Effect of Ephestia kuehniella Eggs on Development and Transcriptome of the Ladybird Beetle Propylea japonica

Simple Summary Improving augmentative biological control relies on the development of a cost-effective and readily available diet for rearing natural enemy insects. This study highlights the effectiveness of Ephestia kuehniella eggs in promoting the development of the predatory ladybird beetle Propylea japonica. It represents the first study in assessing the advantages of E. kuehniella eggs as dietary sources for ladybirds by examining transcriptional regulation. Abstract The eggs of the Mediterranean flour moth, Ephestia kuehniella, are frequently utilized as alternative diets and have demonstrated promising outcomes when consumed by various insects. Nonetheless, the specific reasons for their effectiveness remain unclear. In our study, we assessed the developmental performance of the ladybird Propylea japonica when fed E. kuehniella eggs, alongside 12 factitious prey or artificial diets. Our findings revealed that ladybirds fed E. kuehniella eggs displayed a performance comparable to those fed the natural prey Megoura crassicauda. Transcriptome profiling of larvae raised on E. kuehniella eggs and M. crassicauda revealed that genes upregulated in the former group were enriched in metabolic pathways associated with carbohydrates, lipids, and other essential nutrients. This suggests that E. kuehniella eggs may have a higher nutrient content compared to natural prey. Furthermore, a notable downregulation in the expression of immune effector genes, such as Attacin and Coleoptericin, was observed, which might be attributed to the lower microbial content in E. kuehniella eggs compared to M. crassicauda. We suggest that the difference between E. kuehniella eggs and M. crassicauda as food sources for P. japonica lies in their nutrient and microbial contents. These findings provide valuable insights for the advancement of innovative artificial breeding systems for natural enemies.


Introduction
Augmentative biological control stands as an important method in agricultural pest management, involving the release of large quantities of natural enemies into the field [1,2].The production of these natural enemies relies on a three-tiered artificial rearing system encompassing predators, prey, and the prey's host plants.Nonetheless, challenges such as discontinuity and high rearing facility costs can impede this method's efficacy [3,4].Thus, the development of a cost-effective and easily accessible artificial diet is crucial for enhancing biological control strategies.
Predatory ladybirds are commonly used as natural enemies in augmentative biological control due to their pest-feeding abilities [4].Propylea japonica (Thunberg, 1781) (Coleoptera, Coccinellidae), a native of Asia, predominantly preys on aphids and various agricultural pests.Known for its adaptability to diverse diet regimes and temperature ranges, P. japonica are widely used in augmentative biological control [13].This study aimed to assess the suitability of E. kuehniella eggs as a diet for P. japonica and explore the differences between E. kuehniella eggs and natural prey as food sources for this ladybird.We compared the life history traits of P. japonica when fed on 14 diets, which included natural prey like Megoura crassicauda Mordvilko, 1919 (Hemiptera, Aphididae) aphids, factitious prey such as E. kuehniella eggs, and artificial diets.We also performed transcriptome profiling to identify differentially expressed genes (DEGs) in P. japonica in response to feeding on E. kuehniella eggs.

Preparing of the Ladybird and Diets
The ladybird P. japonica in the experiment was collected from Baiyun Mountain, Guangzhou, China (113 • 17 ′ E, 23 • 11 ′ N) between 2014 and 2018.Before the experiment, P. japonica eggs were harvested from the cages using plastic straws (0.5 cm in diameter, 3 cm in length) and transferred to plastic Petri dishes (15 cm in diameter, 1.5 cm in height).The hatched larvae from these eggs were then used in the study.A total of 14 diet treatments (Table 1) were assessed for the rearing P. japonica.Nine diet materials were formulated into a culture medium based on a previously published recipe [14], comprising 2.5 g of sucrose, 1.5 g of a protein source, 0.8 g of agarose, and 10 mL of distilled water.The eggs of E. kuehniella (FLO) and Spodoptera frugiperda (FAL) were stored in a freezer at −20 • C and thawed at 4 • C before use.

