High-Sucrose Diet Exposure on Larvae Contributes to Adult Fecundity and Insecticide Tolerance in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel)

Simple Summary The oriental fruit fly, Bactrocera dorsalis, is a highly significant invasive pest in agriculture, causing damage to over 600 species of fruits and vegetables. To gain a better understanding of its host adaptation and rapid spread, it is crucial to investigate the effects of changes in nutrient content on the phenotype and gene expression of B. dorsalis. In this study, we examined the developmental duration, adult fecundity, insecticide susceptibility, and gene expression patterns of B. dorsalis by varying the concentration of sucrose in its larval diet. Our findings indicate that a high-sucrose diet during larval stages resulted in a longer developmental period, higher adult fecundity, and greater tolerance to malathion. In contrast, a low-sucrose diet led to smaller body size, shorter developmental duration, and higher sensitivity to beta-cypermethrin. Additionally, we identified differentially expressed genes associated with various metabolisms, hormone synthesis and signaling, and immune-related pathways under different sucrose concentrations in the larval diet. These results suggest that dietary sucrose plays a significant role in phenotypic adjustments and gene expression patterns in B. dorsalis. Abstract Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is one of the broad host ranges and economically-important insect pests in tropical and subtropical areas. A wide range of hosts means they have strong adaptation ability to changes in dietary macronutrients (e.g., sucrose and protein). However, the effects of dietary conditions on the phenotypes and genotypes of B. dorsalis are still unclear. In this study, we aimed to investigate the effects of larval dietary sucrose on the life history traits and stress tolerance of B. dorsalis, and its defense response at the molecular level. The results showed that low-sucrose (LS) induced decreased body size, shortened developmental duration, and enhanced sensitivity to beta-cypermethrin. Otherwise, high-sucrose (HS) diet increased developmental duration, adult fecundity, and tolerance to malathion. Based on transcriptome data, 258 and 904 differentially expressed genes (DEGs) were identified in the NS (control) versus LS groups, and NS versus HS groups, respectively. These yielded DEGs were relevant to multiple specific metabolisms, hormone synthesis and signaling, and immune-related pathways. Our study will provide biological and molecular perspective to understand phenotypic adjustments to diets and the strong host adaptability in oriental fruit flies.


Introduction
Sugars are the major energy source for insects to maintain the basic activities required for life. In many Dipteran flies, diets containing sucrose and yeast hydrolysates (the main source of proteins) have been proven to be more conducive to reproductive development

Insects
The test insects were originally collected from infested fruits in citrus orchards in Hainan Province, China, in 2008. The insects were maintained in the laboratory for more than 100 generations. The larvae were fed on an artificial diet consisting of yeast powder, sucrose, corn and wheat flour, agar, sorbic acid, vitamin C, and linoleic acid, and the adult flies were fed on an artificial diet consisting of sucrose, yeast hydrolysate, vitamin C, and water [15]. Different developmental stages of B. dorsalis were kept in a temperaturecontrolled insectary at 27 • C and 70% relative humidity, with a 14 h:10 h (L:D) photoperiod.

Experimental Diets
The experimental diets of larvae used in this study were slightly modified from the previous study [7], and we varied the amount of sucrose extract in the larvae diet into three groups. Based on the normal culture conditions in the laboratory, 12.0% sucrose (NS) was used as the control [15], and two sucrose concentrations, i.e., 8.0% sucrose and 16.0% sucrose, were set as the low-sucrose (LS) and high-sucrose (HS) conditions, respectively. The sucrose content configuration is shown in Table S1, and the percentage of sucrose was measured by an LB-32T refractometer (Su Wei, Guangdong, China). The adults were fed a normal diet, as previously mentioned.

