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Cytochrome P450 monooxygenase (P450 or CYP) plays an important role in the metabolism of insecticides and plant allelochemicals by insects. CYP321B1, a novel Spodoptera litura P450 gene, was identified and characterized. CYP321B1 contains a 1488 bp open reading frame (ORF) that encodes a 495 amino acid protein. In fourth instar larvae, the highest CYP321B1 expression levels were found in the midgut and fat body. In the tannin feeding test, tannin can significantly induce the expression of CYP321B1 in the midgut and fat body of 4th instar larvae. To verify the function of CYP321B1, RNA interference and metabolome analysis were performed. The results showed that silencing CYP321B1 significantly reduced the rate of weight gain under tannin induction. Metabolome analysis showed silencing affected 47 different metabolites, mainly involved in secondary metabolite biosynthesis and amino acid metabolism, including amino acids, lipid fatty acids, organic acids and their derivatives. Henoxyacetic acid and cysteamine are the most highly regulated metabolites, respectively. These findings demonstrate that CYP321B1 plays an important role in tannin detoxification and metabolism. Functional knowledge about metabolite detoxification genes in this major herbivorous insect pest can provide new insights into this biological process and provide new targets for agricultural pest control.

The cotton-melon aphid Aphis gossypii is a sap-sucking insect that is considered a serious global pest. The species is distributed over a large geographical range and uses a wide variety of hosts, with some populations being specialized to attack different plant species. Here, we provide de novo chromosome-level genome assemblies of a cotton specialist population (Hap1) and a cucurbit specialist population (Hap3). We achieved this by using a combination of third-generation sequencing platforms, namely Illumina and Hi-C sequencing technologies. We were able to anchor a total of 334.89 Mb (scaffold N50 of 89.13 Mb) and 359.95 Mb (scaffold N50 of 68.88 Mb) to four chromosomes for Hap1 and Hap3, respectively. Moreover, our results showed that the X-chromosome of Hap3 (113.01 Mb) was significantly longer than that of Hap1 (100.26 Mb), with a high level of sequence conservation between the aphid species. We also report variation in the number of protein-coding genes and repeat sequences between Hap1 and Hap3. In particular, olfactory and gustatory receptor genes underwent a high level of gene duplication and expansion events in A. gossypii, including between Hap1 and Hap3. Moreover, we identified two glutathione S-transferase genes which underwent single gene duplications in Hap3, and tandem duplication and inversion events affecting the cytochrome P450 monooxygenase between Hap1 and Hap3, all of which include the CYP3 family. Our results illustrate the variance in the genomic composition of two specialized A. gossypii populations and provide a helpful resource for the study of aphid population evolution, host adaption and insecticide resistance.

Propylea japonica has been regarded as one of the most remarkable natural enemies against aphid in China. However, the mechanism of juvenile hormone (JH) regulation of reproduction in P. japonica is still unclear. In this study, we investigated the JH titers of P. japonica and the development of the ovaries. We selected the six different developmental stages of ladybeetle females for transcriptome sequencing. We identified 583 genes involved in insect reproduction regulation, including 107 insect hormone synthesis signaling pathway-related genes and 476 nutrition-sensing signaling pathway-related genes. Transcriptome analysis indicated that a large number JH synthesis- and metabolism-related enzyme genes and some potential nutrient signal sensing- and transduction-related genes were significantly differentially expressed during P. japonica development. We investigated the effects of Met gene silencing on the reproduction of female adults and found that the ovarian maturation, vitellogenesis, and follicular epithelium development in the dsMet treatment group were significantly inhibited.

Background Aphis gossypii, a polyphagous and recurrent pest induced by pesticides, causes tremendous loss in the yield of crops each year. Previous studies of pesticide-induced sublethal effects mechanism mainly focus on gene level. The symbiotic bacteria are also important participants of this mechanism, but their roles in hormesis are still unclear. Results In this study, life table parameters and 16S rRNA sequencing were applied to evaluate the sublethal and transgenerational effects of sulfoxaflor on adult A. gossypii after 24-h LC20 (6.96 mg L-1 ) concentration exposure. The results indicated that the LC20 of sulfoxaflor significantly reduced the finite rate of increase (λ) and net reproductive rate (R0 ) of parent generation (G0), and significantly increased mean generation time (T) of G1 and G2, but not of G3 and G4. Both reproductive period and fecundity of G1 and G2 were significantly higher than those of the control. Furthermore, our sequencing data revealed that more than 95% bacterial communities were dominated by the phylum Proteobacteria, in which the maximum proportion genus was the primary symbiont Buchnera and the facultative symbiont Arsenophonus. Compared to those of the control, the abundance and composition of symbiotic bacteria of A. gossypii for three successive generations (G0-G2) were changed after G0 A. gossypii was exposed to sulfoxaflor: the diversity of the bacterial community was decreased, but the abundance of Buchnera was increased (G0), while the abundance of Arsenophonus was decreased. Contrary to G0, G1 and G2 cotton aphid exhibited an increased relative abundance of Arsenophonus in sublethal treatment group. Conclusion Taken together, our results provide an insight into the interactions among pesticide resistance, aphids, and symbionts, which will eventually help to better manage the resurgence of A. gossypii. This article is protected by copyright. All rights reserved.

