were investigated at the same time. XDR analysis was carried out and the reaction mechanism was analyzed. The results could be useful for brazing AA6061 to Q235 steel while choosing a suitable flux.

## **2. Materials and Methods**

The 6061 aluminum alloy and Q235 steel were used in this work as base metals. The compositions of the base metals were listed in Tables 1 and 2. Zn–15Al alloys were chosen as the filler metal. A CsF-0.5 wt.% RbF-AlF3 flux was prepared by using commercial CsF-AlF3 flux and an RbF of AR purity (Zhejiang Xinrui Welding Materials Co., Ltd., Shengzhou, China). CsF-RbF-AlF3-GaF3 fluxes with different compositions of AR purity GaF3 were confected and the range of GaF3 was 0.0001–0.125 wt.%. Same amounts of ZnF2, and Zn(BF4)2 of AR purity were doped in the same way to obtain CsF-RbF-AlF3-ZnF2 fluxes and CsF-RbF-AlF3-Zn(BF4)2 fluxes. Nano Ga2O3 powder in the same range was doped into the CsF-RbF-AlF3 flux to get the corresponding CsF-RbF-AlF3-Ga2O3 fluxes. After mixing into a liquid, the fluxes were then dried to powder with the oven. The chemical compositions of these four nanoparticles doped into the fluxes has been listed in Table 3.

**Table 1.** Chemical composition of 6061 aluminum alloy (wt.%).

**Table 3.** Chemical composition of nanoparticles doped into the fluxes (wt.%).

Zn–15Al alloys were extruded into a 2 mm diameter wire as an advanced preparation. SiC paper was used for mechanically polishing the specimens and filler metals. Before brazing, these materials were all degreased with acetone and cleaned by ethanol.

In order to prepare for the spreading test, the base metals were processed into plates of 40 mm × 40 mm × 3 mm. The spreading test was performed according to China's National Standard GB 11364-2008. The weights of the solder and flux used in the test should be 100 mg and 15 mg, respectively. The filler metals were placed in the center of the base metals covered with the fluxes prepared previously, as in Figure 1, and then put into the electrical resistance furnace (Zhejiang Xinrui Welding Materials Co., Ltd., Shengzhou, China). The heating temperature was uniformly set at 530 ◦C. The holding time of each test was set as 60 s. After spreading, the test boards were cleaned using ultrasonic wave waits and the spreading areas were calculated by the software Image-ro Plus (Image-Pro Plus Version 6.0). To be specific, we photographed the spreading boards with the graduated ruler and imported the image into Image-Pro Plus. By determining the actual scale in the picture and using chromatic aberration to circle the spreading outline, the spreading areas could be calculated. For the credibility of the results, the above tests of each group were repeated 5 times and the spreading areas of each group were averaged. At last, the residues of the fluxes were collected and the components in the residues were analyzed with a Brucker D8 XRD analyzer (Ningbo Institute of Materials Technology & Engineering, Ningbo, China).

**Figure 1.** Schematic diagram of the spreading test (mm).

For the shear performance tests, the supplied base metals for the brazed joint were processed into plates with the size of 60 mm × 25 mm × 3 mm. Figure 2 shows the schematic illustration of the brazed joint. The shear performance tests of the AA6061/Q235 brazed joints were carried out in strict accordance with China's National Standard GB 11363-2008. The equipment was an SAMS-CMT5105 universal tensile testing machine (Nanjing University of Aeronautics and Astronautics, Nanjing, China), and the loading rate in the tensile process was 3 mm/min. The test results for each group of samples were all averaged from 5 samples. The brazing temperature was detected and controlled at around 530 ◦C.

**Figure 2.** Schematic illustration of the brazed joint (mm).

#### **3. Results and Discussion**

#### *3.1. The Spreadability and Wettability of Zn-15Al Filler Metal*

The influence of GaF3, ZnF2, Zn(BF4)2 and Ga2O3 particles, doped into CsF-0.5 wt.% RbF-AlF3 fluxes, on the spreadability of the Zn-15Al filler metal was studied via spreading tests performed on the surface of the 6061 aluminum alloy and the Q235 steel. The results were as follows.

The relationships between the concentrations of GaF3, ZnF2, Zn(BF4)2 and Ga2O3 and the spreading areas on the 6061 aluminum alloy are shown in Figure 3a. While the content of GaF3 was 0.01 wt.%, the spreading area was maximized to 329 mm2, which was 65% larger than the area without any addition. With 0.0075 wt.%-doped ZnF2, the spreading area was maximized at 312 mm2, and showed a 56% increase compared with the results derived without adding ZnF2. While the content of Zn(BF4)2 was 0.0075 wt.%, the spreading area was maximized at 304 mm2, and was 52% larger than the area without any addition. With 0.009 wt.%-doped Ga2O3, the spreading area was maximized at 321 mm2 and showed a 61% increase compared with the results derived without adding Ga2O3.

**Figure 3.** Spreading areas of Zn-15Al filler metal (**a**) 6061 aluminum alloy; (**b**) Q235 steel.

Figure 3b showed that the spreadability of the Zn-15Al filler metal over Q235 steel was clearly improved with GaF3 doped into the CsF-RbF-AlF3 flux, compared to others. While the content of GaF3 was 0.0075 wt.%, the spreading area was maximized at 189 mm2. With 0.01 wt.%-doped ZnF2, the spreading area was maximized at 155 mm2. While the content of Zn(BF4)2 was 0.01 wt.%, the spreading area was maximized at 149 mm2. With 0.01 wt.%-doped Ga2O3, the spreading area was maximized at 161 mm2. With the addition of GaF3, the spreading areas over Q235 steel underwent a 105% increase, compared with the results derived without adding GaF3 (92 mm2), and this substance showed clear advantages over ZnF2, Zn(BF4)2 and Ga2O3.

The pictures of the best spreadings of Zn-15Al alloys on AA6061 and Q235, with CsF-RbF-AlF3 flux doped with di fferent additions, are shown in Figure 4. Taking the test results from two kinds of base metal together, it could be concluded that the suitable ranges of GaF3, ZnF2, Zn(BF4)2 and Ga2O3 in the CsF-RbF-AlF3 flux respectively were 0.0075–0.01 wt.%, 0.0075–0.01 wt.%, 0.0075–0.01 wt.% and 0.009–0.01 wt.%, and the maximum spreading area of all the tests was obtained via doping with GaF3.

*Crystals* **2020**, *10*, 683

**Figure 4.** Spreading boards of Zn-15Al filler metal on base metals with CsF-RbF-AlF3 flux doped with different additions: (**a**–**d**) 6061 aluminum; (**e**–**h**) Q235 low-carbon steel.

#### *3.2. The Mechanical Properties of Brazed Joints*

The variation of brazed joint shear strength was shown in Figure 5. It could be seen that the addition of GaF3 has a significant impact on the brazed joint shear strength of AA6061/Q235. While the content of GaF3 was 0.075 wt.%, the shear strength reached a maximum at 126 MPa, which was 110% higher than that achieved without GaF3 addition (64.5 MPa). While the content of ZnF2 was 0.01 wt.%, the maximum shear strength was 111 MPa. While the content of Zn(BF4)2 was 0.01 wt.%, the shear strength reached the top at 105 MPa. While the content of Ga2O3 was 0.01 wt.%, the maximum shear strength was 116 MPa.

**Figure 5.** Influence of contents of GaF3, ZnF2, Zn(BF4)2 and Ga2O3 on shear strength of brazed joints.

*Crystals* **2020**, *10*, 683

It could be seen that, only considering the shear strength of the AA6061/Q235 brazed joints, the addition range should be controlled as 0.0075–0.01 wt.%, and this appropriate addition range coincides with the results of the spreading test. Among the four doped ingredients selected, GaF3 showed the best performance as regards the mechanical properties of brazed joints. The second-best performance was achieved with Ga2O3. At the same time, Zn(BF4)2 was inferior to others.

The typical fracture modes of the 6061/Q235 brazed joints were shown in Figure 6. The fracture of the joint occurred mainly at the interface layer of the brazing joint and the Q235 steel. It demonstrated that the interface layer between the filler metal and the Q235 steel was the weakest area of the whole joint, and that when there were layers of brittle compounds at the interface, the brazed joint would crack at the layers of brittle compounds first.

**Figure 6.** Typical fracture modes of 6061/Q235 brazed joints: (**a**) Macromorphology of fracture interface; (**b**) Organization of interface before shear tests; (**c**) Microtopography of fracture interface after shear tests.

#### *3.3. Interfacial E*ff*ect of Di*ff*erent Particles*

## 3.3.1. Effect of Zn(BF4)2

The XRD analysis results for the residues of the fluxes on Q235 low-carbon steel are shown in Figure 7. In inndividually evaluating the spreadability of the Zn-15Al filler metal, and the mechanical properties of the brazed joints with CsF-RbF-AlF3-Zn(BF4)2 over the 6061 aluminum and Q235 steel, it was clear that the addition of Zn(BF4)2 significantly improved the activity of the CsF-RbF-AlF3 flux, with a 52% increase in spreading area and a 92% increase in shear strength.

$$\text{Zn}(\text{BF}\_4)\_2 \to \text{ZnF}\_2 + 2\text{BF}\_3 \tag{1}$$

**Figure 7.** X-ray diffraction analysis results of the residues on Q235 low-carbon steel: (**a**) CsF-RbF-AlF3-Zn(BF4)2; (**b**) CsF-RbF-AlF3-ZnF2 flux; (**c**) CsF-RbF-AlF3- Ga2O3 flux; (**d**) CsF-RbF-AlF3-GaF3 flux.