Effect of Diets on Development
Freshly-laid eggs were collected in 1.5 mL centrifugal tubes filled with orange juice, and incubated in a temperature-controlled insectary as mentioned before. Newly emerged larvae were transferred into 100 mm petri dishes (40 flies per dish, 3 dishes per treatment) filled with the experimental diets. When the late-third instar larvae jumped into the sand basin, the pupation was regarded as the end of the larval stage. The time from egg hatching to pupation (120 flies per treatment) was recorded as the developmental period of the larvae of B. dorsalis. The pre-pupa on the day of pupation was screened out with a sieve pot daily, and placed in a special plastic cage. The bottom of the plastic cup was covered with a layer of wet fine sand about 3 cm thick, and the top of the plastic cup was sealed with gauze until adult emergence. The time from pupation to eclosion (n LS = 76, n NS = 99, and n HS = 97; n is the number of flies) was recorded as the developmental period of pupae. The pupation rate was calculated by comparing the total number of pupae to the total number of larvae in the vial after all insects had pupated. Additionally, the pupae were individually weighed to calculate the average pupal weight under different sucrose concentrations (n LS = 76, n NS = 99, and n HS = 97).
For the eclosion, pupae were placed into plastic cages at around 12 h post-pupation. The emergence rate was calculated by comparing the total number of adults to the total number of pupae in the vial after all insect emergences. Adults that eclosed on the same day were placed in a common cage and fed on an artificial adult diet as previously stated. Adult sexual maturity was considered when eggs laid by females appeared on the wall of the cage. The time from emergence to adult oviposition was recorded as the developmental period of pre-oviposition. In all treatments, three replications were conducted, with 40 insects in each replicate.

Effect of Diets on Ovary Development and Fecundity
Adults were placed into jars (100 mm high × 70 mm diameter), and 5 jars were set up for each of the 3 diets (a total of 15 jars). Ten females (n = 50 females per diet) and ten males were placed in each jar. Food was placed in a small container and replaced daily. To assess ovary development, 9-day-old virgin female flies (n = 30 females per diet) were randomly removed for dissection, and the surface area of individual ovaries was determined. A Leica M205A stereomicroscope (Leica Microsystems, Wetzlar, Germany) was used to capture the images.
Then, 9-day-old female adults were mated with males (fed on normal larval food) in the jar. To investigate the fecundity of female flies, 1.5 mL centrifugal tubes filled with orange juice were used to induce females to lay eggs, and the tubes were replaced daily. The number of eggs was counted from 4 pm to 5 pm every day from 11 to 17 days. Tests on ten pairs of male and female insects per treatment per day were conducted.

Bioassay of Malathion and Beta Cypermethrin
Two insecticides, i.e., malathion (organophosphorus) and beta deltamethrin (pyrethroid), are commonly used for control of B. dorsalis in the field. Therefore, we assessed changes in susceptibility to malathion (98.5% purity) and beta cypermethrin (95.0% purity, Chem Service, West Chester, PA, USA) in adult flies following treatment with different concentrations of sucrose on their corresponding larvae. LD 25 (malathion 150 mg/L; beta cypermethrin 110 mg/L), LD 50 (malathion 200 mg/L; beta cypermethrin 150 mg/L), and LD 75 (malathion 250 mg/L; beta cypermethrin 190 mg/L) of these insecticides were applied to the pronotum of 5-day old adults by a PB600-1 Repeating Dispenser (Hamilton Company, Reno, NV, USA) [15]. The mortality of B. dorsalis was then calculated at 24 h post-treatment. Control flies were treated with the same amount of acetone only. Three replications were conducted for all the treatments, and 30 insects were used in each replicate.

RNA Sequencing
The total RNA was isolated from 5-day-old female adults by using the TRIzol reagent (Life Technologies, Carlsbad, CA, USA). RNA purity and concentration were detected by a NanoVue UV-Vis spectrophotometer (GE Healthcare Biosciences, Uppsala, Sweden). About 6 flies from each sample of LS, NS, and HS groups were collected with three biological replicates. To build sequencing libraries, the Illumina TruSeqTM RNA sample prep kit (Illumina Inc, San Diego, CA, USA) was used, and the libraries were sequenced with pairedend technology using the Novaseq 6000 platform by BioMark Company (Wuhan, China). The original raw reads in fastq format were filtered to remove contaminating adapter fragments, reads containing more than 5% 'N', and low-quality reads. Simultaneously, Q20 (the proportion of reads with quality value ≥ 20) and Q30 (the proportion of reads with quality value ≥ 30) of the clean reads were calculated. The clean reads were then mapped to the reference genome sequence of B. dorsalis (ASM78921v2, GenBank assembly accession: GCA_000789215.2) using Hisat2 (http://ccb.jhu.edu/software/hisat2/index.shtml, accessed on 3 November 2020). Novel transcripts and genes were achieved by StringTie 2.1.2 (https://ccb.jhu.edu/software/stringtie/index.shtml, accessed on 3 November 2020) on the base of the reference genome.

