Molecular basis for the co-evolution of thermophysiology and pigmentation in Habrobracon hebetor (Hymenoptera: Braconidae)

doi:10.21203/rs.3.rs-4897874/v1

Download PDF

Research Article

Molecular basis for the co-evolution of thermophysiology and pigmentation in Habrobracon hebetor (Hymenoptera: Braconidae)

https://doi.org/10.21203/rs.3.rs-4897874/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

In the context of global warming and increasing extreme heat, insect responses to temperature include physiological and biochemical changes such as changes in body color. One challenge in biology is to integrate diverse mechanisms of insect plasticity underlying responses to temperature. Life history and trait trade-offs provide a framework for investigating this issue as it relates to the synergistic optimization of related traits. High temperatures during the pupal stage can induce reductions in melanin levels and affect the body color of Habrobracon hebetor adults, with these changes accompanied by enhanced longevity at the expense of fecundity. In our study, we used transcriptome and metabolome sequencing, antioxidant enzyme (SOD, CAT, POD) assays, and additions of exogenous antioxidants, and we found three important relationships: (1) The establishment of heat tolerance in this parasitoid involved responses of antioxidant systems, biogenic amines, and heat shock protein genes, and this tolerance was induced by heat experienced in the pupal stage, with results in the adult stage. (2) A trade-off existed between survival and reproduction based on energy and resource allocation under heat stress. This relationship was disrupted by feeding adults exogenous antioxidants (glutathione and melatonin), as demonstrated by a simultaneous elevation of survival and reproduction at high temperatures. (3) Parasitoid melanosis-related genes (DDC and AANAT) both participated in the regulation of melatonin synthesis. These results enrich our understanding of the theory of plastic body color differentiation in insects and may provide useful insights for the conservation of parasitoids in tropical regions.

Thermophysiology

body color differentiation

parasitic wasps

life history trade-offs

plasticity

Global climate change is increasing the magnitude, frequency, and duration of heat events, with significant impacts on organisms and ecosystems (González-Tokman et al. 2020). Insects are an important component of biodiversity, providing services to the entire environment, including pest control, pollination and nutrient recycling (Castexae t al. 2018; Lyu et al. 2023). The response of insects to high temperatures is therefore an important ecological issue in the context of global climate change. Insects are known to respond to temperature change in various ways, some of which have been empirically tested, involving enzymatic reactions, metabolic and hormonal changes, cellular stress responses, and morphological changes (e.g., changing body color) (Roulin 2014; Colinet et al. 2015; Ma et al. 2021). High temperatures accelerate the metabolic rate of insects, which subsequently leads to higher oxygen consumption and the production of more free radicals (also known as reactive oxygen species, ROS), such as the O²⁻, H₂O₂, and -OH, which cause oxidative damage to lipids, proteins, and nucleic acids in organisms, potentially affecting insect activities such biological control (Hance et al. 2007).

The antioxidant system response (antioxidant enzyme activity and antioxidant level) mitigates oxidative stress damage in insects and helps maintain normal physiological functions under a certain degree of heat stress (González-Tokman et al. 2020). Superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) are key enzymes in the enzymatic defense system of insects at high temperatures (Zhao et al. 2020; Ning et al. 2024), and these enzymes are in a dynamic mutual equilibrium to prevent the toxic effects of free radicals (González-Tokman et al. 2020). SOD catalyzes the disproportionation reaction of O²⁻ that generates H₂O₂, which can be decomposed by CAT and POD (Zhao et al. 2020).

In general, compounds with antioxidant properties can exert their antioxidant effects either through direct neutralization by electron donation, by promoting the expression of endogenous antioxidant enzymes (agents), and by a balanced effect of both pathways (Vaccaro et al. 2020; Ghani et al. 2024). Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are two important coenzymes in the cells of living organisms, and their reduced forms, NADH and NADPH, relate to oxidative stress (Wang et al. 2014). NADP directly increases glutathione (GSH) content and CAT activity, while NADPH is a coenzyme of GSH reductase (Ghani et al. 2024). GSH, ascorbic acid (ASA), melatonin, pyruvate, and N-acetylcysteine (NAC) all bind directly to free radicals through nonenzymatic reactions (Jena et al. 2013; Tauffenberger et al. 2019; Vaccaro et al. 2020). In addition, GSH and ASA serve as substrates for POD-specific catalysis of H₂O₂, while melatonin increases GSH content and enhances SOD, CAT, and glutathione peroxidase activity (Jena et al. 2013; Altaf et al. 2024). Similar to NADPH, H + supplied by NAC promotes the synthesis of reduced GSH (Wang et al. 2014), and pyruvic acid enhances the citric acid cycle, shifting metabolism to the pentose phosphate bypass and producing NADPH, thus indirectly increasing the capacity of the GSH antioxidant system (Tauffenberger et al. 2019). In addition, trehalose is a typical stress metabolite that can form a unique protective film on the cell surface under harsh environmental conditions such as high temperatures, effectively protecting the structures of biomolecules (Kandror et al. 2002).

The establishment of heat tolerance is also associated with the synthesis of biogenic amines and the expression of heat shock protein genes (Armstrong and Robertson 2006; Xiong et al. 2024). The insect heat stress response begins with the rapid release of biogenic amines such as octopamine, dopamine, and serotonin (Armstrong and Robertson 2006; González-Tokman et al. 2020), which is accompanied by an increase in the activity or concentration of specific biogenic amine catalytic enzymes (tyrosine hydroxylase, dopa decarboxylase, etc.), their precursors, and their metabolites (González-Tokman et al. 2020). For example, octopamine modulates physiological processes such as K + conductance and Na+/K + ATPase in locust tissues, thereby exerting neurofunctional thermoprotection (Armstrong and Robertson 2006). Heat shock proteins (HSPs) are synthesized when cells are stimulated by high temperature, and many studies have found that the levels of HSPs are positively correlated with the survival of insects under heat stress (Xiong et al. 2024). These proteins induce the formation of spatial structures in peptide chains, remove damaged peptide chains that are unable to fold correctly, and play a role in regulating the activity of Na+-K+-ATPase and promoting intracellular glycogen isomerization and gluconeogenesis (González-Tokman et al. 2020). Also, body color change in insects helps them adapt to changes in environmental temperatures (Roulin 2014); for example, the thermal melanization hypothesis suggests that highly reflective (light) coloration facilitates the organism's ability to reduce thermal stress by reflecting radiant heat in hotter environments (Clusella-Trullas et al. 2008). The molecular mechanisms driving biopigmentation have been intensively studied in Drosophila, where the pigment synthesis pathway involves the genes encoding enzymes such as dopa decarboxylase, the yellow enzyme family, and arylalkylamine-N-acetyltransferase, and also genes for pigment precursors such as dopa, dopamine, and N-acetyl dopamine (Kronforst et al. 2012). Moreover, in addition to the thermodynamic benefits that body color can provide, at the genetic level, genes regulating pigmentation are thought to have pleiotropic effects that can limit co-evolution of insects, especially ties to immune defense and other fitness-related traits (Roulin 2014). This issue has not yet been investigated in any species of parasitoids. Also of broad interest is the development of a framework that would integrate the mechanisms of multiple plastic responses to temperature in insects, and life history and trait trade-offs and provide a framework for studying them in relation to the synergistic optimization of related traits (Kvist et al. 2013).