Feeding Experiments of P. japonica on Different Diets
The feeding experiments were performed from March, 2017 to December, 2023.Newly emerged male and female adults were paired in plastic Petri dishes (9 cm in diameter, 1.5 cm in height), and the eggs they laid were collected.The newly hatched larvae were individually placed into plastic Petri dishes (3 cm in diameter, 1 cm in height) and divided into 14 groups with varying diets.Each dish was equipped with a moist cotton to ensure water availability.Throughout the experiment, the total developmental time, adult weight, and survival rate were documented daily, with daily replenishment of food.Adults were weighed on the day of emergence.The experiment included a total of 14 treatments and 24 batches (Table 1).To evaluate the possible batch effect of the main treatments APH and FLO, three batches were conducted for each.For the other treatments, one to three batches were conducted.Each batch comprised 22-99 tested individuals (Table 1).All feeding experiments were performed in climatic chambers at 25 (±1) • C, 75 (±5) % RH under a 16:8 (L: D) photoperiod.Statistical analyses for comparing life history traits among different treatments and batches were carried out using R software v4.2.3.The Shapiro-Wilk normality test and Bartlett's test were employed to assess the normality and homogeneity of variance for the developmental time and adult weight data.Data conforming to these assumptions underwent one-way ANOVA analysis.In cases where a significant difference was observed, Tukey's Honestly Significant Difference (HSD) test was utilized for post hoc comparisons.Alternatively, the Wilcoxon signed-rank test was applied.A significance level of p < 0.05 was adopted for all statistical tests.

Transcriptome Analysis
Fourth-instar larvae (<24 h after molting) of P. japonica from the above batches APH01 and FLO02 were randomly collected for transcriptome analysis.An individual larva was used as a single biological replicate, and three and two biological replicates were set for APH01 and FLO02, respectively.The total RNA of each individual was extracted using TRIzol reagent (CWBIO, Beijing, China).RNA quality and quantity were determined using a Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, NC, USA).Only RNA samples with a 260/280 ratio from 1.8 to 2.0, a 260/230 ratio from 2.0 to 2.5, and an RNA integrity number (RIN) > 8.0 were used for sequencing.Sequencing was performed on the Illumina HiSeq 2500 platform.Adaptors and low-quality sequences were removed using the default settings for Trimmomatic v0.36 [15].The clean reads were then mapped to the published genome of P. japonica [16] using HISAT2 v2.2.1 [17].Abundance estimation was performed using StringTie v2.1.4[18] based on genome annotation.Differential expression analysis between treatments was performed using DESeq2 [19] according to the standard workflow, with a log2(fold change) (log2FC) value > 1 or <−1 and an adjusted p value (Q value) < 0.05 used as the criteria for defining differentially expressed genes (DEGs).Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations were performed using eggnog-mapper v2 (eggNOG database v5.0) [20,21].Functional enrichment of DEGs was performed in the R package clusterProfiler [22].Visualization of GO enrichment was performed using GO-Figure![23].

The Analysis of Life History Traits of P. japonica on Different Diet
In the analysis of life history traits, 9 out of the 14 treatments (APH, FLO, PUP, HON, WHI, POL, BRI, FAL, and YEL) led to individuals reaching the adult stage (Figure 1A).The survival rates of APH were generally lower than those of FLO (Figure 1A).Treatments WHI, POL, BRI, FAL, and YEL had less than 25% of the tested individuals reaching adulthood (Figure 1A), and their developmental time and adult weight data were excluded from further analysis.Figure 1B integrated the parameters of survival rate, developmental time, and adult weight, demonstrating that APH and FLO exhibited superior performance compared to PUP and HON.None of the life history traits were significantly different between the APH and FLO treatments (Table 2).In addition, the performance of APH and FLO exhibited variability among batches.For example, the development time of a batch in FLO (FLO02) was significantly shorter than not only batches in APH (APH02-03) but also FLO (FLO01) (p < 0.05 in Wilcoxon signed-rank test) (Figure 1C).Similarly, the adult weight of a batch in APH (APH03) was significantly higher than batches in both APH (APH02) and FLO (FLO02-03) (p < 0.05 in Wilcoxon signed-rank test) (Figure 1D).In the comparative analysis of transcriptome profiling between APH (as control) and FLO (as treatment), the samples from the treatment group displayed coefficient of determination (r 2 ) values ranging from 0.82 to 0.93, with a mean of 0.88 (Table 3).A total of 479