Differential Gene Expression and Functional Annotation Analyses
In this study, the TPM (Transcripts Per Kilobase of exon model per million mapped reads) approach was used to investigate the expression levels of genes [16]. Differential expression genes between the LS vs. NS and the HS vs. NS were analyzed using DESeq2 software [17]. The BH (Benjamini and Hochberg) approaches were performed to obtain the adjusted p values with the controlling of the false discovery rate (FDR). The absolute value of log 2 (fold change) ≥ 1 and adjusted p-value < 0.05 were used as the threshold to judge significant differences in gene expression. Furthermore, the KOBAS and Goatools software programs were used for the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses, respectively [18,19].

qPCR Detection
Based on DEGs, 7 genes in the insulin signal pathway were selected for validation by qPCR. The qPCR primer is described in Table S2. As previously mentioned, total RNA was isolated from 5-day-old adults. After RNA extraction, genomic DNA was removed using DNase I (Promega, Madison, WI, USA). The cDNA was synthesized with PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Dalian, China). Then, qPCR was conducted using NovoStart ® SYBR qPCR SuperMix (Novoprotein, Shanghai, China) by a CFX96 instrument (Bio-Rad, Singapore), as described previously [20]. The α-tubulin gene (GU269902) was selected as an internal reference gene [21]. The relative expression levels were calculated using qBase [22]. Each qPCR consisted of three biological replicates.

Statistical Analysis
The data were analyzed using SPSS Statistics version 20 software (SPSS Inc., Chicago, IL, USA). The data are presented as mean ± standard error (SE). Student's t-test (p < 0.05) was used to determine differences between the means of ovarian surface area and fecundity. Periods, pupal weight, pupation, and eclosion rates were statistically analyzed using oneway ANOVA followed by Tukey's Honestly Significant Difference tests (significance level: p < 0.05). All figures were produced in GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA).

Biological Parameters of B. dorsalis under Different Sucrose Concentration Diets
Feeding larvae with different concentrations of sucrose diets affects the developmental duration of B. dorsalis (Table 1). Because the flies in the control group were a domesticated population that had adapted to artificial diets, we tended to focus on the effects of the changed sucrose contents in the larval diets imposed on the biological parameters when compared with the standard sucrose contents (control group). The developmental duration of larvae was prolonged with the increase in sucrose concentration in diets. There was a significant difference between the LS and NS groups (p < 0.05). The developmental duration of B. dorsalis larvae fed with a low-sucrose concentration diet was the shortest (10.62 ± 1.306 days), and the HS group was the longest (12.24 ± 1.337 days). In terms of the developmental duration of pupae, it was also slightly prolonged with the increase in sucrose concentration, but the differences were not significantly different. The preoviposition period also showed an extension trend as sucrose concentration increased. The pre-oviposition period of the HS group was significantly longer than that of the NS group (p < 0.05), while there was no significant difference between the LS group and the NS group. The pupation rate, pupal weight, and other biological parameters of B. dorsalis were also affected by sucrose in the diets of larvae. Compared with the NS group, both the HS group and LS group showed a decreasing trend in the pupation rate, pupal weight, and eclosion rate. In terms of pupation rate, a significant difference was detected between the LS group and the NS group (p < 0.05), but there was no significant difference between the HS group and the NS group. Among them, the pupation rate of the larvae in the LS group was the lowest (63.33%), followed by the HS group (80.83%), and the NS group was the highest (82.50%). For pupal weight, there were significant differences between the NS group and the HS group (p < 0.05), and between the NS group and LS group (p < 0.05). However, there was no significant difference in eclosion rate among the LS, NS, and HS groups.