In a previous study, we found that unfavorable high temperatures (32°C, a level inhibiting population survival) if sustained for 3 d during the pupal stage induced almost complete fading of melanin in Habrobracon hebetor (Hymenoptera: Braconidae) adults, especially on the abdomen (Wang et al. 2024). This color change was also associated with an increase in heat tolerance (longevity), but at the expense of fecundity (Wang et al. 2024). This parasitoid wasp is present in Hainan Island, China, where the climate is hot and humid throughout the year, and plays an important role as a larval ectoparasitoid wasp in the integrated control of the important leaf-feeding pests of palm plants, Opisina arenosella (Lepidoptera: Oecophoridae) and Tirathaba rufivena (Lepidoptera: Pyralidae) (Yan et al. 2023). In the tropics, a small increase in the already high baseline temperature may cause a dramatic increase in the metabolic rate of insects, making tropical insects more vulnerable to the effects of global warming (Hance et al., 2007), which also means that selection for heat-tolerant strains of key biological control agents may be crucial for their continued successful in agents pests in the tropics (Castexa et al. 2018).

In this study, we used transcriptome and metabolome sequencing, antioxidant enzyme (SOD, CAT, POD) assays, and exogenous antioxidant supplementation to explore the following issues: 1) the roles of antioxidant systems, biogenic amines, and heat shock protein genes in heat tolerance in parasitoics (and that this tolerance is induced by heat in the pupal stage and transmitted to the adult stage); 2) how the re-allocation of energy and resources under heat stress causes a trade-off between survival and reproduction (and how exogenous antioxidant supplementation can break this trade-off and simultaneously enhance survival and reproduction under high temperature); and 3) how genes related to melanin deposition in parasitoid wasps are concurrently involved in the regulation of antioxidant (melatonin) synthesis. The results of this study enrich understanding of plastic body color differentiation in insects and may provide a useful reference for the conservation of parasitoid wasps under global warming and the resulting occurrence of extreme high temperature events.

2.1 Insect rearing

The H. hebetor colony was reared following the protocol of Wang et al. (2024) at 27 to 28°C, 65–75% RH, and 16:8, L:D photoperiod

2.2 Sample preparation for transcriptional and metabolic profiling

In a previous study, we determined the conditions that induced the complete loss of melanin in the abdomen of H. hebetor adults to be 32°C, during the pupal stage, for more than 3 d (Wang et al. 2024). H. hebetor pupae used for transcriptional and metabolic analysis were selected based on the degree of loss of melanin in the abdomen of the parasitoids and placed in three categories: (1) NT: 28℃ for 3 d that yields85% melanin, (2) HT1: 32℃ for 1 d, followed by 28℃ for 2 d that yields 61% melanin, and (3) HT3: 32℃ for 3 d that yields 3% melanin). For each treatment at least 300 pupae were used, and the treated pupae were frozen immediately after treatment in liquid nitrogen and stored at -80℃. Transcriptional and metabolome analyses were performed with three and five biological replicates, respectively. However, only the three metabolome samples with the bests reproducibility were retained for further analysis.

2.3 RNA sequencing, de novo assembly, and annotation

Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Takara Inc., Kyoto, Japan) for 45 min at 37℃ to remove residual DNA. RNA was quantified with a Nanodrop 2000 device (Thermo Scientific, Waltham, MA, USA), and RNA integrity number was assessed with the RNA 6000 Nano Kit (5067 − 1511; Agilent Technologies Inc, Santa Clara, CA, USA) on a Bioanalyzer system (Agilent Technologies Inc). mRNA with poly A tails was obtained using a NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Beijing, China). Samples with a total RNA concentration > 10 ng/µl were used for library preparation. The first strand of cDNA was synthesized using RNA as template with a TruSeq RNA sample prep kit v2 (Illumina, San Diego, CA, USA) and sequenced by Biomarker Technologies (Beijing, China) on an Illumina HiSeq^™ 2000 platform. After cleaning up the raw reads and discarding low quality reads (Q30 > 0.85 or reads with > 10% unknown bases), clean reads are assembled into transcripts based on Trinity (Yoon et al., 2014). Sequenced reads were first cut into shorter fragments (K-mer), then these small fragments were extended into longer fragments (Contig). The overlap among fragments was used to obtain a collection of fragments (Component), and finally, De Bruijn plots and sequenced reads information were combined to identify transcript sequences in each fragment collection separately. To annotate the sequences obtained by de novo assembly, BLASTX was used to align the assembled unigenes with the Hymenoptera protein dataset of the Kyoto Encyclopedia of Genes and Genomes (KEGG) database by selecting the BLAST parameter E-value ≤ 1e-5 and the HMMER parameter E-value ≤ 1e-10. Unigene's KEGG Orthology results in KEGG were obtained using KOBAS (Chen et al. 2011). The reads obtained from sequencing were compared with the Unigene library using Bowtie (Langmead et al. 2009), and based on the results of the comparison, the expression level was estimated in combination with RSEM (Dewey & Li 2011). FPKM values were utilized to indicate the expression abundance of the corresponding Unigenes. To reduce background noise and focus on genes that are more important for biological impact, we excluded genes that did not have an FPKM value greater than 10 in any of the three temperature models.

2.4 Measurement of metabolites

Measurement of metabolites was also done by Biomarker Technologies (Beijing, China). Pupal samples (50 mg each) were vortex-mixed with 1000 µL of an extraction solution containing an internal standard (methanol acetonitrile water v/v = 2:2:1, internal standard concentration 20 mg/L) for 30 s. Subsequently, steel beads were added, and the sample was processed with a grinder (45 Hz) for 10 min and then ultrasonicated for 10 min in an ice-water bath. A liquid-mass spectrometry system was used for metabolomics analysis of the samples. This analysis consisted of an ultra-high performance liquid chromatography (Acquity I-Class PLUS, Waters, Milford, Connecticut, USA) coupled with a tandem high-resolution mass spectrometer (Xevo G2-XS QTOF, Waters). The columns used were Acquity UPLC HSS T3, 1.8 µm 2.1*100 mm, Waters. The injection volume was 1 ul, and the mobile phases for positive and negative ion mode were 0.1% formic acid aqueous solution and a 0.1% formic acid acetonitrile aqueous solution. Primary and secondary mass spectrometry data acquisition was performed based on the MSe mode under the control of MassLynx V4.2 (Waters). In each data acquisition cycle, dual-channel data acquisition was performed simultaneously for low and high collision energies. The low collision energy was 2 V, the high collision energy interval was 10 ~ 40 V, and the scanning frequency for both was 0.2 s. The ESI ion source parameters were as follows: (1) capillary voltage: 2500 V (positive ion mode) or -2000 V (negative ion mode); (2) conical bore electrode at 30 V; (3) ion source temperature of 100 ℃; (4) temperature of the de-solventizing gas of 500 ℃; and (5) the flow rate of the reverse gas was 50 L/h. The ion source was operated by the Progen and the ESI ion source was operated by the ESI ion source. The metabolite peaks were extracted and aligned by Progenesis QI software (Waters), and the metabolite peaks were annotated by combining with the METLIN database and the KEGG database, and the tolerance range of the peaks was configured to have a retention time of 0.5 min and a mass-to-charge ratio of 50 ppm.