The Analysis of Transcriptome Profiling between APH and FLO
In the comparative analysis of transcriptome profiling between APH (as control) and FLO (as treatment), the samples from the treatment group displayed coefficient of determination (r 2 ) values ranging from 0.82 to 0.93, with a mean of 0.88 (Table 3).A total of 479 differentially expressed genes were identified, with 345 genes upregulated and 134 genes downregulated.Specifically, immune effector genes such as Attacin and Coleoptericin were significantly downregulated (Figure 2).The results of the Gene Ontology (GO) enrichment analysis revealed that the significantly upregulated genes were mainly enriched in nutritional metabolic functions, including catalytic activity, transmembrane transporter activity in the Molecular Function (MF) category, and carbohydrate metabolic processes in the Biological Process (BP) category (Figure 3).Conversely, the downregulated genes were only enriched in two GO terms: 4-hydroxyphenylpyruvate dioxygenase activity and tyrosine catabolic process.The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed similar findings, with upregulated genes being enriched in pathways related to glycogen metabolism (Table 4).differentially expressed genes were identified, with 345 genes upregulated and downregulated.Specifically, immune effector genes such as Attacin and Coleopt significantly downregulated (Figure 2).The results of the Gene Ontology (GO) e analysis revealed that the significantly upregulated genes were mainly enriche tional metabolic functions, including catalytic activity, transmembrane transpo ity in the Molecular Function (MF) category, and carbohydrate metabolic proce Biological Process (BP) category (Figure 3).Conversely, the downregulated g only enriched in two GO terms: 4-hydroxyphenylpyruvate dioxygenase activity sine catabolic process.The Kyoto Encyclopedia of Genes and Genomes (KEG ment analysis showed similar findings, with upregulated genes being enriche ways related to glycogen metabolism (Table 4).

Discussion
In the feeding experiment, most of the artificial diets resulted in a poorer performance of P. japonica than the natural prey pea aphids M. crassicauda.Some diets could only support a few ladybirds to mature into adults, such as whiteflies (WHI treatment) and the pollen of Brassica campestris (POL treatment).Other diets, like the pupae of the honeybee Apis mellifera (PUP treatment) and bee honey (HON treatment), could sustain over 50% of larvae to reach the adult stage, but at the cost of a significantly decreased larval development time and adult weight.Hence, none of these diets were deemed suitable for P. japonica.Among all the tested artificial diets, the natural prey pea aphids M. crassicauda (APH treatment) and E. kuehniella eggs (FLO treatment) exhibited the best performance for feeding P. japonica.Although there was variability in performance among batches in our experiments, the life history traits of P. japonica reared on both E. kuehniella eggs and aphids showed relatively similar patterns.This finding aligns with previous reports that identified E. kuehniella eggs as a viable alternative diet for the development and reproduction of P. japonica [8].Therefore, E. kuehniella eggs are suitable for use as an alternative food source in the artificial feeding system of P. japonica.
By comparing the transcriptome of P. japonica fed on E. kuehniella eggs with that of aphids, we identified numerous upregulated metabolism-related genes that were enriched in several sugar metabolism pathways.This outcome parallels findings in previous studies on other ladybird species, indicating that changes in life history traits and transcriptome regulation may reflect variations in the nutritional composition of the E. kuehniella eggs [14,24].Similar changes in life history traits and differentially expressed metabolism-related genes in response to varied nutritional compositions of diets have been observed in other insects.For example, nutrient-sensing and metabolic pathways in A. mellifera are activated by feeding on honey, which contains higher levels of proteins and amino acids, compared to sugar feeding [25].These results underscore the importance of nutrient and biochemical composition in assessing the suitability of an artificial diet [26].However, we did not test the nutritional content differences between E. kuehniella eggs and pea aphids M. crassicauda.Additionally, we cannot rule out the possibility that other factors also contributed to the differential expression of metabolism-related genes.
Aphids are known to harbor various symbiotic bacteria, such as Buchnera aphidicola, Serratia symbiotica, Hamiltonella defensa, Regiella insecticola, Rickettsia, Rickettsiella, Spiroplasma, Wolbachia, and Arsenophonus [27].Some of these bacteria may have adverse effects on the natural enemies to protect their host [28,29].These bacteria can change the gene expression in the natural enemies [30].In contrast, E. kuehniella eggs contain fewer microbes that could elicit changes in gene expression in ladybirds.Previous studies have reported that the >99% microbiota composition of these eggs belonged to an intracellular bacterium, Wolbachia [24].In this study, two immune effector genes of P. japonica were significantly downregulated when feeding on E. kuehniella eggs, suggesting that these genes may play a crucial role in eliminating bacteria from aphids.Therefore, feeding on E. kuehniella eggs may result in less immune stress, contributing to the superior performance of P. japonica feeding on this diet.
Our research highlights that E. kuehniella eggs are a suitable artificial diet for the massrearing of P. japonica, surpassing other tested alternative prey or artificial diets.Through the transcriptome profiling analysis, we suggest that the difference between E. kuehniella eggs and M. crassicauda as food sources for P. japonica lies in their nutrient and microbial contents.These findings also offer valuable insights for developing improved systems for the captive breeding of other natural enemies.However, the effects of feeding E. kuehniella eggs to multiple successive generations of P. japonica remain unclear and need further research for clarification.