Fecundity and Development of the Ovary under Different Concentrations of Sucrose Diets
The ovary size of B. dorsalis in the HS group (t = 7.627; df = 82; p = 0.0006) was significantly smaller than that in the NS group ( Figure 1A). However, there was no obvious change in the ovary size in B. dorsalis between the LS and NS groups. In the HS group, the cumulative (t = 2.472; df = 18; p = 0.0237) and average daily (t = 2.198; df = 18; p = 0.0430) egg production per adult of B. dorsalis was significantly increased when compared to the NS group ( Figure 1B,C), while there was no significant change in egg laying amount between the LS and NS groups.

The Susceptibility of B. dorsalis to Insecticides in Different Sucrose Diet Concentrations
The mortality of various dosages of two insecticides against B. dorsalis adults was determined after their corresponding larvae were supplied with diets containing different concentrations of sucrose ( Figure 2). The mortality of the HS group (F = 14.778; df = 2, 8; p = 0.005) was significantly lower than that in NS group under the treatment of malathion at LD 50 . However, there was no significant difference in the mortality under LD 25 and LD 75 between the LS or HS and NS groups (Figure 2A). On the contrary, the mortality of the LS group (F = 6.686; df = 2, 8; p = 0.034) was significantly higher than that of the NS group under the treatment of beta-cypermethrin at LD 50 . The same as for malathion, there were no significant differences in the mortality of LD 25 and LD 75 between each of the treatments and the NS group ( Figure 2B).  The cumulative egg production of each female adult at the age of 11-17 days. NS, normal sucrose; LS, low-sucrose; HS, high-sucrose. *** denotes significance at p < 0.001, * denotes significance at p < 0.05, and ns denotes no significance.

The Susceptibility of B. dorsalis to Insecticides in Different Sucrose Diet Concentrations
The mortality of various dosages of two insecticides against B. dorsalis adults was determined after their corresponding larvae were supplied with diets containing differen concentrations of sucrose ( Figure 2). The mortality of the HS group (F = 14.778; df = 2, 8; p = 0.005) was significantly lower than that in NS group under the treatment of malathion at LD50. However, there was no significant difference in the mortality under LD25 and LD7 between the LS or HS and NS groups (Figure 2A). On the contrary, the mortality of the LS group (F = 6.686; df = 2, 8; p = 0.034) was significantly higher than that of the NS group under the treatment of beta-cypermethrin at LD50. The same as for malathion, there were no significant differences in the mortality of LD25 and LD75 between each of the treatments and the NS group ( Figure 2B).

Transcriptome Assembly and Differentially Expressed Genes (DEGs) Analysis
Comparing the clean reads to the genome sequences of B. dorsalis, the error rate of sequencing reads was between 0.0246 and 0.0254% for the tested samples (Table 2). In total, 71.48 GB of clean data was obtained, with an average Q20 of 98.09% and a Q30 of 94.15% (Table 2). In total, 15,212 genes were detected in the whole insects from 9 samples

Transcriptome Assembly and Differentially Expressed Genes (DEGs) Analysis
Comparing the clean reads to the genome sequences of B. dorsalis, the error rate of sequencing reads was between 0.0246 and 0.0254% for the tested samples (Table 2). In total, 71.48 GB of clean data was obtained, with an average Q20 of 98.09% and a Q30 of 94.15% (Table 2). In total, 15,212 genes were detected in the whole insects from 9 samples of B. dorsalis under different sucrose concentration exposures, and there were 13,843 (91.00%) genes that could be identified based on genome data. The remaining 1369 unidentified genes were viewed as novel genes. A total of 258 differentially expressed genes, comprising 116 down-regulated (44.96%) and 142 up-regulated (55.04%), were identified between the NS and LS groups ( Figure 3A

Functional Annotation and Validation of DEGs
In the GO analysis, the DEGs were enriched into three ontologies, and the results are shown in Figure 3C,D. Interestingly, the "amino sugar metabolic process" (1.03%), "chitin binding" (0.99%), and "chitin metabolic process" (0.95%) terms were significantly enriched in the NS versus LS groups (Table S3). The expression level of Cht10 (LOC105232357) in the LS group was down-regulated by 7.71-fold compared to the NS control group ( Figure S1). We also found that the "reproductive process" (6.29%), "chromosome organization" (1.33%), and "spindle organization" (0.63%) terms were significantly enriched in the NS versus the HS group (Table S4). The expression of Vm26Aa (LOC105234031), Vg1 (LOC105232170), and Vg2 (LOC105222970) from the HS group were increased by 866.49-, 81.28-, and 5.85-fold compared to the NS control group, respectively ( Figure S2).