2.5 Quantitative real-time PCR

We determined the relative expression levels of nine Unigenes at room temperature (NT: the pupal stage exposed to 28°C for 3 d) and high temperature (HT3: the pupal stage exposed to 32°C for 3 d) to verify the accuracy of the transcriptome results. Samples for QRTPCR were prepared at the same time as those used for transcriptome sequencing. RNA was extracted, and cDNA was synthesized as described in section 2.3 above. qRT-PCR was performed in an AriaMx real-time PCR system (Agilent Technologies Inc.) using a 2×T5 Fast qPCR Mix kit (SYBR Green I, Beijing Tsingke Biotech Co., Ltd, China). Reactions were carried out at 95°C for 2 min; followed by a 40 cycles of three-step PCR for 10 s at 95°C, 15 s at 60°C, and 20 s at 72°C. The final lysis phase lasted 5 s at 95°C, 1 min at 60°C, and a warm-up of 0.11°C/s at 95°C. RNA levels of target genes were normalized to the level of microtubule protein mRNA and relative concentrations were determined using the 2^-ΔΔCt method. A stabley expressed mitochondrial 28S ribosomal protein S24 (MRP S24) gene was selected as the reference gene. To verify the specificity of amplification, dissociation curves were included at the end of each qPCR run at 60 to 95°C. All primers used for qRTPCR in this study are listed in Supplementary Table 1.

2.6 Antioxidant enzyme activities

To measure the antioxidant enzyme activities of the black and yellow-type adults of H. hebetor, larvae were reared at 28°C until the pupal stage and then the pupae were divided into two groups and were either reared as pupae at 28°C (= the pupal control treatment [PC]) or 32°C (the pupal high-temperature treatment [PH]), until emergence of adult parasitoids. The H. hebetor adults that developed from the 28°C PC group were divided into two subgroups, one held at 34°C for 1 day (AC1) or for three days (AC3), during which adults were provided with a 15% (w/v) honey solution that was replaced daily. Those wasps that had developed from pupae reared at 32°C were similarly treated the same way as the 28°C PC group, being marked as AH1 and AH3. At least 500 individuals (pupae or adults) were used for each of the six treatments. There were three replicates for each treatment.

Kits from Nanjing Jiancheng Biological Engineering Research Institute Co. (Nanjing, China) were used to determine the protein concentrations and the activity levels of superoxide dismutase, catalase, and peroxidase. Each H. hebetor adult was weighed (fresh weight) using analytical scales. A saline solution (0.9% NaCl) was then added as per the following ratio: weight of the parasitoid (mg) : volume of salt solution (ml) = 1 mg: 9 ml. Then the parasitoid tissues were crushed using a manual grinder, and the homogenate was prepared in an ice water bath and centrifuged at 2500–3000 rpm for 10 min. We then used the supernatant and analyzed it at a wavelength of 595 nm and an optical path 1 cm long for protein. Similarly, we used values of 550 nm and 1 cm pathway for SOD, 405 nm and 0.5 cm for CAT, and 420 nm and 1 cm for POD. Double-distilled water was used to set zero and then we measured the absorbance OD value of each tube with a microplate reader (ThermoFisher Scientific 51119180, Thermo Fisher Technology, China Co. Ltd).

2.7 Antioxidant supplementation

To prepare the supplemental antioxidant solutions, 10 mg each of melatonin and glutathione were weighed out using an analytical scale and dissolved in separate 10 mL aliquots of an aqueous solution at 40°C. These solutions – a 1 mg/mL melatonin solution and a similar solution of glutathione were then diluted 100-fold with additional water to 10 µg/mL. We then collected 100 mL of each of these diluted solutions and added 5 mg of honey to each, as well as to 100 mL of the original aqueous solution (as the control). Ninety 24 h-old H. hebetor females (that had successfully mated) were placed individually in glass cylinders (10 cm dia × 20 cm long) and then divided into three equal groups. Thirty cylinders for each group were then plugged with sterile cotton soaked in either the melatonin, glutathione, or aqueous solutions. These cylinders with wasps and solutions were then held at 34°C in a climatic chamber. Parasitoid females were each provided with 30 hosts (O. arenosella late instar larvae of uniform size). Solutions and hosts were replaced daily, and the process repeated until each parasitoid died. Exposed hosts from each daily exposure were observed under a microscope, and the number of unhatched eggs and larvae were counted in each host. We also counted the number of paralyzed, moribund hosts (ones without motor response to a light touch with a brush ), and we recorded the longevity of the parasitoid wasps.

2.8 Data analysis

For our statistical analyses we used RStudio (R v.3.6.3; R Foundation for Statistical Computing, Vienna, Austria) with the packages lme4 (Bates et al. 2015), emmeans (Lenth et al. 2018), ggeffects (Lüdecke, 2018a), sjPlot (Lüdecke, 2018b), and DHARMa (Hartig, 2019).

Metabolic profiles in positive and negative ion scanning patterns were combined and normalized by generalized logarithmic transformation for mRNA expression (FPKM values) and metabolite content. Principal component analysis was used to determine the degree of intra- and inter-group variation of transcriptomic and metabolomic samples under different temperature treatments. Clustered heatmaps were used to show trends in the expression of mRNAs (FPKM values) and metabolites between temperature patterns, using k-means clustering with Euclidean distance.

Data were tested for normality using the Shapiro-Wilk test, homogeneity of variances was assessed with Levene's Test, and outliers were evaluated via a box plot diagram. We removed some outliers before statistical analysis. One-way ANOVA was used to determine the main effect of temperature patterns on RNA-seq gene expression (FPKM values), relative mRNA expression, metabolite content, and antioxidant enzyme activity, and differences in life history parameters (longevity, fecundity, and number of paralyzed hosts) across antioxidant supplementation treatments, followed by Tukey’s test for post hoc comparisons (α = 0.05). Non-normally distributed data were analyzed using a gamma-distributed GLM, and post hoc comparisons were done using the Tukey’s test (α = 0.05). Before using GLMs, we ran the diagnostic tool provided with the DHARMa package (Hartig, 2019) to determine whether the model's residuals were either uniformly distributed or overdispersed.

3.1 Principal component analysis of the transcriptome and metabolome

The melanin content of the abdomen of H. hebetor adults was closely related to the temperature pattern it experienced during the pupal stage, with the melanin percentage decreasing by 28.2% and 96.5% under the 1 d (HT1) and 3 d (HT3) treatments at 32°C, respectively, compared with the 28°C treatment (NT). PCA analyses revealed good agreement between intragroup duplicates of the transcriptome and metabolome of H. hebetor pupal tissues across the three temperature patterns, with the first two principal components accounting for 85.5% (transcriptome) and 83.3% (metabolome) of the total variance, respectively. For the transcriptome (Fig. 1A), the samples under NT treatment and NH1 treatment were not highly separated, indicating that the physiological state of pupal tissues embodied in the expression at the mRNA level was similar, but these two samples were highly separated from the samples under HT3 treatment. Also, the metabolome samples under the three temperature patterns were highly separated from each other, which proved that the metabolite species and contents in pupal tissues had a large tendency to change between temperature patterns (Fig. 1B).

