The impact of three thioxothiazolidin compounds on trehalase activity and development of Spodoptera frugiperda larvae

Insects

The S. frugiperda, offered by the Zhejiang Academy of Agricultural Sciences (Zhejiang, Hangzhou), was subsequently reared by our team in the Key Laboratory of Animal Adaptation and Evolution, School of Life and Environmental Sciences, Hangzhou Normal University (Zhejiang, Hangzhou). The specific rearing conditions and methods were consistent with those of Pinto et al. (2019); Tang et al. (2024).

Thioxothiazolidin compounds

The experimental TRE inhibitors 1G, 2G, and 11G were provided by the PMDD laboratory of China Agricultural University (Beijing). To achieve a concentration of 2 × 10⁻³ mmol/mL, dissolve these inhibitors in 2% DMSO , referring to the minimum effective concentration of TRE inhibitors on S. frugiperda measured by Wang et al. (2022b); Tang et al. (2024). Detailed information on the molecular weights and formulas of the TRE inhibitor can be found in Table 1, and their structural formulas are depicted in Fig. 1.

Microinjection of trehalase inhibitors

Using Sutter Instrument (Novato, CA, USA) to convert glass capillaries into microinjection glass needles, and subsequently installed these glass needles into the injection joint of the Eppendorf TransferMan^®4r microinjection system and securely attached to the robotic arm. The S. frugiperda larvae were paralyzed on ice on the first day of the third instar. They were later then transferred to the loading platform of the microinjection system with a pointed brush and inserted 300 nL of the potential inhibitors into the thinner area between the second and third pairs of thoracic feet with needles compared to the control group treated with the equal volume of DMSO (2%). Treated larvae were reared in artificial climate boxes (Junyuan Experimental equipment store, Nantong, China).

Determination of trehalase activities

Samples were collected 48 h later for subsequent analyses after injecting 1G, 2G, 11G, and 2% DMSO. Approximately 15 larvae were collected randomly from each group and then mixed with 200 µL of phosphate-buffered saline (PBS; pH 7.0) for 30 min of sonication. The samples added 800 µL PBS were centrifuged at 1,000 × g at 4 °C for 20 min, followed by collection of approximately 350 µL of supernatant for another 60 min ultracentcentrifugation. The activity of soluble trehalase was determined with another 300 µL of the resulting supernatant, and the rest precipitate was mixed with 300 µL of PBS for subsequent determination about the activity of membrane-bound trehalase, following the Gglucose (Go) Assay Kit instructions (Lot No. SLCD8160, Sigma, St. Louis, MO, USA). Three biological replicates were performed for each group.

Determination of chitin content

Collecting larvae in Eppendorf (EP) tubes 48 h after treatment. Three biological replicates and three technical replicates were contained in each experiment. The samples, added with 500 µL of 6% KOH solution, were incubated at 80 °C for 90 min and then centrifuged at 12,000 × g for 20 min at 4 °C after 5 min of shaking. The resulting precipitate was resuspended in 200 µL McIlvaine’s buffer (Macklin, China). The chitin was hydrolyzed by adding 10 µL of chitinase from Streptomyces griseus (Sigma-Aldrich, USA), and the mixture was incubated at 37 °C and 150 rpm for 72 h in a shaker.

After completion of the hydrolysis reaction, the samples were centrifuged at 12,000 × g for 1 min at 25 ° C. The collecting supernatant (60 µL) was added with an equal volume of sodium borate. Stirring and incubating the mixture in a water bath (99.9 °C) for 10 min, followed by cooling to 25 °C. A total of 600 µL of 1 × DMAB was added into the mixture for later incubation in a water bath at 37 °C for 20 min. Sample (200 µL) were added to the wells of enzyme-linked plates for determination of the absorbance at 585 nm. The standard curve was drawn by measuring the absorbance value of the standard solution for calculating the chitin concentration. This procedure was referred to the determination of chitin content by Yao et al. (2010).