Functional Annotation and Validation of DEGs
In the GO analysis, the DEGs were enriched into three ontologies, and the results are shown in Figure 3C,D. Interestingly, the "amino sugar metabolic process" (1.03%), "chitin binding" (0.99%), and "chitin metabolic process" (0.95%) terms were significantly enriched in the NS versus LS groups (Table S3). The expression level of Cht10 (LOC105232357) in the LS group was down-regulated by 7.71-fold compared to the NS control group ( Figure S1). We also found that the "reproductive process" (6.29%), "chromosome organization" (1.33%), and "spindle organization" (0.63%) terms were significantly enriched in the NS versus the HS group (Table S4). The expression of Vm26Aa (LOC105234031), Vg1 (LOC105232170), and Vg2 (LOC105222970) from the HS group were increased by 866.49-, 81.28-, and 5.85-fold compared to the NS control group, respectively ( Figure S2).

Discussion
For holometabolous insects, the larval stage is critical, and the nutrients obtained during this stage are directly related to subsequent individual growth and development [23]. In this study, we found that high sucrose concentrations in the larval diets resulted

Discussion
For holometabolous insects, the larval stage is critical, and the nutrients obtained during this stage are directly related to subsequent individual growth and development [23]. In this study, we found that high sucrose concentrations in the larval diets resulted in the extension of the development duration of the immature stage (e.g., the larval and pupal periods) of B. dorsalis. Our results are somewhat consistent with previous research that has shown that diets with increased sugar content prolong the development period, and reduce the egg-to-adult survival rate and female fecundity of Drosophila species [3,24,25]. High sugar feeding induces systemic metabolic changes associated with carbohydrate metabolic imbalance, resulting in delayed development and reduced fresh weight in Drosophila [26,27]. On the contrary, some studies have shown that insect larvae will shorten their lifespan to avoid insufficient dietary sucrose conditions, and they usually emerge as smaller body size adults earlier [28]. The reason behind this phenomenon may be explained by the fact that low concentrations of sucrose cause abnormal glucose metabolism in insects, which shortens their developmental period [29]. Furthermore, the pre-oviposition period of female adults on the high-sucrose larval diet is obviously longer than that on the normal sucrose larval diet.
In many insects, vitellogenesis is a nutrient-dependent process that is promoted by the juvenile hormone (JH) [30,31]. We discovered that high sucrose intake during the larval stage has an effect on ovarian development during the adult stage, and that ovarian development is significantly delayed. However, lower sucrose intake in larvae does not affect adults' reproductive organ development (Figure 1). Previous studies indicated that changes in dietary sucrose had an important influence on oxidative damage and oxidative stress in insects [7,32]. Under high-sucrose conditions, excessive sugar intake may result in insufficient glucose metabolism by B. dorsalis, which subsequently produces large amounts of reactive oxygen species (ROS). We speculated that the increase in sucrose metabolism in the early stage of B. dorsalis under high glucose stress restricted the development of the ovary by affecting the antioxidant system.
Interestingly, after higher sucrose consumption at the larval stage, the egg-laying capacity of B. dorsalis female adults was significantly higher than that in the control (NS) groups ( Figure 1B). Therefore, the high sucrose intake in the larval stage enhanced the fecundity in the adult stage of B. dorsalis. We also found that the genes (Vm26Aa, Vg1, and Vg2) related to vitellogenesis and the genes (CYP315a1/shadow and CYP307a1/spook) associated with ecdysone synthesis were up-regulated in the adult stage in the transcriptomic data. Among these genes, Vm26Aa, Vg1, and Vg2 were three key genes that played important roles in vitellogenesis and oocyte development [33,34]. Here, we inferred that high-sucrose diet intake in the larval stage may enhance reproductive capacity by increasing the expression of the above genes in their corresponding adult stages. Our results indicated that a good nutrition supply in the larval stage will play an important role in promoting the fecundity of the adult stage, but there is a balance between the ovarian development time and egg-laying amount.
In this study, relatively high-and low-sucrose larval diets altered the sensitivity of B. dorsalis adults to insecticides. Intriguingly, our results showed that high sucrose concentration consumption in larvae could significantly reduce the sensitivity of B. dorsalis to the LD 50 of malathion ( Figure 2A). However, low sucrose consumption in larvae can significantly increase the sensitivity of B. dorsalis adults to the LD 50 of beta-cypermethrin ( Figure 2B). Generally, bioassay data indicated that the adult flies formed by the larvae of the HS group have enhanced tolerance levels to insecticides when compared to the NS and LS groups (Figure 2). In B. dorsalis, resistance mechanisms may involve complex interactions of various genes, such as the detoxification enzymes, i.e., cytochrome P450s (P450s), glutathione S-transferases (GSTs), and carboxylesterases (CarEs) [35]. Based on transcriptomic data, we found some detoxification genes (e.g., CYP6a14, CYP307a1, and GST1) up-regulated in adult flies with their larvae fed a high-sucrose diet. The function of these above paralogous genes was confirmed to be associated with insecticide toler-ance. For example, the knockdown of CYP6A14-1, CYP307A1, and GST1-1-1 in a resistant aphid strain by RNAi resulted in increased susceptibility to insecticides [36]. Additionally, some more up-regulated detoxification genes, i.e., three P450 genes (CYP313a4, CYP304a1, CYP6t1) and one carboxylesterase gene (CarE6), may also contribute to the tolerance of insecticide in B. dorsalis adults after their larvae were exposed to high-sucrose diets. The expression level of BdCYP437A3 was significantly up-regulated after 36 h of avermectin treatment [37]. Furthermore, CYP6 family genes are involved in the metabolism of highly effective beta cypermethrin [38]. Indeed, the genes CYP304a1 and CarE6 have been linked to the detoxification of multiple insecticides in insects [39][40][41].
In the NS vs. LS, DEGs were significantly enriched for "chitin metabolic process" and "chitin binding" terms, suggesting that B. dorsalis could adapt to the changes in sucrose content by regulating the expression of chitin metabolism-related and binding activity genes, which might be involved in the ability of B. dorsalis to cope with insecticide susceptibility. Indeed, cuticular alterations can also affect the intensity and spectrum of insecticide resistance or tolerance [42,43]. In this study, the chitin-related genes from the LS group were down-regulated, especially Cht10, and this result indicated that penetration resistance might be one of the important factors that resulted in the decreased insecticide tolerance of the LS group to exogenous harmful substances, i.e., beta-cypermethrin.