3.2. Heat stress activates endogenous antioxidant synthesis pathways

The glutathione metabolism pathway (ko00480) was activated in H. hebetor pupal tissues after heat stress. Both L-glutamate and L-cysteine are precursors for reduced glutathione synthesis in the pathway, and both are generated by glutamate-cysteine ligase catalytic subunit (GCLC) (FPKM: NT = 10.72; HT1 = 10.97; HT3 = 20.09) (F₂ = 32.106, P = 0.001) to produce L-gamma-Glutamylcysteine, which is then catalyzed by glutathione synthase to synthesize Glutathione. Compared with the control (28°C for 3 days), both L-glutamate and glutathione contents increased significantly and the L-Cysteine content declined strongly after 3 days of heat stress (32℃). Also, oxidized glutathione could be converted to glutathione by phospholipid-hydroperoxide glutathione peroxidase (GPX4), as evidenced by a significant up-regulation of GPX4 expression in the 32°C-3d treatment (FPKM: NT = 4.55.72; HT1 = 3.31; HT3 = 27.93) (F₂ = 17.516, P = 0.003). Also, there was a significant reduction in oxidized glutathione under prolonged heat stress duration (NT = 13726.011 ; HT1 = 7318.364; HT3 = 2997.023) (F₂ = 54.859, P = 0.000) (Fig. 2A and B).

In the starch and sucrose metabolism pathway (ko00500), we focused on glycogen degradation and trehalose synthesis (Fig. 2C and D). Glycogen can generate D-Glucose 6-phosphate (D-Glucose-6P) in the presence of hexokinase (HK), D-Glucose-6P is then catalyzed by phosphoglucomutase (PGM) to generate D-Glucose 1-phosphate (D-Glucose-1P), followed by D-Glucose-1P. Then, uridine diphosphate (UDP)-glucose was catalyzed by trehalose 6-phosphate synthase/phosphatase (TPS) to produce Trehalose 6-phosphate and Trehalose in two sequential steps. Compared with the control, the expression of genes encoding HK (FPKM: NT = 66.00; HT1 = 58.36; HT3 = 233.61) (F₂ = 219.048, P = 0.000), PGM (FPKM: NT = 19.77; HT1 = 29.42; HT3 = 84.12) (F₂ = 134.701, P = 0.000), and TPS (FPKM: NT = 42.91; HT1 = 36.24; HT3 = 262.24) (F₂ = 388.338, P = 0.000) increased significantly in the 32°C-3 day treatment, accompanied by a significant increase in D-Glucose-6P (NT = 487.00; HT1 = 2031.467; HT3 = 15371.454) (F₂ = 4501.562, P = 0.000) and D-Glucose-1P (NT = 383.76; HT1 = 2031.467; HT3 = 2220.103) (F₂ = 348.236, P = 0.000). However, there was a significant decrease in the glycogen content (NT = 730.845; HT1 = 557.09; HT3 = 459.62) (F₂ = 47.141, P = 0.000), suggesting that the conversion of glycogen to trehalose occurred in the pupal tissues after heat stress.

In the nicotinate and nicotinamide metabolism pathway (ko00760), our focus was on nicotinamide adenine dinucleotide (NAD) synthesis (Fig. 2E and F). In the temperature pattern change for the treatments NT-HT1-HT3, the level of the five precursors of NAD. (1) Nicotinamide-beta-riboside (NR) (NT = 416.40; HT1 = 616.28; HT3 = 680.33) (F₂ = 45.531, P = 0.000) and (2) Nicotinate D-ribonucleotide (NT = 0.03; HT1 = 2.06; HT3 = 118.57) (F₂ = 2915.553, P = 0.000), gradually increased, but the level of (3) Nicotinate D-ribonucleoside (NT = 588.58; HT1 = 355.06; HT3 = 323.74) (F₂ = 211.024, P = 0.000) and (4) Deamino-NAD+ (NT = 588.58; HT1 = 355.06; HT3 = 323.74) (F₂ = 56.364, P = 0.000) gradually decreased. Meanwhile, the level of (5) Nicotinamide D-ribonucleotide (NMN) first decreased and then rebounded back to the initial level (NT = 164.18; HT1 = 71.14; HT3 = 159.63) (GLM gamma, df = 2, P < 0.001). Also, the level of NAD (NT = 4382.72 ; HT1 = 4594.82; HT3 = 3422.99) (F₂ = 45.531, P = 0.000) and nicotinamide adenine dinucleotide phosphate (NADP) (NT = 383.42; HT1 = 375.02; HT3 = 559.57) (F₂ = 8.771, P = 0.017) showed an opposite trend, whereas the contents of the reduced forms of these two coenzymes, i.e., NADH (NT = 330.99; HT1 = 550.25; HT3 = 223.85) (F₂ = 26.900, P = 0.001) and NADPH (NT = 178.24; HT1 = 138.83; HT3 = 163.09) (F₂ = 5.591, P = 0.043), showed an opposite trend between NT-HT1, and later both returned to their initial levels in the HT3 treatment.

In addition, all four annotated metabolites were involved in the insect antioxidant response and acted as positive promoters (Fig. 3), namely pyruvic acid (Fig. 3A), NAC analog (S-[2-(N7-Guanyl)ethyl]-N-acetyl-L-cysteine) (Fig. 3B), ASA analog (ascorbic acid 2-sulfate) (Fig. 3C), and octopamine (Fig. 3D), all of which were highly expressed in the NHT3 treatment. Also, two of the genes encoding the protein lethal(2) were down-regulated (Fig. 3E and F), while one other such gene (Fig. 3G) was up-regulated under heat stress (32℃ for 3 d). All of the genes encoding the heat shock protein 68-like (hsp 68-like) (Fig. 3H and I) were significantly up-regulated.

3.2 Simultaneous involvement of DDC and AANAT encoding genes in melanin and melatonin synthesis

Ecdysone content was first unchanged and then significantly reduced in all three (NT-HT1-HT3) treatments (NT = 31183.52; HT1 = 33730.12; HT3 = 12682.25) (GLM gamma, df = 2, P < 0.001) (Fig. 4A and B), whereas the mRNA levels of genes annotated to the JH Ⅲ synthesis pathway were, overall, highly expressed in HT3 treatments. In addition, the precursors of the previous step of JH Ⅲ synthesis, i.e., JH Ⅲ acid (NT = 296.59; HT1 = 11061.62; HT3 = 426.25) (F₂ = 1262.833, P = 0.000) and methyl farnesoate (NT = 276.50; HT1 = 129.33; HT3 = 121.73) (F₂ = 1262.833, P = 0.000), the level of the former first increased and then significantly decreased. This lower level, however, remained higher than the initial level, while the level of the latter decreased significantly (Fig. 4C and D).