Zn(BF4)2 could produce BF3 as shown Equation (1). BF3 could react with oxides such as FeO, Fe2O, NiO, Cr2O3 and ZnO, thus playing the role of removing the oxide film and promoting the wetting, spreading and flowing of the molten filler metal in the brazed part [16]. However, BF3 could not react with oxides such as Al2O3 and MgO on the surface of t he aluminum alloy, thus limiting the activity of the CsF-RbF-AlF3-Zn(BF4)2 flux.

3.3.2. Effect of ZnF2

It was found in the study [13] that adding ZnF2 to the flux could produce a mass transfer effect, and improve the activity of the flux. This was because the ZnF2 was reduced by the base metal, and molten Zn has great solubility in aluminum. It was also observed that adding ZnF2 could reduce the initial temperature of the flux, which was also related to the reduction of the precipitation of molten Zn.

$$\text{ZnF}\_2 \xrightarrow{\text{A}} \text{Zn} \downarrow + 2\text{F}^- \tag{2}$$

$$\rm{F}^{-} + \rm{Al}\_{2}\rm{O}\_{3} \rightarrow \rm{AlF}\_{3} + \rm{O}^{2-} \tag{3}$$

$$\rm{F}^{-} + \rm{Fe}\_{2}\rm{O}\_{3} \rightarrow \rm{FeF}\_{3} + \rm{O}^{2-} \tag{4}$$

When heated, ZnF2 would react as shown Equation (2). The F- generated by the reaction would react with the oxide on the surface of the base metals, as shown in Equations (3) and (4), respectively, thus enhancing the membrane removal effect of CsF-RbF-AlF3 flux. The Zn atoms precipitated from the reaction would react with the aluminum atoms on the surface of the aluminum alloy and the Fe atoms on the surface of steel, and the wetting and spreading would be promoted on the surface of the base metals [17].

The presence of ZnSiO3, ZnFe2O4, AlPO4 and FePO4 in the residue on the one hand indicated that the ZnF2 in the flux did participate in the reaction, and the Zn atoms from the reduced precipitation participated in the reaction and played a role in improving the activity. On the other hand, it indicated that the enrichment phenomenon of P occurred, which resulted in the formation of compounds such as AlPO4 and FePO4, which also played an important role in the removal of the oxide film from the steel surface.

## 3.3.3. Effect of Ga2O3

The "skin effect" of high-frequency currents is a well-known natural phenomenon in physics, especially in electromagnetism. However, the "skin effect" of some oxides or halides in brazing has rarely been reported. It was found that [18] adding a very small amount of Ga2O3 to the CsF-RbF-AlF3 flux could increase the brazed area by about 50~90%. Observing the surface of the spreading filler metal via its spectrum showed that much Ga2O3 was enriched at the spreading surface and at the edge of the spreading area. Further, the relative amount of Ga2O3 was higher here than in the middle part, from which it could be concluded that the chemical reaction mechanism resulted from the skin effect of the Ga2O3, which enabled the Ga2O3 to flow fast and then promote the CsF-RbF-AlF3 flux flow, so as to improve the spreadablity of the filler metal.

$$\text{MgO} + \text{Ga}\_2\text{O}\_3 \to \text{MgGa}\_2\text{O}\_4\tag{5}$$

Extremely small amounts of Ga2O3 were enriched to participate in the reaction, and played a nonnegligible role. The appearance of MgGa2O4 indicated that the reaction between the flux and the MgO, which was more stable than Al2O3, was also liable to react as in Equation (5). However, RbF did not appear as a compound phase in the residue, which indicated that the compound of Rb was diffused-distributed, and acted as catalysis and welding aid. CsF always appeared in the form of Cs11O3, which indicated that CsF did undergo chemical reactions with Al2O3 and MgO, and replaced the O atoms in Al2O3 and MgO. This result explains why the spreading area increased with the increased addition of Ga2O3, shown in Figure 3.

## 3.3.4. Effect of GaF3

From the above it was demonstrated that the CsF-RbF-AlF3 flux doped with GaF3 obtained the maximum spreading area, both on 6061 aluminum alloy and Q235 steel, and reached the highest shear strength of 126MPa. The CsF-RbF-AlF3 flux doped with GaF3 showed remarkable superiority to that doped with ZnF2, Zn(BF4)2 and Ga2O3.

The mechanism of GaF3 could be summarized as a "synergistic effect" on the oxide removal of steel. When GaF3 was doped, the flux could not only remove the oxide films on the surface of the aluminum alloy, but could also remove those on the surface of the steel at the same time, thus making it superior to brazed aluminum-steel heterogeneous materials.

$$\rm{CaF\_3} \rightarrow \rm{Ga^{3+}} + \rm{3F^-} \tag{6}$$

$$2\text{ZnO} + 2\text{GaF}\_3 \rightarrow \text{ZnGa}\_2\text{O}\_4 + 3\text{ZnF}\_2\tag{7}$$

The ionic compound GaF3 consists of cations Ga3+ and anions F−, and makes it such that Ga3+ and F− ions can be more easily dissociated from GaF3 than from Ga2O3, as in Equation (6), while been heated. This flux was competent in removing the oxides of the base metal and decreasing the interfacial tension, in virtue of the activity of Ga3+, which has "skin effect", as well as F−, which reacts as in Equations (3) and (4). In addition, as Equation (7) showed, the production of ZnF2 enhanced the activity of the flux.


**Author details**

Albert Sabban1,2

1 Kinneret Academic College, Galilee, Israel

2 Ort Braude College, Karmiel, Israel

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


*2*.*3*. *Infarct and Edema Assessment*

After 24 h of reperfusion, the rats were subjected to general halothane anesthesia and decapitated. The brain was immediately removed and placed in ice-cold saline.  The brain slices were immediately immersed in 2% 2,3,5-triphenyl tetrazolium chloride (TTC, Wako Pure Chemicals Industries, Osaka, Japan) at 37 °C for 15 min and then in 4% formaldehyde [26,27]. Infarct areas were identified using an image analysis system (Scion Image 1.62, Frederick, MD, USA) and were combined to obtain the infarct volumes per brain according to the following formula: corrected infarct volume (%) = (left hemisphere volume − (right hemisphere volume − the infarct volume)) × 100/left hemisphere volume. Edema in the ischemic hemisphere was also calculated as follows: edema (%) = (right hemisphere volume − the infarct volume)/left hemisphere volume × 100.


**4.2 Exosomes and immune cells**

*Immune Response Activation and Immunomodulation*

and Treg responses [69–71].

from degradation.

many more [78].

**4.1 Exosome formation to secretion**

depending upon their maturation stage and specific DC subset. Immature DCs (iDCs) express low levels of HLA-II but no co-stimulatory molecules. The interaction of MSCs with DCs leads to the inhibition of maturation of monocytes and CD34+ precursor cells. Moreover, the direct activation of DCs leads to the release of PGE-2, IL-6, TSG-6, MCSF, and jagged-2 mediated signaling. Tolerogenic phenotype occurs when DC secretome of pro-inflammatory cytokines (TNF-α and IL-12) shifts toward anti-inflammatory IL-10 in which further downstream induces Th2

Advances in stem cell technology have opened interesting perspectives within the realm of regenerative medicine. As reported, MSCs participate in repair and regenerative processes via different mechanisms like homing and transdifferentiation and immunomodulation, which depends on paracrine mechanisms [72, 73]. The initial studies using MSCs were based on local engrafting of MSCs and differentiating into multiple tissue types. However, with the in-depth study of different mechanisms of MSC action, it has been reported that <1% MSCs are able to survive transiently after systemic administration [74]. This suggests that paracrine mechanisms through secretion of various molecules called secretome might be the possible mechanism for MSC regenerative potential. This has attracted significant attention for the potential use of MSC secretome in tissue repair and regeneration. The secretome released by MSCs includes various biologically active growth factors and cytokines which aid in immunomodulatory properties of MSCs [75]. However, we cannot neglect the fact that the secretome released in the milieu of ECM of the cells is an easy target for denaturation due to the presence of proteases and other enzymes in the microenvironmental niche. Therefore, these growth factors and cytokines have been shown to be packed into small vesicles called exosomes

which are secreted by MSCs in the extracellular milieu of cell along with the

secretome [76]. They function through encapsulation of biological active molecules such as miRNA, proteins, and immunomodulatory molecules and protect them

Exosomes are lipid membrane-bound extracellular vesicles which possess a diameter of 30–120 nm and a density of 1.09–1.18 g/mL and are secreted by all cell types. These exosomes carry cellular components like proteins and nucleic acids and aid in cell-cell communication. The exosome was first discovered in 1984 by Johnstone in sheep reticulocytes [77]. It was initially believed that exosomes remove unwanted proteins from cells. Later on, it was demonstrated that many other cell types also secrete exosomes including immune cells, cancer cells, stem cells, and

Exosomes are endosomal in origin, formed within multivesicular endosomes (MVEs). These vesicles are being released when membranes of MVEs fuse with that of the cellular plasma membrane. These exosomes express various surface markers like CD63, CD81, and CD9. They carry surface molecules that are present on the parent cell which aids in identification of exosomes and their parent cell source as

Moreover, the exosomes secreted by stem cells carry various proteins (growth factors and cytokines) and nucleic acids (mRNA and miRNA) that can influence their mode of action [79]. The content carried by these vesicles depends on the type

**4. Paracrine interaction with in the niche (exosomes)**

**114**

well [77].