Determination of chitinase activities

Collecting larvae in EP tubes after 48 h of treatment, with three biological replicates and three technical replicates for each experiment. The tissue (approximately 0.1 g) was homogenized in one mL of ice-cold extraction solution for 20 min centrifugation at 10,000 × g at 4 °C. The 400 µL of supernatant was collected on ice for chitinase activity testing with a chitinase reagent kit (Suzhou Keming Biotechnology Co., Ltd). Chitinase activity (mg/h/g fresh weight) was calculated according to the formula of Tang et al. (2024): ${\displaystyle \text{Chitinase activity}=\left(\Delta A+0.2753\right)÷6.4108\times V_{\mathrm{total reaction system}}÷\left(V_{\mathrm{sample}}÷V_{\mathrm{total sample}}\times W\right)÷T\times 10=3.899\times \left(\Delta A+0.2753\right)÷W}$

(A, absorbance; ΔA =A_{measured value} - A_{control value}; W, sample weight).

Observation of the feeding, growth, and development of treated S. frugiperda larvae

The initial treatment numbers for the 2% DMSO, 1G, 2G, and 11G groups were all 90. Larvae were selected and fed separately every 2 days with an artificial diet in a feeding box on the first day of the third instar. The larval shells were used for determination of the larval age and dislodged after data recording to avoid confusion. Every 24 h, the weight, emergence, and mortality rate of the larvae were recorded on a daily basis and record the remaining number of larvae for each treatment on a record sheet. Calculate the survival rate based on the recorded data and observe the phenotype of larvae after pupation.The formula for calculating survival rate is as follows: ${\displaystyle \text{Survival rate at different ages}=\text{number of survivors at that age}÷\text{number of initial treatments}\times 100\text{\%}.}$ Larvae were observed daily to record their growth and development since the first day after injecting TRE inhibitors. The weights of larvae were measured, as well as the survival, deformity, pupation, and emergence rates were recorded when pupae formed. The images of abnormal larvae were taken with a Canon EOS 50D to record phenotypic changes, and categorized into lethal and non-lethal phenotypes based on the lethality of the observed abnormalities.

Quantification and statistical analyses

The IBM SPSS Statistics 20 software (SPSS v. 20, Armonk, NY, USA) was used to perform statistical analyses. The differences between control and treatment groups were compared by one-way analysis of variance (ANOVA) or Student’s t-test. Post-hoc tests were performed with the Tukey. Statistical significance was set at p < 0.05. The data were expressed as mean ± standard error. After data analysis, GraphPad Prism software, version 8.0, was used to generate graphs. Data analysis methods were based on those previously described by Tang et al. (2024).

Thioxothiazolidin compounds inhibit trehalase activity in S. frugiperda larvae

Thioxothiazolidin compounds (1G, 2G, and 11G) were injected into the third instar larvae of S. frugiperda at a concentration of 2 × 10⁻³ mmol/mL, and samples were analyzed for the detection of TRE activity at 24 h. The results showed that 1G, 2G, and 11G inhibited TRE2 activity compared to the control group, but had no significant effect on TRE1 (Figs. 2A, 2C). After a 48-hour injection period, both TRE1 and TRE2 activities were downregulated in the larvae treated with three inhibitors (Figs. 2B, 2D). Of the tested compounds, 1G and 2G displayed the most pronounced inhibitory effects on TRE2 activity, suggesting their potential as strong inhibitors (Fig. 2D).

Thioxothiazolidin compounds affect chitin content and chitinase activity in S. frugiperda larvae

The chitin content in the 11G group was significantly lower than that in the control group, whereas there was no significant difference between the control and 1G and 2G groups after 24 h injection (Fig. 3A). Compounds 2G and 11G significantly reduced the chitin content of S. frugiperda larvae injecting 48 h later, whereas 1G had no significant effect (Fig. 3B). The activity of chitinase was significantly upregulated in the 1G group treated 24 h later. In contrast, at the 48-hour injection point, the chitinase activity of 1G and 11G groups decreased sharply, while that of 2G group increased significantly compared with the control group (Figs. 3C, 3D).