The synthesis of eumelanin in the pathway (tyrosine metabolism, ko00350) (Fig. 5A) begins with the conversion of tyrosine to L-dopa, a step catalyzed by tyrosinase (TYR) and 3-monooxygenase (TH), as follows. (1) Part of L-dopa continues to be catalyzed by TYR to generate dopaquinone, which is followed by two steps to produce leucodopachrome and L-dopachrome sequentially. TYR can also directly catalyze Tyrosine to generate dopaquinone, while dopaquinone can be combined with leucodopachrome and then converted back to L-dopa. (i) A portion of L-dopachrome is converted to 5,6-dihydroxyindole (5,6-DHI), which is subsequently catalyzed by TYR to produce indole-5,6-quinone and finally eumelanin. (ii) Part of the L-dopachrome was catalyzed by dopachrome tautomerase (DCE) to generate 5,6-dihydroxyindole-2-carboxylate (DHICA), which was followed by tyrosinase-related protein 1 (TYRP1) to produce 5,6-indolequinone-2-carboxylic acid (5,6-IQCA) and then eumelanin. (2) The other part of L-dopa is catalyzed by dopa decarboxylase (DDC) to produce dopamine, followed by arylalkylamine-N-acetyltransferase (AANAT) to produce N-acetyl dopamine (NADA), which is the melatonin precursor of the colorless scleroprotein in the insect epidermis (Kronforst et al. 2012).

Melatonin synthesis (tryptophan metabolism, ko00380) (Fig. 5B) begins with the tryptophan 5-monooxygenase (TPH1/2) catalyzation o of tryptophan to 5-hydroxy-L-tryptophan, followed by a three-step process in which the conversion generates serotonin, N-acetylserotonin and melatonin. This stepis catalyzed sequentially by DDC, AANAT, and acetylserotonin O-methyltransferase (ASMT), respectively.

In the temperature pattern (NT-HT1-HT3), the mRNA level of genes encoding TYR (FPKM: NT = 106.99; HT1 = 49.50; HT3 = 13.46) (F₂ = 113.519, P = 0.000) exhibited a gradual down-regulation; The expression of genes encoding TH (FPKM: NT = 10.03; HT1 = 9.97; HT3 = 335.39) (F₂ = 300.615, P = 0.000) was first unchanged and then up-regulated; The expression of genes encoding DCE showed a mixed regulation, with six instances of up-regulation (FPKM: NT = 106.99; HT1 = 49.50; HT3 = 13.46) (F₂ = 113.519, P = 0.000) and two instances of down-regulation (FPKM: NT = 10.87; HT1 = 13.41; HT3 = 4.22) (F₂ = 113.519, P = 0.000). Also, the level of precursors for eumelanin synthesis eventually exhibited either an increase or a lack of change between the three temperature patterns (NT, HT1, NT3), i.e., L-dopa (NT = 196.08; HT1 = 753.35; HT3 = 692.00) (F₂ = 1220.763, P = 0.000), dopaquinone (NT = 11943.57; HT1 = 9873.35; HT3 = 13066.89) (GLM gamma, df = 2, P = 0.007), 5,6-DHI (NT = 5.44; HT1 = 1.84; HT3 = 56.76) (F₂ = 1544.200, P = 0.000), and 5,6-IQCA (NT = 36.21; HT1 = 52.41; HT3 = 63.38) (F₂ = 78.416, P = 0.000). Nevertheless, there is a reduction in dopamine levels (NT = 816.60; HT1 = 647.72; HT3 = 540.09) (F₂ = 1544.200, P < 0.001) and in the expression of genes encoding DDC (NT = 25.42; HT1 = 38.60; HT3 = 0.75) (F₂ = 1544.200, P = 0.000) that regulate its production (Fig. 4C and D).

In general, with the exception of DDC and serotonin (NT = 416.67; HT1 = 461.34; HT3 = 440.60) (F₂ = 1.694, P = 0.261), genes encoding enzymes and metabolites of the melatonin synthesis pathway were in an up-regulated state following heat stress, i.e., TPH1/2 (FPKM: NT = 0.95; HT1 = 1.11; HT3 = 10.09) (F₂ = 719.469, P = 0.000), 5-hydroxy-L-tryptophan (NT = 3902.52; HT1 = 4760.57; HT3 = 4635.24) (F₂ = 77.785, P = 0.000), AANAT (FPKM: NT = 24.87; HT1 = 29.42; HT3 = 63.58) (F₂ = 32.726, P = 0.001), and melatonin (NT = 157.50; HT1 = 172.53; HT3 = 231.93) (F₂ = 6.627, P = 0.030) (Fig. 4C and D).

3.3 Transfer of pupal antioxidant enzymes to the adult

Individuals reared at 28 or 32°C during the pupal stage induced black- and yellow-type H. hebetor adults, respectively. Pupae reared at 32°C had a significantly lower protein concentration [28°C (black) = 25.68 g/L; 32°C (yellow) = 20.04 g/L; T₁ = 7.894, P = 0.001]. Heat stress in the pupal stage was also associated with lower peroxidase (POD) activity in the pupae: 28°C (black) = 0.45 U/mg; 32°C (yellow) = 0.36 U/ mg prot (T₁ = 5.780, P = 0.004). The heat stress treatment was, however, associated with significantly elevated pupal levels of superoxide dismutase (SOD) [28°C (black) = 2.93 U/mg prot; 32°C (yellow) = 4.95 U/mg prot) (T₁ = -10.183, P = 0.001) and catalase (CAT) activity [28°C (black) = 18.93 U/mg prot; 32°C = 25.82 (yellow) U/mg prot) activity (T₁ = -5.552, P = 0.005) (Fig. 5).

Black- and yellow-type H. hebetor parasitoids in the adult stage showed similar concentrations as did pupae of the same color. As in pupae, the protein concentration was higher in black adults (black-type adults = 23.30 g/L, yellow-type adults = 20.46 g/L, T₁ = 6.253, P = 0.001), as was POD activity (black-type adults = 0.53 U/mg prot; yellow-type adults = 0.41 U/mg prot; T₁ = 2.926, P = 0.043). However, yellow adults had higher SOD levels (black-type adults = 3.73 U/mg prot; yellow- type adults = 4.53 U/mg prot; T₁ = -4.880, P = 0.008) and higher CAT activity (black-type adults = 20.25 U/mg prot; yellow-type adults = 23.67 U/mg prot; T₁ = -5.355, P = 0.006) (Fig. 5).

3.4 Exogenous antioxidant supplementation increases female H. hebetor longevity, fecundity, and the number of hosts paralyzed

Compared to the control (honey water), supplemental melatonin and glutathione solutions 10 ug/mL per day significantly enhanced the longevity (Fig. 6A), fecundity (Fig. 6B), and number of paralyzed hosts of female parasitoid wasps (Fig. 6C) at 34°C. This enhancement was not significantly different between the two antioxidants.

In this study, we found that heat stress activated the antioxidant system of H. hebetor in the pupal stage, including levels of three antioxidant enzymes (SOD, CAT, POD) and the synthesis of three antioxidants (GSH, melatonin, trehalose, NAD)., Heat stress also induced the release of biogenic amines (octopamine, dopamine, serotonin, ecdysone) and the expression of heat shock protein genes, increasing the organism's thermotolerance. Furthermore, these heat tolerance changes induced in H. hebetor pupae were transmitted to adults. This outcome was strongly supported by data on antioxidant enzyme activity ( i.e., the activity levels of SOD, CAT, and POD) in H. hebetor tissues, which could be induced by heat stress in the pupal stage, but maintained during the adult stage. Adult lifespan was, as a consequence, significantly longer that adults from non-exposed pupae when adults were exposed to high temperatures. However, this thermal tolerance came at the expense of fecundity. Synovigenic parasitoid wasps such as H. hebetor have incomplete egg maturation in their ovaries at adult emergence and need to feed on glycogen in support oogenesis (Ou et al. 2021). Thus, decreased fecundity in adults whose pupae were exposed to unfavorable heat stress is most likely due to impaired the development of the ovaries in the pupal stage.