The exosomes were studied for their multifaceted application in antigen presentation, and vastly studied immune cell was dendritic cells. The clinical studies have been conducted to evaluate the dendritic cell derived exosomes for their therapeutic potential. However, compared to preclinical studies, only a few clinical trials have been conducted using exosomes. Reported studies were conducted where dendritic cell-derived exosomes were evaluated for their safety, tolerability, and efficacy in cancer patients. Exosomes carry parental cell surface marker expression. In this regard, DC derived exosomes are HLA-II positive as a result they can only be used in patient specific studies [80, 81]. In contrast, MSCs are immunologically naïve as they express only HLA class I molecules and lack HLA class II, CD40, CD80 and CD86 expression on their cell surface. Also, they are capable of immune escape and fail to induce an immune response by the transplanted host. Similarly, exosomes secreted by them are also immunologically naïve [82]. Considering all of the above properties, several recent studies have focused their research on evaluation of stem cell-derived exosomes in the area of immunomodulation with fewer reports.

In a recent study, exosomes derived from MSCs were specifically identified to mimic the effect of MSCs, and this paved the way to cell-free therapeutic approach using exosomes instead of the cell itself [83]. The first report using MSC exosomes were in cardiovascular diseases where Lai et al. [85] identified exosomes as the cardioprotective components in MSC paracrine secretion [84]. This was followed by several other studies where exosomes isolated from tissue-specific MSCs were studied for their therapeutic potential in various diseases.

Initial studies were performed on bone marrow-derived exosomes for evaluating their regenerative potential in cardiovascular diseases [85], acute kidney injury [86], bone defects, etc. [87]. By 2013, only researchers started exploring the regenerative potential of exosomes derived from adipose tissue and Wharton's jelly sources. These studies have explored the various mechanisms by which these exosomes mimic MSCs. The content of these exosomes was evaluated by using various techniques like RNA sequencing, mass spectrometry, etc., to identify different molecules and their target effect.

Conforti et al. reported the effect of MSC-derived vesicles on B-cell proliferation which was further confirmed by Di Trapani's group in 2016 [88, 89] . They observed that exosomes had higher levels of miRNAs compared to MSCs and induce inflammatory priming via increasing levels of miR-155 and miR-146. These are two miRNAs involved in the activation and inhibition of inflammatory reactions. Similar studies were reported where MSC-derived exosomes were shown to increase the ratio between regulatory and effector T cells along with the increase in cytokine such as IL-10 [90]. Similarly, Chen et al*.* [91] has also reported immunomodulatory effects of MSC-derived exosomes toward peripheral blood mononuclear cells (PBMNCs) focusing specifically on T cells. It was observed that there was significant inhibition of pro-inflammatory cytokines, IL-1β and TNF-α, but enhancement of the expression of anti-inflammatory cytokine, TGF-β1. This cytokine profile in their study mimics the immunomodulatory effect of MSCs [91]. Zhang et al. showed that these exosomes may polarize monocytes toward M2-like phenotype, which in turn induces CD4+ T-cell differentiation into regulatory T cells [92].

Blazquez et al. demonstrated AD-MSC-derived exosomes as a therapeutic agent for the treatment of inflammation-related diseases. They showed that exosomes

exerted an inhibitory effect on the differentiation of activated T cells, reduced T-cell proliferation, and IFN-γ secretion in an in vitro stimulatedT-cell model [93]. Favaro et al. has shown the effect of BM-MSC-derived exosomes on PBMNCs isolated from type I diabetic patients. These exosomes were able to inhibit the IFN-γ production and significantly increased the production of immunomodulatory mediators such as PGE-2, TGF-β, IL-10, and IL-6 [94]. The in vitro studies were complemented by the in vivo studies which confirmed the immunosuppressive effect of exosomes in mouse allogeneic skin grafting models [95]. Bai et al 2017 have subcutaneously administered exosomes isolated from human embryonic stem cellderived MSCs and showed that there was delayed occurrence of GvHD for 2days, concomitant with increasing Treg polarization. In continuation, the author has also demonstrated that exosomes released from WJ-MSCs can effectively ameliorate experimental autoimmune uveoretinitis (EAU) in rats by inhibiting the migration of inflammatory cells [95]. Moreover, there is only single case study on humans in which MSC exosomes have been tested in the treatment of resistant grade IV acute GvHD patient which experienced improvement in symptoms for 5 months. There were no side effects reported, and the decrease of pro-inflammatory cytokines such as TNF-α, IL-1β, and IFN-γ was observed. The anti-inflammatory molecules IL-10, TGF-β1, and HLA-G contained in the exosome preparations were believed to contribute to the immunosuppressive effect of MSC-Exo [96].

Although there are limited studies available using MSC-derived exosomes, future advancements into research and gain in in-depth knowledge of immunomodulatory properties of these MSC exosomes could be seen. These nano-vesicles can be developed as cell-free therapies. The use of these exosomes as cell-free therapies provides following key advantages:

Despite these advantages of MSC-derived exosomes, there has been a lack of manufacturing process that is required to generate exosomes with clinically relevant quantities. Therefore, there is an urgent need for technological advancements. Nevertheless, regulatory requirements will be necessary to establish the safety and efficacy profile of these exosome products.


**4. Conclusions**

The combination of proteomics, lipidomics, and isotopic profiling experiments allowed us to uncover a profound reprogramming of lipid metabolism in IDH1 mutant AML cells through a simultaneous increase of both FA oxidation and de novo lipogenesis. This reprogramming is—at least partly- dependent on 2-HG production, which controls FAS expression. Integration of all of these omics data in an AML metabolic network could allow fluxes calculations to gain even more insight into the metabolic regulation of IDH WT and R132H cells.

#### **Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/19/11/3325/ s1.

**Author Contributions:** Conceptualization, J.-E.S., J.-C.P. and J.B.-M.; methodology, J.B.-M., F.R. and L.S.; software, P.M.; validation, F.R., A.B. and L.S.; formal analysis, M.S. and F.R.; investigation, F.R, A.B., F.V., L.T., S.Z., E.S., L.K.L. and L.S.; resources, L.S., P.M. and E.S.; data curation, P.M.; writing—original draft preparation, L.S. and J.B.-M.; writing—review and editing, L.S, P.M., J.-C.P., J.-E.S. and J.B.-M.; visualization, L.S., M.S. and J.B.-M.; supervision, J.-E.S. and J.B.-M. project administration, J.B.-M. and L.S.; funding acquisition, J.-E.S. and J.-C.P.

**Funding:** This work was supported by grants from the Région Midi-Pyrénées (CRLE; J.-E.S.), Plan Cancer 2014-BioSys (FLEXAML; J.-E.S.) and the Institut national de la santé et de la recherche médicale (Inserm). MetaToul is part of the national infrastructure MetaboHUB (The French National infrastructure for metabolomics and fluxomics, www.metabohub.fr) and is supported by MetaboHUB-ANR-11-INBS-0010, by the Région Occitanie, the European Regional Development Fund, the SICOVAL, the Infrastructures en Biologie Sante et Agronomie (IBiSa, France), the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Recherche Agronomique (INRA) and Inserm.

**Acknowledgments:** MetaToul (Metabolomics & Fluxomics Facitilies, Toulouse, France, www.metatoul.fr) is gratefully acknowledged for carrying out metabolome et lipdiomic analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.


Author details

5. Conclusions

330 Adaptive Robust Control Systems

The dynamic model of overhead crane with distributed mass and elasticity of handling cable is formulated using the extended Hamilton's principle. Based on the model, we successfully analyzed and designed two nonlinear robust controllers using two versions of Lyapunov candidate functions. The first can steer the payload to the desired location, while the second can maintain payload fluctuation in a defined span. The proposed controllers well stabilize all

Figure 5. System responses in the case of L = 9 m and m = 9 kg with conventional Lyapunov function approach.

Tung Lam Nguyen\* and Minh Duc Duong

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Industrial Automation, School of Electrical Engineering, Hanoi University of Science and Technology, Hanoi, Vietnam


*Article* **Ingestional Toxicity of Radiation-Dependent Metabolites of the Host Plant for the Pale Grass Blue Butterfly: A Mechanism of Field Effects of Radioactive Pollution in Fukushima**

**Akari Morita 1, Ko Sakauchi 1, Wataru Taira 2 and Joji M. Otaki 1,\***

**Abstract:** Biological effects of the Fukushima nuclear accident have been reported in various organisms, including the pale grass blue butterfly *Zizeeria maha* and its host plant *Oxalis corniculata*. This plant upregulates various secondary metabolites in response to low-dose radiation exposure, which may contribute to the high mortality and abnormality rates of the butterfly in Fukushima. However, this field effect hypothesis has not been experimentally tested. Here, using an artificial diet for larvae, we examined the ingestional toxicity of three radiation-dependent plant metabolites annotated in a previous metabolomic study: lauric acid (a saturated fatty acid), alfuzosin (an adrenergic receptor antagonist), and ikarugamycin (an antibiotic likely from endophytic bacteria). Ingestion of lauric acid or alfuzosin caused a significant decrease in the pupation, eclosion (survival), and normality rates, indicating toxicity of these compounds. Lauric acid made the egg-larval days significantly longer, indicating larval growth retardation. In contrast, ikarugamycin caused a significant increase in the pupation and eclosion rates, probably due to the protection of the diet from fungi and bacteria. These results suggest that at least some of the radiation-dependent plant metabolites, such as lauric acid, contribute to the deleterious effects of radioactive pollution on the butterfly in Fukushima, providing experimental evidence for the field effect hypothesis.