Conclusions
In summary, our work revealed many considerable differences in life history traits, fecundity, insecticide tolerance, and gene expression level associated with different sucrose concentrations of larval diets, which indicate that the phenotypic plasticity, reproductiontolerance trade-offs, and gene expression of B. dorsalis are varied in response to the different dietary environments. These data also suggest that B. dorsalis can respond to food sucrose content by regulating developmental duration and fecundity, and inappropriate sucrose levels in the larval diet will also alter the tolerance of subsequent adult flies to insecticides. The findings of this study will contribute to a better understanding of the effect of sucrose on phenotypic adjustments, and the biological mechanism of rapid adaptation to new hosts in B. dorsalis.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/insects14050407/s1: Figure S1. Expression levels of down-regulated genes between the NS and LS groups in Bactrocera dorsalis. Figure S2. Expression levels of up-regulated genes between NS and HS groups in Bactrocera dorsalis. Table S1. Composition of experimental diets for Bactrocera dorsalis larvae. Table S2. The information on qPCR primers for selected genes in Bactrocera dorsalis. Table S3. The gene ontology (GO) enrichment analysis between NS and LS groups in Bactrocera dorsalis. Table S4. The gene ontology (GO) enrichment analysis between NS and HS groups in Bactrocera dorsalis. Table S5. The up-regulated pathways of KEGG enrichment analysis between NS and LS groups in Bactrocera dorsalis. Table S6. The up-regulated pathways of KEGG enrichment analysis between NS and HS groups in Bactrocera dorsalis. Table S7. The down-regulated pathways of KEGG enrichment analysis between NS and LS groups in Bactrocera dorsalis. Table S8. The down-regulated pathways of KEGG enrichment analysis between NS and HS groups in Bactrocera dorsalis.