This damage to adults whose pupae were exposed to heat shock may have been related to the expression of heat shock protein genes, which is metabolically expensive (Xiong et al. 2024). For example, Transgenic Drosophila adults that overexpressed hsp70 overexpressing were more heat-tolerant than wild-type flies, but exhibited reduced growth, survival, and egg hatch (Krebs & Feder 1997). Similarly, short-term heat stress increased the subsequent longevity of Liriomyza huidobrensis (Diptera: Agromyzidae) adults at high temperatures but suppressed egg production accompanied by high expression of hsp20 and hsp70 genes (Huang et al. 2007). Also, in our present study, hsp 68-like genes were up-regulated in H. hebetor pupae after 3d at 32°C. On the other hand, the establishment of heat tolerance may have depleted the resources needed by the parasitoids for reproduction. Glycogen is an important energy source needed for insect growth, development and reproduction (González-Tokman et al. 2020). In this study, we found that heat stress during the pupal stage induced a shift away from glycogen towards trehalose synthesis, and the decrease in glycogen levels may have been detrimental to the development of the parasitoid wasps' ovaries and oogenesis (Ou et al. 2021). In contrast, higher trehalose levels may, to a certain extent, have prevented the denaturation of cell membranes and membrane proteins by free radicals (Kandror et al. 2002). This effect would have prolonged the parasitoid wasp's survival under heat stress. Also, exogenous antioxidant feeding experiments confirmed that this trade-off disappeared in the presence of glutathione and melatonin supplementation, resulting in a simultaneous increase in longevity (and numbers of hosts paralyzed) and fecundity. In Drosophila adults, exogenous melatonin, NAD, and NAC food supplements increased ROS scavenging and increased survival under oxidative stress stress conditions (Vaccaro et al. 2020). However, whether it would be necessary to set up antiglycogen + oxidant feeding sites for parasitoid wasps under extreme heat in actual field environments needs to be carefully considered. The extent to which natural enemies and lepidopteran hosts benefit from such diet supplements is not yet known. Alternatively, perhaps a device could be set up to allow only parasitoids to enter and feed, excluding the pests based on a size difference between them and the parasitoid.

Melanin is the predominant pigment that makes up insect body color and markings (Kronforst et al., 2012), and it is formed by the oxidation of dopa or dopamine. Tyrosinase (TYR), tyrosine hydroxylase (TH), dopa decarboxylase (DDC), and the dopa pigment interconvertase (DCE) are the key enzymes necessary for epidermal melanization in insects (Kronforst et al., 2012), with TH and DDC catalyzing the two most important precursors of epidermal melanin in insects (i.e., levodopa and dopamine, respectively). Futahashi and Fujiwara (2007) treated Papilio xuthus (Lepidoptera: Papilionidae) with a TH inhibitor (3-iodo-tyrosine) to suppress epidermal darkening of its larvae. Compared with the wild type, more melanin was deposited on the body surface of larvae and adults of darkened Bombyx mori (Lepidoptera: Bombycidae) (mln mutant), and the dopamine content in the head, thorax and legs of the mutants was significantly higher than that of the wild type larvae. Localized cloning revealed that the AANAT-encoding gene was variable in the mutant, resulting in the loss of AANAT activity and allowing dopamine to accumulate in large quantities and be metabolized for synthesis of melanin (Dai et al., 2010). In addition, N-acetyl dopamine (NADA), which is generated from AANAT-catalyzed dopamine, is an important substance involved in insect epidermal tanning (a process in which insect epidermal or oocyst proteins crosslink with quinones to become dehydrated and hardened), in which the β-carbon atoms in the NADA of insect epidermis are crosslinked with the side chains of proteins, undergoing a β-tanning reaction that results in the formation of a light-colored epidermis (Kronforst et al., 2012).

This study showed an opposite trend in the expression of transcript levels of genes encoding TH and TYR under 32°C treatment compared to the control (28°C). These two enzymes together catalyze the conversion of tyrosine to L-dopa. Given that the level of L-dopa is elevated after high-temperature treatment, the TH-encoding genes perhaps play a dominant role. DCE catalyzes the conversion of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a precursor of true melanin. It has been shown that there is antagonism between DCE and TYR, e.g., in Manduca sexta (Lepidoptera: Sphingidae), and therefore the two enzymes could inhibit each other's activity (Sugumaran, 2000). The reason we found both up- and down-regulation of several genes encoding DCE under high temperature treatment in the present study may be related to this antagonistic effect. Also, the level of precursors for eumelanin synthesis either increased or remained unchanged between the temperature treatments. That outcome might have been the result of sequential production of metabolites given that the degree of developmental of pupae was inconsistent between the 32 and 28°C treatments for the same period of time in our study. That in turn was due to the fact that high temperatures accelerated the transformation of the pupa to the adult. These increases in the levels of the above substances may be necessary for epidermal pigment synthesis. The significant down-regulation of TYR, which participates in several parts of the melanin synthesis pathway and is the rate-limiting enzyme that regulates melanogenesis, is likely the key factor causing temperature-dependent melanin fading in H. hebetor. In conclusion, the enzymes and substrates in the pigment synthesis pathway are in dynamic equilibrium, and the promotion or inhibition of a pigment substrate, catalase, or gene expression in the pathway will cause metabolism to proceed in the direction of specific pigment synthesis (Kronforst et al., 2012).

As to why the expression of the genes encoding the above enzymes was affected by high temperature in this study, it may be that insect endogenous hormones play a key role. Juvenile hormone (JH) and ecdysone (20-hydroxyecdysone, 20E) are the most important hormones present in insects (Futahashi & Fujiwara, 2008), which together regulate physiological functions such as growth, development, metamorphosis, and reproduction of insects. Both of these hormones respond to temperature (Futahashi and Fujiwara, 2008). Zhu et al. (2019) used CRISPR/Cas 9 genome editing to knock out the Aedes aegypti’s (Diptera: Culicidae) Met gene (JH receptor) and induced a melanized larval phenotype. JH can inhibit M. sexta’s TYR activity (Sugumaran, 2000), and the expression of DDC in this species can also be induced by the 20E cascade response, which in turn affects the darkening of the larval body color. Futahashi and Fujiwara (2007) found that a high titer of 20E promoted the yellow gene and repressed the expression of TH, DDC, and tan genes, thus affecting melanin production in the epidermis of P. xuthus larvae. This study revealed that differentially expressed genes (metabolites) in H. hebetor’s pupal tissues between temperature patterns were significantly enriched in the JH and 20E biosynthetic pathway. Therefore, it is hypothesized that temperature-mediated endocrine signaling produces plastic regulation of melanin synthesis-related enzyme activities in this parasitoid.