**Keywords:** radioactive pollution; Fukushima nuclear accident; lauric acid; alfuzosin; ikarugamycin; plant secondary metabolite; artificial diet; *Zizeeria maha*; *Oxalis corniculata*; low-dose exposure

#### **1. Introduction**

Anthropogenic impacts on wild organisms have been an important scientific and political issue worldwide in this century. Human activities often involve local and global scale pollution of air, water, soil, and ocean, leading to climate changes and human health disorders [1,2]. For example, anthropogenic radionuclides from nuclear bomb tests and nuclear power plant accidents can be found worldwide [3–7]. It is thus important to understand precisely how severely wild organisms are affected by human activities and in what ways. To this end, butterflies have often been used as ecological indicators because of their advantages over other organisms [8,9]. For example, (1) butterflies are often conspicuous and abundant in the field and easy to identify at the species level, (2) a wealth of information on life history is available, (3) rich museum specimens are often available, and (4) many amateur lepidopterists may join field studies covering a wide geographic range. These advantages of using butterflies are invaluable for field studies. Not surprisingly, changes in butterfly species in abundance, range, phenology, and diversity have been used as key factors to understand recent environmental influences in many studies [10–16]. Occasionally, studies have focused on a single or a few indicator species [17–23]. An advantage of a single-species approach is to couple field surveys and laboratory experiments to understand what occurs in the field.

**Citation:** Morita, A.; Sakauchi, K.; Taira, W.; Otaki, J.M. Ingestional Toxicity of Radiation-Dependent Metabolites of the Host Plant for the Pale Grass Blue Butterfly: A Mechanism of Field Effects of Radioactive Pollution in Fukushima. *Life* **2022**, *12*, 615. https://doi.org/ 10.3390/life12050615

Academic Editors: Fabrizio Ambrosino and Supitcha Chanyotha

Received: 30 March 2022 Accepted: 19 April 2022 Published: 20 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The pale grass blue butterfly, *Zizeeria maha*, has been used as a field indicator and laboratory model species to understand evolutionary and developmental plasticity in response to environmental changes [17,24–28]. In this butterfly, an environmentally induced plastic phenotype was genetically assimilated in the laboratory experiment and in the field, which was probably one of the best pieces of evidence for genetic assimilation [29,30]. Just after the establishment of the pale grass blue butterfly as a laboratory model species that can also be used as a field indicator species, the Fukushima nuclear accident occurred in March 2011. Anthropogenic radioactive materials from the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) heavily polluted the east side of Tohoku district in Japan. The northern range margin of the pale grass blue butterfly was located 380 km away from the FDNPP [17], and the polluted area in Fukushima is completely covered by the distribution range of the pale grass blue butterfly. Without question, the pale grass blue butterfly was the logical choice for studying the biological effects of the Fukushima nuclear accident.

The Fukushima nuclear accident was reported to have caused various biological and ecological effects on animals, such as birds [31–33], insects [34–39], Japanese monkeys [40–42], and intertidal invertebrates [43], plants such as rice [44,45], fir trees [46], red pine trees [47], and the creeping wood sorrel *Oxalis corniculata* [48–50], and soil microbes [51]. A series of our studies [31–34,52–63] demonstrated that the pale grass blue butterfly has been severely affected by the Fukushima nuclear accident. One of the pieces of important evidence was provided by the internal exposure experiment, in which the contaminated host plant leaves collected from Fukushima were given to larvae from Okinawa (where radioactive contamination is minimal), resulting in high mortality and abnormality rates. However, when an artificial diet containing pure radioactive cesium (137Cs) was given to larvae, no change in the survival rate was observed [64]. A similar discrepancy between field and laboratory results has been observed in the case of the Chernobyl nuclear accident [65,66]. This field-laboratory paradox was explained by the field effect hypothesis: the host plant in the field responded to low-level radiation stress by upregulating metabolites that were toxic to larvae as a part of plant defense mechanisms [59,67]. Subsequent studies have reported upregulated and downregulated metabolites and nutrients in plant leaves [48–50], supporting this field effect hypothesis.

In a previous metabolomic study [49], the creeping wood sorrel in Okinawa was irradiated by contaminated soil collected from Fukushima, and the leaf samples (the edible part for larvae) were subjected to GC–MS (gas chromatography–mass spectrometry) and LC–MS (liquid chromatography–mass spectrometry) analyses. Under the acute low-dose radiation conditions, 5.7 mGy (34 μGy/h for seven days), many peaks were significantly upregulated, although most of them were annotated as multiple compounds or not annotated at all. One of the upregulated peaks was singularly annotated as lauric acid by targeted GC–MS analysis, and two of the upregulated peaks were singularly annotated as alfuzosin and ikarugamycin by LC–MS analysis. Therefore, the potential toxic effects of these three compounds are of great interest.

Lauric acid is a saturated fatty acid, also called dodecanoic acid, that can be found widely in plants. Lauric acid shows a wide variety of bioactivities as a plant defense, volatile against *Staphylococcus* [68,69], *Mycobacterium tuberculosis* [70], fungus [71], and *Phytophthora sojae*, an agriculturally important plant pathogen that belongs to Protista [72]. Notably, extracts from *Vitex* species containing lauric acid have larvicidal activity against a mosquito species, *Culex quinquefasciatus* [73]. Lauric acid is likely sensed at least by an insect, *Holotrichia parallela* [74], as an odorant. Accordingly, it is reasonable to hypothesize a larvicidal activity of lauric acid in *O. corniculata* against larvae of the pale grass blue butterfly.

Alfuzosin is a synthetic α1-adrenergic receptor antagonist used widely for the treatment of benign prostatic hyperplasia [75–78]. Because it is synthetic, it is unlikely to be present naturally in the plant. However, because the LC–MS peak annotated as alfuzosin has very similar (virtually identical) elution time and exact mass with alfuzosin, examination of MS/MS (mass spectrometry/mass spectrometry, i.e., tandem mass spectrometry) spectrograms was required to differentiate alfuzosin and this unknown plant metabolite

(called the alfuzosin-related compound hereafter) [49]. Faced with the fact that the exact identity of the alfuzosin-related compound cannot be determined easily, we tested the toxicity of alfuzosin itself in this study, assuming that alfuzosin and its related metabolite may have similar biological effects on larvae of the pale grass blue butterfly.

Ikarugamycin is an antiprotozoal agent isolated originally from the soil bacterium *Streptomyces phaeochromogenes* var. *ikaruganensis* [79]. Importantly, ikarugamycin has also been detected from an endophytic actinomycete, *Streptomyces harbinensis*, from soybean root [80]. Endophytic bacteria have been widely observed in plants [81–86], including *O. corniculata* [87,88]. Accordingly, ikarugamycin detected from leaves of *O. corniculata* is likely from an endophytic *Streptomyces* sp. in leaves or roots, which responded to low-level radiation [49,50]. Ikarugamycin and its derivatives are antifungal [89] and antibacterial [89,90] agents and inhibit clathrin-mediated endocytosis in eukaryotic cell lines [91].

These three upregulated metabolites have been hypothesized to function as toxicants for larvae of the pale grass blue butterfly under low-level radiation stress. In other words, these compounds are candidate causal substances for the ecological field effects of lowlevel radiation pollution in Fukushima. In this study, we tested the above hypothesis by investigating the ingestional toxicity of these compounds using a novel artificial diet that has a reduced leaf content of *O. corniculata*.

To detect their potential toxicity, we examined three aspects of development: metamorphosis rates, developmental periods, and adult wing size. The metamorphosis rates were used to detect the number of surviving individuals after metamorphosis and included the following: the pupation rate, the eclosion (survival) rate, and the normality rate. The developmental periods were used to detect developmental retardation or acceleration and included the following: the egg-larval period, the pupal period, and the immature period. Adult wing size included both male and female forewing sizes. In this way, we examined high mortality and abnormalities, growth retardation, and smaller forewing size, which have been detected as biological effects of the Fukushima nuclear accident in previous studies [34–37].

#### **2. Materials and Methods**

#### *2.1. Egg Collection and Larval Rearing*

Egg collection and rearing were performed according to Hiyama et al. (2010) [24] and other related publications [34,52,56] with some minor modifications, as described briefly below. Adults of the pale grass blue butterfly *Z. maha* and its host plant, the creeping wood sorrel *O. corniculata*, were collected at the University of the Ryukyus and its vicinity. The whole plant was placed in a pot and set in a glass tank (300 mm × 300 mm × 300 mm) in which approximately three female butterflies and a few male butterflies were confined at a time. A single trial of egg collection was performed for a period of four days. All rearing processes were executed under the conditions where light was automatically turned on from 6:00 a.m. to 10:00 p.m. (L16:D8) and room temperature was set at 27 ◦C.

After eggs were deposited on the leaves of the host plant, the plant pot was removed from the glass tank and covered with a plastic bag. When the leaves were eaten enough by newly hatched larvae, they were transferred to a transparent plastic container (150 mm × 150 mm × 55 mm), to which a new bunch of the host plant leaves was supplied every day. Larvae were reared with fresh leaves for 14 days from the beginning of egg collection. Larvae were then randomly divided into different treatment groups: a fresh leaf group, an artificial diet group with no test additive (0 mg/g), and a few artificial diet groups with different concentrations of a test additive (0.01 mg/g, 0.1 mg/g, and 1 mg/g). One group was reared in one container that housed 15–25 larvae, depending on the total number of larvae that were obtained simultaneously from a single egg collection trial. The larvae obtained from a single trial were all siblings; they constitute a sibling group. In this way, genetic bias was minimized. The artificial diet was given as four small square lumps (10 mm × 8 mm × 3 mm per lump) at four corners in a container.