Thioxothiazolidin compounds affect the development of S. frugiperda larvae

No significant differences were observed in the developmental time of the fourth, fifth, sixth instar larvae, as well as the pupal stage. Nevertheless, within the experimental cohorts, the pre-pupal larval stage exhibited a marked extension in development time specifically within the 1G, 2G, and 11G groups. Throughout the transformation of S. frugiperda larvae from the third instar to the adult stage, there was no statistically significant disparity in the cumulative growth and development period when comparing larvae subjected to inhibitors with the control group. However, the total developmental duration was significantly different between 1G and 2G treatments (Table 2).

Larval weight changes were assessed from the fourth instar to the pupal stage in S. frugiperda. The figure indicated that larval weight in the fourth instar was observably different between 1G and 11G treatments, but none of the inhibitors altered the larval weight compared with the control (Fig. 4A). On the contrary, there was a significant increase in larval weight at the sixth stage in the inhibitor treatment groups (Fig. 4C). The weight of the 2G and 11G groups was significantly higher than that of the other two groups in the fifth instar (Fig. 4B). Larval weight at the pre-pupal and pupal stage, treated with 1G and 2G, was significantly higher than in the 11G and control groups (Figs. 4D, 4E).

Thioxothiazolidin compounds decrease the survival rate of S. frugiperda larvae

The survival rate underwent a decline from the third instar to the pre-pupal stage, culminating in a 67.78% survival rate in the control group. Subsequently, a notable decrease in survival rate was observed from the pre-pupal stage to adulthood, yielding a 54.44% survival rate among adult moths (Fig. 5A).The death of larvae in the 2G group mainly happened between the sixth instar and emergency stage, resulting in a survival rate of 32.22%, less than half compared to that at initial treatment (Fig. 5A). It is noteworthy that the larvae belonging to the 1G group demonstrated significantly high mortality rates throughout their developmental stages, spanning from the third instar to the pupal stage. Consequently, at the pupal stage, the survival rate of these larvae amounted to a mere 34.44%. Furthermore, among the larvae in the 1G group that successfully progressed into adult moths, they constituted a mere 28.89% of the initially treated larvae, which is a notably lower proportion compared to that observed in the 2G, 11G, and control groups. (Fig. 5A). However, the final fatality rate of the 11G group reached 60%, higher than that of the control group, indicating there was no remarkable effect on larvae mortality in the 11G group (Fig. 5A). The pupation rate exhibited significant variations in the 1G group compared to the other groups, with the exception of the 2G group. Conversely, no discernible differences were observed among the remaining groups (Fig. 5B). Despite the increased larval mortality observed in the 1G and 2G groups, no statistically significant differences were detected among the groups in terms of emergence rates (Fig. 5C).

Thioxothiazolidin compounds increase the rate of abnormal phenotypes and deformity in S. frugiperda larvae

In all treatment groups, from the fourth instar stage to the pupal stage, observations were made revealing abnormal phenotypes of larvae (Fig. 6). Furthermore, the aberration rates were notably elevated in comparison to those observed in the control group (Table 3). Abnormal phenotypes, compared to normally developing larvae, occurred during the development in groups 1G, 2G, and 11G, resulting in what was termed an abnormal non-lethal phenotype (Fig. 6A). In particular, larvae in the 1G group exhibited a ruptured outer epidermis with exposed contents at the fourth instar, accompanied by a darkening abdomen at the pre-pupal period. A similar condition, manifesting as distinct invagination or folding of the outer epidermis, was also observed in the fourth instar larvae of group 2G (Fig. 6B). This phenotype of death due to molting failure was considered as an abnormal lethal phenotype.