The genes encoding DDC and AANAT were involved in both melanin and melatonin biosynthetic pathways. This may be a clue to resolving the mechanism by which body color differentiation in H. hebetor is accompanied by increased thermotolerance. Chen et al. (2022) clarified that both TH and DDC encoding genes are pleiotropic by using gene silencing techniques. The dopamine melanin synthesis pathway, regulated by TH and DDC, is the main pathway for sheath-wing melanization in Harmonia axyridis (Coleoptera: Coccinellidae); furthermore, TH is an essential gene in the growth and development of this ladybug. Also, DDC influences this ladybug’ fecundity by regulating the maturation process of female oocytes. In addition to its role as a precursor substance for melanin, the metabolism of dopamine, which is a direct source of reactive oxygen species (ROS) in the central nervous system, may also regulate the respiratory rate by controlling vital activity and indirectly producing ROS (Vermeulen et al. 2006). The balance between ROS production and clearance can have antagonistic effects on the survival of living organisms, as demonstrated in a strain of Drosophila in which individuals with a darker body coloration also had higher dopamine levels, greater mobility and an increased respiration rate, but shorter lifespan (Vermeulen et al. 2006). In this study, we found that dopamine levels were significantly reduced in the tissues of H. hebetor pupae under high temperature treatment, which may be a strategy to reduce the level of oxidative stress.

In summary, high temperature inhibited the expression of genes encoding TYR and DDC, which are key enzymes needed for melanin synthesis, thus affecting melanin production in the epidermis of H. hebetor adults. These genes may also multidirectionally regulate pigment synthesis and antioxidant processes, leading ng to the co-evolution of coloration patterns and thermophysiology in this parasitoid (True, 2003). Light body coloration (high reflectance) may exert selective pressure on insect body coloration by enhancing survival or reproduction in hot environments through a thermodynamic effect (Clusella-Trullas et al., 2008), i.e., reflecting a portion of radiant heat from the sun and reducing the probability of heat stress losses occurring. In this case, polymorphic effects of genes may be the pathway through which selection pressure acts (True, 2003). The next studies in this line of investigation should use RNAi or gene editing techniques to validate the function of specific genes and tap into upstream signaling pathways to establish a relatively complete regulatory network.

Author contributions

Sanqiang Yan and Baoqian Lyu contributed to methodology; Sanqiang Yan, Hui Lu, Qikai Zhang, Jihong Tang, Bin Jiao, and Yuxin Wu contributed to sampling; Sanqiang Yan and Baoqian Lyu contributed to writing-original draft preparation; Baoqian Lyu to funding acquisition. All authors commented on previous versions of the manuscript and read and approved the fnal manuscript.

Funding We thank National Science Foundation of China (32360703), Hainan International Science and Technology Cooperation R&D Project (GHYF2022002), National Key Research and Development Program (2021YFD1400705), and Ministry of Agriculture and Rural Affairs Financial Project (102125201630040009001) for funding.

Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflict of interest The authors declare no conflict of interest.