We cleaned containers and changed old lumps of the artificial diet for fresh ones every day, but unexpected deaths that were apparently unrelated to the toxicity of the test additives could not be entirely avoided. This is probably partly because we use fresh leaves collected from the field without sterilization because the sterilization process may breakdown or vaporize some ingredients in fresh leaves important for larvae to initiate eating behavior (such as oxalic acid as an eating initiator [92]). Pupation and eclosion were checked every morning, roughly from 8:30 am to 1:00 pm, so that the data on the developmental periods (days) could be obtained later. We set a criterion that the eclosion (survival) rate of the sibling group of the artificial diet without a test additive should be more than 45% to be considered a successful rearing trial. Sibling groups with eclosion rates below this criterion were considered technical failures and excluded from subsequent analyses. This threshold was set as the gap based on our rearing experience. After pupation, pupae inside the container were transferred to small petri dishes individually. Soon after eclosion, adult butterflies were frozen until subsequent analyses.

#### *2.2. Artificial Diet Preparation*

Larvae require leaves (or some plant chemicals) as a component of an artificial diet to eat, but the leaf content in an artificial diet should be minimized to test toxicological effects of a metabolite in leaves themselves. Furthermore, the process of collecting fresh leaves is the most laborious and time-consuming process for preparing an artificial diet. To meet these demands, we developed a novel artificial diet for the pale grass blue butterfly for the feeding experiments in this study. We used a commercially available diet, Silk Mate L4M (Nosan Corporation, Yokohama, Kanagawa, Japan), for rearing silkworm. This diet was supplied as powder containing defatted soybean, starch, sugar, cellulose, formative agent, citric acid, mulberry leaf powder, vitamins, minerals, preservative, and antibiotics (diet additives), according to the manufacturer's specification. For the current study, fresh leaves of the creeping wood sorrel, Silk Mate L4M, and deionized water were mixed at a weight ratio of 1:3:5. Thus, this new diet was named AD-FSW-135.

A possibility that Silk Mate L4M contains lauric acid, the alfuzosin-related compound, and ikarugamycin from mulberry leaves was not considered in this study based on the following reasons, although it cannot be excluded completely. First, ikarugamycin and alfuzosin (and thus the alfuzosin-related compound) have never been reported from mulberry leaves to the best of our knowledge. Besides, their concentrations in the creeping wood sorrel were low (see below). Second, lauric acid is known to be contained in mulberry leaves [93,94]. However, because lauric acid is volatile, it may be minimized during an autoclave sterilization process of Silk Mate L4M. The basal levels of the three compounds that were carried from the fresh leaves in AD-FSW-135 (prepared in this study) were shown to be low (see Section 4).

#### *2.3. Lauric Acid, Alfuzosin, and Ikarugamycin*

Lauric acid (catalog No. 042-23281, Wako Special Grade; FUJIFILM Wako Chemicals, Tokyo, Japan), alfuzosin (catalog No. PHR1638, Pharmaceutical Secondary Standard, Certified Reference Material; Sigma–Aldrich, St. Louis, MO, USA), and ikarugamycin (catalog No. 15386; Cayman Chemical, Ann Arbor, MI, USA) were purchased. They were in powder form and were added to the diet preparation directly. They were then mixed well manually or using an electric mixer until the diet preparation was visually judged to be homogeneous. In this way, we assumed that the test additives were incorporated evenly into the diet and became solubilized. However, they might not have been solubilized completely at higher concentrations (see Section 4). Concentrations of these test additives were expressed in milligrams per gram of artificial diet (mg/g) throughout this paper. For lauric acid, we tested 0 mg/g (control), 0.01 mg/g, 0.1 mg/g, and 1 mg/g. This range covered the estimated concentration of lauric acid in leaves (see below). For alfuzosin, we tested 0 mg/g (control), 0.01 mg/g, and 0.1 mg/g. For ikarugamycin, we tested 0 mg/g (control) and 0.01 mg/g. These ranges of alfuzosin and ikarugamycin were much greater

than the concentrations of the alfuzosin-related compound and ikarugamycin in leaves (see below). Nevertheless, we tested these concentrations because excessive doses are often necessary to obtain the median toxic dose TD50 and the median lethal dose LD50 and because comparison among the three compounds at the same doses may provide us with valuable information on diverse effects of leaf compounds. Basal levels of the compounds from leaves in AD-FSW-135 were not taken into account for analyses due to uncertainty of the estimated leaf concentrations.

#### *2.4. Concentration of Lauric Acid in Leaves*

The concentration of lauric acid in leaves was roughly estimated as follows. Lauric acid in leaves was discovered by the targeted method of GC–MS [49] (Appendix A). Because this metabolite was targeted based on previously known chemical information, the identification and peak area data were more credible than the nontargeted method. Although each compound has a different detection efficiency in GC–MS, it is possible to roughly compare peak area values among targeted metabolites detected simultaneously from the same samples. One of the targeted metabolites was oxalic acid. The concentration of oxalic acid in leaves of *O. corniculata* has been reported to be 16.9 mg/g (leaf) [95]. On the other hand, the mean peak area value of oxalic acid (No. 15) in nonirradiated samples in the targeted GC–MS analysis was 6,409,017 (*n* = 3) (Figure A1) [49]. Similarly, the mean peak area value of lauric acid (No. 175) in nonirradiated samples in the targeted GC–MS analysis was 18,899 (*n* = 3) (Figure A1) [49]. Therefore, the concentration of lauric acid in nonirradiated leaves was calculated to be 0.050 mg/g (leaf). When irradiated, the mean peak area value of lauric acid was 23,977 (*n* = 3) [49], and it increased approximately 1.27 times to 0.063 mg/g (leaf) under the previous experimental conditions [49].

#### *2.5. Concentration of the Alfuzosin-Related Compound in Leaves*

The concentration of the unknown alfuzosin-related compound in *Oxalis* leaves was estimated by HPLC spectrograms of LC–MS newly performed in this study (Figure A2). Leaf samples for the previous study (Figure A1) [49] and the current study (Figure A2) were identical. Sample preparation procedures followed a previous LC–MS study [49]. Washed fresh leaves of *O. corniculata* (100 mg) were frozen, ground, and thoroughly homogenized with methanol (300 μL). After a brief centrifugation, 200 μL was recovered, from which 10 μL was subjected to LC–MS analysis. This extraction process can be considered a total volume increase to 400 μL, assuming that leaf density is close to that of water (1.0 g/mL). The experimental conditions for the LC–MS analysis in the present study are described in Appendix A.

The alfuzosin-related compound in the leaf extract had a mean peak area value of 201.81 in triplicate of a sample (Figure A2a). This peak area value was similar to that of the alfuzosin standard (Sigma–Aldrich), 199.36, when 0.10 ng/mL methanol extract was analyzed (Figure A2b). Although the alfuzosin standard spectrum showed an additional peak, this peak was found to be an impurity peak in methanol (Figure A2c). Considering the volume conversion factor, ×4.0, the concentration of the alfuzosin-related compound in nonirradiated leaves was estimated to be 0.40 ng/g (leaf). In a previous metabolomic study, the mean peak area values of the alfuzosin-related compound (No. 4746) were 133,169 (*n* = 3) (without irradiation) and 534,069 (*n* = 3) (irradiated) (Figure A1) [49]. Thus, when irradiated, the area value increased 4.01 times to 1.6 ng/mg (leaf) under the previous experimental conditions.

#### *2.6. Concentration of Ikarugamycin in Leaves*

The concentration of ikarugamycin in leaves was estimated based on the previous peak area values of LC–MS (Figure A1) [49]. Alfuzosin-related compound (No. 4746) had a mean peak area value of 133,169 (*n* = 3) in nonirradiated samples, whereas ikarugamycin had a peak area value of 76,713 (*n* = 3) from the same samples [49]. Assuming that it is possible to roughly compare peak area values among metabolites detected simultaneously

from the same samples, the concentration of ikarugamycin was calculated to be 0.20 ng/g (leaf) based on the estimated concentration of the alfuzosin-related compound in leaves. When irradiated, the mean peak area value of ikarugamycin was 99,905. Thus, the area value increased 1.30 times to 0.26 ng/g (leaf) under the previous experimental conditions.

#### *2.7. Toxicological Output Data*

To understand the toxicity of the three metabolites, we recorded three metamorphosisrelated data as the number of individuals as follows: the number of individuals that successfully pupated (the number of pupae), the number of individuals that successfully eclosed (the number of adults), and the number of individuals that successfully eclosed without wrinkled wings (the number of normal adults), as shown in Appendix B (Tables A1–A4). These three numbers were used for calculating the three metamorphosis rates: the pupation rate, the eclosion rate, and the normality rate. For calculations, these numbers were divided by the starting number of larvae, and the results were expressed as a percentage. The eclosion rate was also called the "survival rate".

We also recorded three developmental period data as the number of days: the number of days from the time point when egg collection started to pupation (the egg-larval days), the number of days from pupation to eclosion (the papal days), and the number of days from the time point when egg collection started to eclosion (the immature days). The immature days are simple summation of the egg-larval days and the pupal days. The egg-larval days included prepupal days.