Figure 1 .
Figure 1.Effects of different diet treatments and batches on life history traits of Propylea japonica.(A) Survival rate of batches in APH, FLO, PUP, HON, WHI, POL, BRI, FAL, and YEL treatments.(B) Survival rate (point size), development time (X-axis), and adult weight (Y-axis) of batches in APH, FLO, PUP, and HON treatments.(C) Development time and (D) female adult weight of batches in APH and FLO treatments.Different colors indicate different diet treatments.Bars with the same letter are not significantly different (p ≥ 0.05).

Figure 1 .
Figure 1.Effects of different diet treatments and batches on life history traits of Propylea japonica.(A) Survival rate of batches in APH, FLO, PUP, HON, WHI, POL, BRI, FAL, and YEL treatments.(B) Survival rate (point size), development time (X-axis), and adult weight (Y-axis) of batches in APH, FLO, PUP, and HON treatments.(C) Development time and (D) female adult weight of batches in APH and FLO treatments.Different colors indicate different diet treatments.Bars with the same letter are not significantly different (p ≥ 0.05).

Figure 3 .
Figure 3. Gene Ontology (GO) enrichment analysis of differentially expressed genes in Propylea japonica when feeding on Ephestia kuehniella eggs (FLO treatment) compared to pea aphids Megoura crassicauda (APH treatment).Each bubble represents a cluster of similar GO terms summarized by a representative term reported in the legend and sorted by the average p values of the representative GO term across the module of (A) Molecular Function and (B) Biological Process.Bubble size indicates the number of GO terms in each cluster, and the color is the average p value of the representative GO term across the gene modules.Similar clusters plot closer to each other.

Figure 3 .
Figure 3. Gene Ontology (GO) enrichment analysis of differentially expressed genes in Propylea japonica when feeding on Ephestia kuehniella eggs (FLO treatment) compared to pea aphids Megoura crassicauda (APH treatment).Each bubble represents a cluster of similar GO terms summarized by a representative term reported in the legend and sorted by the average p values of the representative GO term across the module of (A) Molecular Function and (B) Biological Process.Bubble size indicates the number of GO terms in each cluster, and the color is the average p value of the representative GO term across the gene modules.Similar clusters plot closer to each other.

Table 1 .
Information of the treatments, batches, number of individuals tested, and dates of the experiments conducted in this study.

Table 2 .
Comparison of life history traits in Propylea japonica when feeding on Ephestia kuehniella eggs (FLO treatment) compared to pea aphids Megoura crassicauda (APH treatment).

Table 2 .
Comparison of life history traits in Propylea japonica when feeding on Ephestia kuehniella eggs (FLO treatment) compared to pea aphids Megoura crassicauda (APH treatment).
3.2.The Analysis of Transcriptome Profiling between APH and FLO

Table 3 .
Coefficient of determination (r 2 ) values of gene expression between the studied transcriptomes.

Table 3 .
Coefficient of determination (r 2 ) values of gene expression between the studied transcriptomes.

Table 4 .
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes in Propylea japonica when feeding on Ephestia kuehniella eggs (FLO treatment) compared to pea aphids Megoura crassicauda (APH treatment).

Table 4 .
Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes in Propylea japonica when feeding on Ephestia kuehniella eggs (FLO treatment) compared to pea aphids Megoura crassicauda (APH treatment).