Altaf M M, Awan ZA, Ashraf S, Altafd, M A, Zhu Z G, Alsahli A A, Ahmad P (2024) Melatonin induced reversibility of vanadium toxicity in muskmelon by regulating antioxidant defense and glyoxalase systems. J Hazard Mater 473:134452. https://doi.org/10.1016/j.jhazmat.2024.134452
Armstrong GAB, Robertson RM (2006) A role for octopamine in coordinating thermoprotection of an insect nervous system. J Therm Biol 31(1):149-158. https://doi:10.1016/j.jtherbio.2005.11.022
Bates D, Maechler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using lme4.J Stat Softw 67(1): 1-48. https://doi.org/10.48550/arXiv.1406.5823
Castexa V, Benistona M, Calanca P, Fleury D, Moreau J (2018) Pest management under climate change: the importance of understanding tritrophic relations. Sci Total Environ 616:397-407. https://doi.org/10.1016/j.scitotenv.2017.11.027
Xie C, Mao XM, Huang JJ, Ding Y, Wu JM, Dong S, Kong L, Gao G, et al (2011) KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res 39(01):316-22.
Chen X, Tian RB, Xu QX, Li S, Wang S, Zang LS, Xiao D (2022) Regulatory mechanism of DOPA decarboxylase (DDC) on fecundity in Harmonia axyridis (Coleoptera: Coccinellidae). Acta Entomol Sin 65(1):1-9. https://doi.org/10.16380/ j.kcxb.2022.01.001
Clusella-Trullas S, Terblanche JS, Blackburn TM, Chown SL (2008) Testing the thermal melanism hypothesis: a macrophysiological approach. Funct Ecol 22:232-238. https://doi: 10.11116.1365-2435.20407.01377.x
Colinet H, Sinclair BJ, Vernon P, Renault D (2015) Insects in fluctuating thermal environments. Annu Rev Entomol, 60:123-140. https://10.1146/ annurev-ento-010814-021017
Dai FY, Qiao L, Tong XL, Cao C, Chen, P, Chen J, Xiang ZH. (2010). Mutations of an arylalkylamine-N-acetyltransferase, Bm-iAANAT, are responsible for silkworm melanism mutant. J Biol Chem 285(25): 19553-19560. https://doi.org/10.1074/jbc.M109.096743
Futahashi R, Fujiwara H (2007) Regulation of 20-hydroxyecdysone on the larval pigmentation and the expression of melanin synthesis enzymes and yellow gene of the swallowtail butterfly, Papilio xuthus. Insect Biochem Molec 37(8): 855-864. https://doi.org/10.1016/j.ibmb.2007.02.014
Futahashi R, Fujiwara H (2008) Juvenile hormone regulates butterfly larval pattern switches. Science 319(5866):1061-1061. https://doi.org/10.1126/science.1149786
Ghani M U, Yang Z, Feng T, Chen J, Khosravi Z, Wu Q, Cui H (2024) Comprehensive review on glucose 6 phosphate dehydrogenase: A critical immunometabolic and redox switch in insects. Int J Biol Macromol 273:132867. https://doi.org/10.1016/j.ijbiomac.2024.132867
González-Tokman D, Córdoba-Aguilar A, Dáttilo W, Lira-Noriega A, Sánchez-Guillén RA, Villalobos F (2020). Insect responses to heat: physiological mechanisms, evolution and ecological implications in a warming world. Biol Rev 95(3):802-821. https://doi.org/10.1111/brv.12588
Hance T, van Baaren J, Vernon P, Boivin G (2007) Impact of extreme temperatures on parasitoids in a climate change perspective. Annu Rev Entomol 52(1): 107-126. https://doi.org/10.1146/annurev.ento.52.110405.091333
Hartig F (2019) DHARMa: Residual Diagnostics for Hierarchical (Multilevel/Mixed) Regression Models. R package version 0.4.3. https://cran.r-project.org/web/packages/ DHARMa/vignettes/DHARMa.html
Huang LH, Chen B, Kang L (2007). Impact of mild temperature hardening on thermotolerance, fecundity, and Hsp gene expression in Liriomyza huidobrensis. J Insect Physiol 53(12):1199-1205. https://doi.org.10.1016/j.jinsphys.2007.06.011
Kandror O, DeLeon A, Goldberg AL (2002) Trehalose synthesis is induced upon exposure of Escherichia coli to cold and is essential for viability at low temperatures. P Natl Acad Sci Usa 99(15):9727-9732. https://doi.org/10.1073/pnas.14231409
Krebs RA, Feder ME (1997) Deleterious consequences of Hsp70 overexpression in Drosphilla melanogaster larvae. Cell Stress Chaperon 2(1):60-71. https://doi.org.10.1379/1466-1268(1997)002<0060:dcohoi>2.3.co;2
Kronforst MR, Barsh GS, Kopp A, Mallet J, Monteiro A, Mullen SP, Rosenblum EB (2012) Unraveling the thread of nature’s tapestry: the genetics of diversity and convergence in animal pigmentation. Pigm Cell Melanoma R. 25(04):411-433. https://doi:10.1111/j.1755-148X.2012.01014.x
Kvist J, Wheat CW, Kallioniemi E, Saastamoinen M, Hanski I, Frilander MJ (2013) Temperature treatments during larval development reveal extensive heritable and plastic variation in gene expression and life history traits. Mol Ecol 22(3):602-619. https://doi/10.1111/j.1365-294X.2012.05521.x
Langmead B, Trapnell C, Pop M, Trapnell SL (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:1-10. https://doi.org.10.1186/gb-2009-10-3-r25
Lenth R, Singmann H, Love J, Buerkner P, Herve M (2018) emmeans: Estimated Marginal Means, Aka Least-Squares Means. R Package v.1.3. https://CRAN.Rproj ect.org/ /package=emmeans
Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. Bmc Bioinformatics 12:1-16. https://doi.org/10.1186/1471-2105-12-323
Lüdecke D (2018a) ggeffects: Tidy data frames of marginal effects from regression models. J Open source Softw 3(26):722. https://doi.org.10.21105/joss.00772
Lüdecke D (2018b) sjPlot: Data Visualization for Statistics in Social Science. R Package v.2. https://stren gejac ke.Github.io/sjPlo t/
Lyu BQ, Wang SC, Wyckhuys KAG, Liu Z (2023) Biological pest control protects pollinators. Science 380(6642):251. https://10.1126/science.adh3467.
Ma CS, Ma G, Pincebourde S (2021) Survive a warming climate: insect responses to extreme high temperatures. Annu Rev Entomol 66(1): 163-184. https://doi.org/10.1146/annurev-ento-041520-074454
Ning J, Liang P, Wu X, Wang W, Wu Q, Wang S, Xie W (2024) Silencing of catalase reduces unfavorable low-temperature tolerance capacity in whiteflies. Pest Manag Sci 80(7):3116-3125. https://doi.org/10.1002/ps.8016
Ou HD, Atlihan R, Wang XQ, Li HX, Yu XF, Jin X, Yang MF (2021) Host deprivation effects on population performance and paralysis rates of Habrobracon hebetor (Hymenoptera: Braconidae). Pest Manag Sci 77(4):1851-1863. https://doi.org.10.1002/ps.6210
Roulin A (2014) Melanin-based colour polymorphism responding to climate change. Global Change Biol 20(11):3344-3350. https://doi.org/10.1111/gcb.12594
Sugumaran M, Nellaiappan K (2000) Characterization of a new phenoloxidase inhibitor from the cuticle of Manduca sexta. Biochem Bioph Res Co 268(2):379-383. https://doi.org/10.1006/bbrc.2000.2138
Tauffenberger A, Fiumelli H, Almustafa S, Magistretti PJ (2019) Lactate and pyruvate promote oxidative stress resistance through hormetic ROS signaling. Cell Death Dis 10(9):653. https://doi.org/10.1038/s41419-019-1877-6
True JR (2003) Insect melanism: the molecules matter. Trends Ecol Evol 18(12):640-647. https://doi.org/10.1016/j.tree.2003.09.006
Vaccaro A, Dor YK, Nambara K, Pollina EA, Lin C, Greenberg ME, Rogulja D (2020) Sleep loss can cause death through accumulation of reactive oxygen species in the gut. Cell 181(6):1307-1328. https://doi.org/10.1016/j.cell.2020.04.049
Vermeulen CJ, Cremers TIFH, Westerink BHC, Van De Zande L, Bijlsma R (2006) Changes in dopamine levels and locomotor activity in response to selection on virgin lifespan in Drosophila melanogaster. Mech Ageing Dev 127(7):610-617. https://doi.org/10.1016/j.mad.2006.02.004
Wang B, Ma YX, Kong XN, Ding XT, Gu HC, Chu TQ, Ying WH (2014) NAD+ administration decreases doxorubicin-induced liver damage of mice by enhancing antioxidation capacity and decreasing DNA damage. Chem-Biol Interact. 212:65-71. http://dx.doi.org/10.1016/j.cbi.2014.01.013
Wang SC, Yan SQ, Lyu BQ, Wu QQ, Lu H, Zhang QK, Qiu HY, Wu YX (2024) Pre-adult thermal experience induces body color differentiation of Habrobracon hebetor and increases its potential to control hosts under high temperatures. Entomol Gen 44(1): 181-189. https://doi.org/10.1127/entomologia/2023/2231
Xiong SJ, Yu KL, Lin HW, Ye XH, Xiao H, Yang Y, Ye GY (2024) Regulatory network in heat stress response in parasitoid wasp focusing on Xap5 heat stress regulator. Iscience 27(1):108622. https://doi.org/10.1016/j.isci.2023.108622
Yan SQ, Lin NF, Xiang KP, Kuang FP, Lyu BQ, Lu H, Tang JH (2023) Performance and population prediction of Opisina arenosella on four species of palm plants. Entomol Exp Appl 171(3):227-237. https://doi/10.1111/eea.13267
Yoon SK, Park EJ, Choi YI, Bae EK, Kim JH, Park SY, Lee HS (2014) Response to drought and salt stress in leaves of poplar (Populus alba x Populus glandulosa): expression profiling by oligonucleotide microarray analysis. Plant Physiol Bioch 84:158-168. https://doi.org/10.1016/j.plaphy.2014.09.008
Zhao Y, Li Y, He M, Yun Y, Peng Y (2020) Antioxidant responses of the pest natural enemy Hylyphantes graminicola (Araneae: Linyphiidae) exposed to short-term heat stress. J Therm Biol 87:102477. https://doi.org/10.1016/j.jtherbio.2019.102477
Zhu GH, Jiao Y, Chereddy SC, Noh MY, Palli SR (2019) Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, Aedes aegypti. P Natl Acad Sci Usa 116(43):21501-21507. https://doi.org/10.1073/pnas.190572911

No competing interests reported.

Supplementaryfile.docx

Download PDF

Editor assigned by journal
13 Aug, 2024
Submission checks completed at journal
13 Aug, 2024
First submitted to journal
12 Aug, 2024

You are reading this latest preprint version

Molecular basis for the co-evolution of thermophysiology and pigmentation in Habrobracon hebetor (Hymenoptera: Braconidae)

Status:

Version 1

Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Insect rearing

2.2 Sample preparation for transcriptional and metabolic profiling

2.3 RNA sequencing, de novo assembly, and annotation

2.4 Measurement of metabolites

2.5 Quantitative real-time PCR

2.6 Antioxidant enzyme activities

2.7 Antioxidant supplementation

2.8 Data analysis

3 Results

3.1 Principal component analysis of the transcriptome and metabolome

3.2. Heat stress activates endogenous antioxidant synthesis pathways

3.2 Simultaneous involvement of DDC and AANAT encoding genes in melanin and melatonin synthesis

3.3 Transfer of pupal antioxidant enzymes to the adult

3.4 Exogenous antioxidant supplementation increases female H. hebetor longevity, fecundity, and the number of hosts paralyzed

4 Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1