Additionally, we measured adult forewing size from the wing base to the apex using a desktop digital microscope SKM-2000 with its associated software SK-measure (Saito Kogaku, Yokohama, Kanagawa, Japan). Because male and female forewing sizes are known to be different in this species [24,64], forewing size data were compiled sexdependently. Individuals with wrinkled wings were not subjected to size measurements. The developmental period data and the forewing size data were compiled in Table S1. The numbers of individuals in repeated biological trials were also shown in Table S1.

#### *2.8. Statistical Analysis*

A treatment group (0.01 mg/g, 0.1 mg/g, or 1 mg/g of a test additive of interest) was statistically compared to a corresponding no treatment (control) group (0 mg/g). We performed the χ2 test for the data on the number of individuals that produced the pupation rate, the eclosion rate, and the normality rate. The χ2 test was also performed for evaluating the performance of the artificial diets and for comparing the normalized eclosion and normality rates between lauric acid and alfuzosin. Yates' correction was not performed. We performed either Student's *t*-test (equal variance) or Welch's *t*-test (unequal variance) for the egg-larval days, the pupal days, the immature days, and the forewing size, assuming that they were normally distributed. Bonferroni or other correction was not performed. Statistical analyses were performed using Microsoft Excel (Office 365), JSTAT (Yokohama, Kanagawa, Japan), and MetaboAnalyst [96]. MetaboAnalyst was also used to produce box plots of metabolites obtained in a previous study [49].

#### **3. Results**

#### *3.1. Performance of the Artificial Diet AD-FSW-135*

The new artificial diet AD-FSW-135 developed for this study was compared with the previous diets, AD-F (artificial diet with fresh leaves) [24] and AD-FSI-112 (artificial diet with fresh leaves, soy powder, and Insecta F-II (Nosan Corporation)) [64], in terms of ingredients (Figure 1a). The most important difference among the three artificial diets was the leaf content. *Oxalis* leaves occupied 58.7% of the diet in AD-F [24] and 32.2% in AF-FSI-112 [64]. In contrast, *Oxalis* leaves occupied only 11.1% in the new diet AD-FSW-135. In other words, AD-FSW-135 (this study) contained approximately one-fifth and one-third of fresh leaves of the previous diets AD-F [24] and AD-FSI-112 [64], respectively. The reduced leaf content in the new diet AD-FSW-135 was considered important for toxicological

tests (see Section 2). This leaf-content reduction was achieved by the introduction of Silk Mate L4M (Nosan Corporation), a commercially available artificial diet for silkworms. In a previous diet, AD-FSI-112, Insecta F-II from the same manufacturer was used [64]. Simplification of the contents for quick and easy preparation with just three ingredients was also an important advantage of the new artificial diet AD-FSW-135 developed for this study.

**Figure 1.** Ingredients and performance of artificial diets. (**a**) Ingredients and their weight percentages. AD-FSW-135 has a reduced leaf content. It also has simplified contents with just three ingredients. (**b**) Survival rate (eclosion rate). AD-FSW-135 shows a higher survival rate than AD-D [24] and a lower rate than AD-F [24] and AD-FSI-112 [64]. The *p*-values obtained from the χ2 test are shown. The AD-FSW-135 results were obtained from ten biological repeats (see Supplementary Table S1). (**c**) Male (top) and female (bottom) forewing size. The *p*-values obtained from the *t*-test between the natural diet and AD-FSW-135 are indicated. Both natural diet and AD-FSW-135 results were obtained from ten biological repeats (see Supplementary Table S1).

Throughout the rearing experiments with the new artificial diet AD-FSW-135 containing a compound of interest (called a test additive), we always simultaneously reared a group of larvae with natural fresh leaves of the creeping wood sorrel and another group of larvae with AD-FSW-135 without a test additive (0 mg/g) (Appendix B; Table A1). To evaluate the performance of the new diet AD-FSW-135, the survival (eclosion) rates of larvae without an additive were compared among the previous and present diets (Figure 1b). AD-FSW-135 (this study) had a significantly higher survival rate than AD-D (artificial diet with dried leaves) [24] and a significantly lower rate than AD-FSI-112 [64] and AD-F [24], indicating that the survival rate of AD-FSW-135 (this study) was not very high but was not very low. AD-FSW-135 (this study) was thus considered acceptable for toxicological tests, as long as the majority of larvae could eat AD-FSW-135 and grow.

The forewing size of adult individuals reared with the new diet AD-FSW-135 was reduced in comparison to that of fresh plant leaves (Figure 1c). Males from the natural diet and AD-FSW-135 groups showed forewing sizes of 12.16 ± 0.71 mm (mean ± standard deviation) and 10.88 ± 0.65 mm, respectively. Females from the natural diet and AD-FSW-135 groups showed forewing sizes of 12.67 ± 0.68 mm and 11.68 ± 0.92 mm, respectively. In both sexes, the forewing size was reduced significantly. However, these results were essentially similar to those of the previous diets in terms of size distributions [24,64]; this level of size reduction seems to be inherent in rearing butterflies in artificial diets. Therefore, the forewing size reduction in the AD-FSW-135 results was considered acceptable for toxicological tests in this study.

#### *3.2. Lauric Acid*

We prepared three concentrations of lauric acid in the artificial diet: 0.01 mg/g, 0.1 mg/g, and 1 mg/g, in addition to the diet without it (0 mg/g). These concentrations covered an estimated concentration of lauric acid in the irradiated leaves of *O. corniculata* of

0.063 mg/g. The number of pupae, eclosion, and normal adults were recorded (Appendix B; Table A2). We examined the toxicity of lauric acid from three different viewpoints: metamorphosis rates (the pupation rate, eclosion rate, and normality rate), developmental periods (egg-larval days, pupal days, and immature days), and adult forewing size.

The normality rate linearly decreased in response to the concentration of lauric acid (Figure 2a). The pupation rate and the eclosion rate were largely similar to the normality rate except at 0.1 mg/g, which showed an increase (Figure 2a). In comparison to the diet without lauric acid (0 mg/g), the diet with 1 mg/g showed a significant decrease in the pupation rate, eclosion rate, and normality rate, indicating the toxicity of lauric acid. At 0.1 mg/g and 1 mg/g, the egg-larval days appeared to be significantly longer than the control (0 mg/g) (Figure 2b). The immature days of the 0.01 mg/g and 0.1 mg/g treatments were significantly longer than those of the control (0 mg/g). These results indicate that developmental retardation occurred in the larval periods at all three concentrations of lauric acid. The forewing size of females at 0.1 mg/g was reduced significantly in comparison to the control (0 mg/g), although such a reduction was not observed at other concentrations (Figure 2c).

**Figure 2.** Results of the toxicity test for lauric acid. Asterisks indicate levels of statistical significance in comparison to the control (0 mg/g); \*, *p* < 0.05; \*\*, *p* < 0.01. These results were obtained from four biological repeats (see Supplementary Table S1). (**a**) Pupation rate (green), eclosion rate (brown), and normality rate (red). The *p*-values obtained from the χ2 test are indicated. The pink vertical broken line indicates a rough position of the estimated concentration of lauric acid in irradiated leaves, 0.063 mg/g. (**b**) Egg-larval days (blue), pupal days (brown), and immature days (gray). The mean values (±standard deviation) are shown as bar height. The *p*-values obtained from the *t*-test are indicated. (**c**) Male (blue green) and female (pink) forewing size. The mean values (±standard deviation) are shown as bar height. The *p*-values obtained from the *t*-test are indicated.

#### *3.3. Alfuzosin*

We prepared two concentrations of alfuzosin in the artificial diet, 0.01 mg/g and 0.1 mg/g, in addition to the diet without it (0 mg/g), to compare the results with those of lauric acid, although the lowest concentration used in the present study, 0.01 mg/g, was 6.3 × 103 times higher than an estimated concentration of the alfuzosin-related compound in irradiated leaves, 1.6 ng/g. As in the case of lauric acid, we examined the metamorphosis rates, developmental periods, and adult forewing size (Appendix B; Table A3).

The pupation rate, eclosion rate, and normality rate all decreased significantly in response to alfuzosin, but not linearly (Figure 3a). Reasons for lower rates at 0.01 mg/g than those at 0.1 mg/g were uncertain but may be technical (see Section 4). The egg-larval days and the immature days were significantly different from those without alfuzosin (0 mg/g) (Figure 3b). Somewhat surprisingly, these differences showed developmental acceleration instead of retardation. The forewing size did not differ from that of the control (0 mg/g), but at 0.1 mg/g in females, the forewing size tended to increase, although the increase was not statistically significant (Figure 3c).

**Figure 3.** Results of the toxicity test for alfuzosin. Asterisks indicate levels of statistical significance in comparison to the control (0 mg/g); \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001. These results were obtained from three biological repeats (see Supplementary Table S1). (**a**) Pupation rate (green), eclosion rate (brown), and normality rate (red). The *p*-values obtained from the χ2 test are indicated. The pink vertical broken line indicates a rough position of the estimated concentration of the alfuzosin-related compound in irradiated leaves, 1.6 ng/g. (**b**) Egg-larval days (blue), pupal days (brown), and immature days (gray). The mean values (±standard deviation) are shown as bar height. The *p*-values obtained from the *t*-test are indicated. (**c**) Male (blue green) and female (pink) forewing size. The mean values (±standard deviation) are shown as bar height. The *p*-values obtained from the *t*-test are indicated.

#### *3.4. Ikarugamycin*

We prepared one concentration of ikarugamycin in the artificial diet, 0.01 mg/g, in addition to the diet without it (0 mg/g) to compare the results with those of lauric acid, although the lowest concentration used in the present study, 0.01 mg/g, was 3.8 × 104 times higher than an estimated concentration of ikarugamycin in irradiated leaves, 0.26 ng/g. As in the cases of lauric acid and alfuzosin, we examined the metamorphosis rates, developmental periods, and adult forewing size (Appendix B; Table A4).

Surprisingly, the pupation rate and the eclosion rate increased significantly in response to ikarugamycin, although an increase in the normality rate was not significant (Figure 4a). These results indicate mild drug efficacy of ikarugamycin instead of toxicity. In contrast to lauric acid and alfuzosin, the egg-larval days, pupal days, and immature days at 0.01 mg/g were not different from those without ikarugamycin (0 mg/g) (Figure 4b). The forewing size at 0.01 mg/g did not differ from those without ikarugamycin (0 mg/g) (Figure 4c).

#### *3.5. Comparison of Three Compounds*

Here, we compared the results of the three compounds tested above. The eclosion (survival) rates and the normality rates were normalized so that they became 100% when no compound was added to the diet (0 mg/g) (Appendix B; Tables A5 and A6) as shown in Figure 5. The eclosion rates (Figure 5a) and the normality rates (Figure 5b) were not very different, but lauric acid exhibited a smooth and gradual dose-dependent decrease in the normality rate curve, although not in the eclosion rate curve, as seen previously (Figure 2a). It is remarkable that the normality rate curves of the three compounds showed different behaviors; in response to concentration, the lauric acid curves decreased dose-dependently, the alfuzosin curves decreased more sharply and not linearly, and the ikarugamycin curves increased (Figure 5b). At the concentration of 0.01 mg/g, where three compounds were able to be compared, the normality rates of lauric acid, alfuzosin, and ikarugamycin were 69.5%, 23.4%, and 125.0%, respectively (Appendix B; Table A6). The differences between lauric acid and alfuzosin appeared to be more significant at the concentration of 0.01 mg/g than 0.1 mg/g in both the eclosion and normality rates, but this may be because of a low solubility of alfuzosin at 0.1 mg/g (see Sections 2 and 4).

**Figure 4.** Results of the toxicity test for ikarugamycin. Asterisks indicate levels of statistical significance in comparison to the control (0 mg/g); \*, *p* < 0.05. These results were obtained from three biological repeats (see Supplementary Table S1). (**a**) Pupation rate (green), eclosion rate (brown), and normality rate (red). The *p*-values obtained from the χ2 test are indicated. The pink vertical broken line indicates a rough position of the estimated concentration of ikarugamycin in irradiated leaves, 0.26 ng/g. (**b**) Egg-larval days (blue), pupal days (brown), and immature days (gray). The mean values (±standard deviation) are shown as bar height. The *p*-values obtained from the *t*-test are indicated. (**c**) Male (blue green) and female (pink) forewing size. The mean values (±standard deviation) are shown as bar height. The *p*-values obtained from the *t*-test are indicated.

**Figure 5.** Comparison of the results of the three compounds. The *p*-values obtained from the χ2 test between lauric acid and alfuzosin are shown. (**a**) Eclosion (survival) rate (normalized). (**b**) Normality rate (normalized).

As a convention of toxicological analysis, a regression line was determined using the normality rates as *y* = −24.9*x* + 79.8 (*R*2 = 0.40) for lauric acid, and at the normality rate of 50% (*y* = 50), lauric acid concentration *x* was determined as 1.2 mg/g. This is considered equivalent to the median toxic dose, TD50. Similarly, a regression line was determined as *y* = −185*x* + 64.6 (*R*2 = 0.069) for alfuzosin, and at the normality rate of 50% (*y* = 50), the alfuzosin concentration *x* was determined to be 0.079 mg/g, which is 15 times smaller than that of lauric acid. Just to be sure, if the value at 0.1 mg/g in alfuzosin was erroneously high due to technical reasons, such as low solubility (see Section 4), the LD50 value of alfuzosin should be much lower.

Likewise, using the eclosion (survival) rates, a regression line was determined as *y* = −24.8*x* + 90.8 (*R*2 = 0.69) for lauric acid, and at the normality rate of 50% (*y* = 50), lauric acid concentration *x* was determined as 1.6 mg/g. This is considered equivalent to the median lethal dose, LD50. Similarly, a regression line was determined as *y* = −126*x* + 66.9 (*R*2 = 0.038) for alfuzosin, and at the normality rate of 50% (*y* = 50), the alfuzosin concentration *x* was determined as 0.13 mg/g, which is 12 times smaller than that of lauric acid. As in the case of TD50, the LD50 value of alfuzosin should be much lower if the value at 0.1 mg/g was technically erroneous.

#### **4. Discussion**

We tested the ingestional toxicity of three compounds, namely, lauric acid, alfuzosin, and ikarugamycin, which were significantly upregulated in *O. corniculata* and annotated by a previous metabolomic study [49]. For convenience, concentration data are compiled in Table 1. In this study, we employed a new artificial diet, AD-FSW-135, which contained a relatively small amount of host plant leaves, occupying just 11% of the entire diet. It is important to keep the leaf content as low as possible in the artificial diet due to an experimental addition of a testing compound. Indeed, the basal levels of the three compounds in the new diet AD-FSW-135 (Table 1) were considered low enough for the current study. This new diet showed acceptable performance based on the survival rate and forewing size, two indexes to evaluate artificial diets [64]. Thus, we believe that the use of AD-FSW-135 in the present study is justifiable but that there is still much room for further improvement of artificial diets.

**Table 1.** Concentrations of the three metabolites of interest.

\*1: For irradiation conditions, see Sakauchi et al. (2021) [49]. \*2: Concentrations in toxicity tests ignore the basal levels of these metabolites from leaves in AD-FSW-135. \*3: Coverage indicates if leaf concentration was covered by the tested concentration range. \*4: Alfuzosin was tested, but the leaf concentrations shown here are those of the alfuzosin-related compound. NA: Not applicable.

With this new diet AD-FSW-135, we demonstrated that lauric acid was toxic to larvae dose-dependently in terms of metamorphosis rates, although the larval response was mild. Lauric acid is present in leaves without radiation, and larvae are certainly tolerant to lauric acid at the leaf level of 0.050 mg/g, explaining the gradual dose–response curves. The mild toxicity of lauric acid is expressed in its TD50, 1.2 mg/g, in contrast to the TD50 of alfuzosin, 0.079 mg/g. LD50 values also indicated such a relationship. We observed some toxicity even at the level of 0.01 mg/g, but this may be because larvae were exposed to a sudden rise in lauric acid concentration when the artificial diet was first given. In addition to the changes in the metamorphosis rates, growth retardation was detected at the egg-larval period in response to lauric acid. Furthermore, the forewing size reduction was observed, although only at 0.1 mg/g in females. These results indicate the toxicity of lauric acid on the developmental physiology of the butterfly and appear to be biologically significant in the field because the estimated concentration of lauric acid in irradiated leaves, 0.063 mg/g, was covered by the current study.

In a previous study, the fold change values in the upregulation of lauric acid was 1.27 at low-level radiation exposure; the cumulative dose to the plant was 5.7 mGy (34 μSv/h in a period of seven days) [49]. It is somewhat surprising that the plant significantly responded to this low-level exposure, and we expect that the fold change value may increase further in response to higher levels of radiation exposure. According to Nohara et al. (2014) [52], the ground radiation dose rate in Iitate was 18.9 μSv/h, which is indeed lower than the experimental dose rate used in our study, 34 μSv/h. However, experimental irradiation in the present study was only by external exposure during a very limited period of time (seven days), but in the wild, both external and internal exposures are expected for much longer periods of time throughout the entire life span of the butterfly. Importantly, the present results are reminiscent of those found in previous exposure experiments [34–36,52–56] and may also explain the spatiotemporal dynamics of the abnormality rates and collection efficiency (an indicator of population density) in 2011–2013 in Fukushima [37]. Therefore, we conclude that lauric acid acts as a potent toxicant (larvicide) for the pale grass blue butterfly not only in the laboratory but also in wide polluted areas in Fukushima in the field.

This conclusion is consistent with previous studies on lauric acid as a plant defense chemical [68–74]. More tolerance may evolve in larvae in the field, and this scenario may explain the adaptive evolution of the butterfly shown in the polluted areas in Fukushima [56]. Interestingly, lauric acid has been reported to be a feeding stimulant for the silkworm at the concentration of 0.013% in an artificial diet [94]. This percentage corresponds to the 0.1 mg/g level in the present study. In lepidopteran insects, a feeding stimulant for a given species is often toxic to other organisms [97]. Thus, it is reasonable that a feeding stimulant for the silkworm moth, lauric acid, is toxic to the pale grass blue butterfly. Conversely, a feeding stimulant for the pale grass blue butterfly, oxalic acid [92], is probably toxic to other insects including the silkworm moth.

Alfuzosin was also demonstrated to be toxic, but its toxicity was not linearly dose dependent in the metamorphosis rates. We do not understand this nonlinearity, but it might have originated from a technical reason regarding low solubility; alfuzosin might not have been dissolved well in the diet at the relatively high concentrations. Surprisingly, in addition to the reduced metamorphosis rates, alfuzosin appeared to act on the egglarval period to accelerate growth and tended to increase the forewing size. These results are in sharp contrast to those of lauric acid, indicating different toxic pathways in these two compounds. Because alfuzosin is an antagonist of the α1-adrenergic receptor [75–78], it may act on insect receptors for biogenic amines, such as octopamine and tyramine [98]. Nonetheless, both alfuzosin and lauric acid seem to affect the larval period but not the pupal period.

Because the alfuzosin concentrations tested in AD-FSW-135 were much higher than those in leaves and because the biological effects of alfuzosin and its related compound are not necessarily similar, direct extrapolations of the alfuzosin results to the alfuzosin-related compound were difficult. However, there may be a possibility that the alfuzosin-related compound was as toxic as alfuzosin due to their structural similarities. If so, the alfuzosinrelated compound is 15 times as toxic as lauric acid (based on the TD50 values) and 12 times as lethal as lauric acid (based on the LD50 values), but the concentration of the alfuzosinrelated compound in leaves was much lower than that of lauric acid. Therefore, the presence of the alfuzosin-related compound in leaves would not affect larvae in the field.

In contrast, ikarugamycin showed mild drug efficacy instead of toxicity. This may be simply because it is an antibiotic that inhibits bacterial or fungal growth in the artificial diet, although some antibiotics were contained in Silk Mate L4M, a commercially available ingredient of AD-FSW-135. In that case, ikarugamycin may protect the plant in the wild from fungal and bacterial infection. However, this drug efficacy of ikarugamycin for larvae may not be evident in the field because of the low concentration of ikarugamycin in leaves. Therefore, ikarugamycin would not nullify the toxicity of plant larvicides, such as lauric acid, in the field. Importantly, the present results of ikarugamycin suggest a possible contribution of metabolites from endophytic bacteria to plant and larval immunity under radiation stress. Practically, further addition of ikarugamycin or other antibiotics into AD-FSW-135 may improve its performance in the future.

In reality, in the wild, lauric acid and other upregulated unknown metabolites probably function together to ward off insects. Indeed, in response to radiation exposure, 24 upregulated peaks (*p* < 0.05) were obtained in LC–MS, among which only two of them (alfuzosin and ikarugamycin) were annotated singularly [49]. Only one upregulated peak (*p* < 0.05) was obtained in targeted GC–MS, which was lauric acid [49]. Additionally, 10 upregulated peaks (*p* < 0.05) were obtained in nontargeted GC–MS [49].

It is not possible, at least at this point, to demonstrate collective effects of many upregulated compounds with an artificial diet containing them. On the other hand, the "collective effects" have already been known by internal exposure experiments using contaminated leaves collected from Fukushima, resulting in lower survivorship and growth retardation [34–36,52–56]. We also have evidence that external exposure resulted in similar outcomes [34]. Therefore, the present finding that at least one upregulated metabolite, lauric acid, is larvicidal, is important. It is reasonable to conclude that the intricate balance between the plant and the larva through chemical interactions was affected by the Fukushima nuclear accident.

In addition to revealing the importance of plant-insect interactions in evaluating the biological effects of the Fukushima nuclear accident, this study opened new perspectives. Because ikarugamycin is likely produced by an endophytic bacterium, bacterial, fungal, or other microbial communities in plants and soil may play a role in amplifying the biological effects of low-dose radiation pollution.

#### **5. Conclusions**

We demonstrated within a reasonable concentration range (0.01 mg/g to 1 mg/g) that lauric acid is able to function as a toxicant for the pale grass blue butterfly at the leaf concentration (0.063 mg/g with radiation) by lowering metamorphosis rates and by causing growth retardation. Based on its TD50 and LD50 values (1.2 mg/g and 1.6 mg/g, respectively), lauric acid may be considered a mild larvicide. In the field, lauric acid probably acts as one of the larvicides in leaves in response to radiation exposure. Interpretations of alfuzosin results are not straightforward, but its relatively low TD50 and LD50 values (0.079 mg/g and 0.13 mg/g, respectively) imply that the alfuzosin-related compound may also be toxic, although it may be irrelevant in the field because of its low leaf concentration (1.6 ng/g with radiation). Because ikarugamycin is an antibiotic likely from endophytic bacteria, its drug efficacy on increasing the metamorphosis rates of larvae may be secondary; it may function to prevent the artificial diet from fungal and bacterial growth. As an extrapolation, ikarugamycin may function to protect leaves from fungi and bacteria under radiation stress. The case of ikarugamycin suggests a contribution of endophytic bacteria to the process of radiation-stress management in the plant.

In conclusion, the present results provide experimental evidence for the field effect hypothesis that concentration changes in radiation-induced metabolites, such as lauric acid, in the host plant leaves of the pale grass blue butterfly caused deterioration of the butterfly at the individual and population levels in radioactively polluted areas in Fukushima.

**Supplementary Materials:** The following file is available online at https://www.mdpi.com/article/ 10.3390/life12050615/s1, Table S1: Toxicological Output Data.

**Author Contributions:** Conceptualization, J.M.O.; methodology, J.M.O.; W.T., K.S. and A.M.; software, A.M. and J.M.O.; validation, J.M.O.; formal analysis and investigation, A.M.; resources, A.M., K.S. and W.T.; data curation, J.M.O.; writing—original draft preparation, J.M.O.; writing—review and editing, J.M.O., A.M., K.S. and W.T.; visualization, J.M.O. and A.M.; supervision, project administration, and funding acquisition, J.M.O. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Asahi Glass Foundation (Tokyo) and the basic fund from the University of the Ryukyus. The APC was funded by the basic fund from the University of the Ryukyus.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study and the source data are available in this article and in the Supplementary Materials.

**Acknowledgments:** We thank S. Gima (Center for Research Advancement and Collaboration, University of the Ryukyus) for the LC–MS analysis; Y. Yona, Y. Iraha, A. Hiyama, and R.D. Gurung for their technical assistance for preparing artificial diets; and other members of the BCPH Unit of Molecular Physiology for suggestions and technical help.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

#### **Appendix A**

To estimate the concentrations of the three metabolites of interest, lauric acid, alfuzosinrelated compound, and ikarugamycin in leaves, the peak area values for these metabolites and oxalic acid obtained in the previous GC–MS and LC–MS analyses were compared (Figure A1). Exact values are mentioned in the Section 2. For the alfuzosin-related compound, LC–MS analyses were newly performed in the present study in triplicate to estimate its concentration in leaves using an alfuzosin standard (Sigma–Aldrich) as a reference material (Figure A2). Leaf samples used for Figures A1 and A2 were identical.

We used a Shimadzu Prominence UFLC XR System (Kyoto, Japan) equipped with a Shimadzu solvent delivery unit LC-20ADXR and a Shimadzu autosampler SIL-20ACXR using a reverse-phase column Inertsil ODS-4 (2.1 mm × 150 mm) (GL Sciences, Tokyo, Japan). Mobile phase A was a 0.1% aqueous solution of formic acid, and mobile phase B was acetonitrile with a time program of its concentrations as follows: 20% (0 min) → 40% (10 min) → 98% (10.01–15 min) → 20% (15.01–23 min). The injection volume was 10 μL, and the flow rate was 0.2 mL/min.

A peak of the alfuzosin-related compound was obtained at 6.7 min from the leaf extract (Figure A2a). The alfuzosin standard also showed a peak at 6.7 min but with an additional peak at 2.1 min (Figure A2b). The latter peak was an impurity from methanol (Figure A2c). MS/MS analyses were also performed simultaneously to confirm the identities of these compounds (not shown).

**Figure A1.** Peak area values of oxalic acid and lauric acid in the targeted GC–MS analysis [49] (left) and peak area values of alfuzosin-related compound and ikarugamycin in the LC–MS analysis [49] (right). Box plots for lauric acid, alfuzosin, and ikarugamycin are also shown in Sakauchi et al. (2021) [49]. IR (shown in red) and NC (shown in green) indicate irradiated samples (*n* = 3) and nonirradiated control samples (*n* = 3), respectively. A single black dot represents the mean value of triplicate of a sample. FC indicates fold change of mean values from nonirradiated to irradiated samples. These plots were produced using MetaboAnalyst [96]. Asterisks indicate levels of statistical significance; \*, *p* < 0.05; \*\*, *p* < 0.01 (*t*-test).

**Figure A2.** Identification of the alfuzosin-related metabolite by HPLC. (**a**) Leaf extract. A peak at 6.74 min was observed. This is one of the triplicate results. The peak area value here is 224.17. (**b**) Alfuzosin standard (Sigma–Aldrich) 0.1 ng/mL in methanol. The peak at 6.69 min is attributed to alfuzosin, and the peak at 2.09 min is attributed to an impurity in methanol. The peak area value of alfuzosin, 199.36, is similar to that of the alfuzosin-related compound. (**c**) Methanol only. A peak at 2.12 min was observed, demonstrating impurity.

#### **Appendix B**

The exact numbers and percentages of individuals obtained after the feeding experiments are shown below for leaf and AD-FSW-135 (without any test additive) controls (Table A1), lauric acid (Table A2), alfuzosin (Table A3), and ikarugamycin (Table A4). Normalized eclosion rates (survival rates) (Table A5) and normalized normality rates (Table A6), used for Figure 5, are also shown. Further information can be found in Table S1.

**Table A1.** Number of individuals after rearing with live leaves or AD-FSW-135.

**Table A2.** Number of individuals after oral administration of lauric acid.

**Table A3.** Number of individuals after oral administration of alfuzosin.

**Table A4.** Number of individuals after oral administration of ikarugamycin.

**Table A5.** Normalized eclosion rates (survival rates).

NA: not applicable.

**Table A6.** Normalized normality rates.

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