The genetically modified cotton holding Bt proteins, is noxious to bollworms larvae but very little is known about its impact on population of non-target spiders within the field. Studies conducted with the aim to identify spiders along with their abundance, prevalence pattern, preying habit on insect pests in Bt cotton (L280, Cry2Ab4) and commonness of symbiotic bacteria in these spiders. Spiders collections were made via vial tapping/jarring, from May to end of October (2018). In total, 13,342 spiders were recorded during the entire cropping season. Least relative abundance was in May (0.28%), highest in August (30.39%) while ahead decline was observed, reached to 12% in October. The 27 species were verified, least richness was in May (3 species), high in August (19 species), later in the end, reduced to 7 species. High abundance was of Hylyphantes graminicola (68.56%) and Neoscona theisi (19.98%). Existence of H. graminicola and Pardosa astrigera observed during the whole cropping season. Presence of seven guilds among spiders had a diverse nature of hunting insect pests and on number of species existed in the crop, maximum was from direct hunting habit. Composition and community structure of microbiota varied as of spider species. Proteobacteria was the most abundant bacterial phylum and topmost genus was Wolbachia in observed spiders. Five groups of bacteria distinguished across 4 clusters of spiders depending on core community of symbiotic bacterial genera. This effort is an initial step to get knowhow about spiders in Bt cotton, which will facilitate future research studies concerning spiders.

The complete mitochondrial genome of Aphis gossypii Glover cucumber biotype was sequenced using traditional PCR amplification coupled with Sanger sequencing. The genome is 15,870 bp long, with 83.7...

Parasitism usually causes considerable changes in lipids and fatty acids by redirecting the development of the host. In this study, changes in weight and in free fatty acid content of cotton aphids were recorded after aphids had been parasitized. Results showed that the weight of parasitized Aphis gossypii was increased compared to nonparasitized aphids, and significantly increased weights were detected at 1, 2, and 3 instars after parasitization by Lysiphlebia japonica. Free fatty acid test kits and GC-MS showed that the fatty acid content increased in the early stage of parasitization but decreased after 3 days of parasitization. Seven genes related to the fatty acid synthesis pathway were significantly upregulated in the parasitized aphids, where they were 1.96-10.97 times greater. Our data described the change that occurs in the fatty acid content of parasitized A. gossypii.

Bacteria and insects have a mutually beneficial symbiotic relationship. Bacteria participate in several physiological processes such as reproduction, metabolism, and detoxification of the host. Adelphocoris suturalis is considered a pest by the agricultural industry and is now a major pest in cotton, posing a serious threat to agricultural production. As with many insects, various microbes live inside A. suturalis. However, the microbial composition and diversity of its life cycle have not been well-studied. To identify the species and community structure of symbiotic bacteria in A. suturalis, we used the HiSeq platform to perform high-throughput sequencing of the V3–V4 region in the 16S rRNA of symbiotic bacteria found in A. suturalis throughout its life stages. Our results demonstrated that younger nymphs (1st and 2nd instar nymphs) have higher species richness. Proteobacteria (87.06%) and Firmicutes (9.43%) were the dominant phyla of A. suturalis. At the genus level, Erwinia (28.98%), Staphylococcus (5.69%), and Acinetobacter (4.54%) were the dominant bacteria. We found that the relative abundance of Erwinia was very stable during the whole developmental stage. On the contrary, the relative abundance of Staphylococcus, Acinetobacter, Pseudomonas, and Corynebacterium showed significant dynamic changes at different developmental stages. Functional prediction of symbiotic bacteria mainly focuses on metabolic pathways. Our findings document symbiotic bacteria across the life cycle of A. suturalis, as well as differences in both the composition and richness in nymph and adult symbiotic bacteria. Our analysis of the bacteria in A. suturalis provides important information for the development of novel biological control strategies.

The predatory ladybird beetle, Harmonia axyridis, is a predominant natural enemy of pest insects in cotton fields. Commercialization of genetically modified crops has promoted the increased use of the herbicide glyphosate. In this study, to assess potential negative effects of glyphosate on beneficial non-target organisms in cotton fields, we first examined how glyphosate exposure affected the development and endosymbiotic bacterial community of H. axyridis. The results showed that the survival rate, development duration, pupation rate and emergence rate of H. axyridis under low and high concentrations of glyphosate exposure were not significantly changed, but glyphosate did significantly reduce the body weight of H. axyridis. Based on 16S rRNA sequencing, there were no significant differences in the diversity or richness of the endosymbiotic bacteria of H. axyridis before and after glyphosate exposure. The dominant bacterial phyla Firmicutes and Proteobacteria and genera Staphylococcus and Enterobacter remained the same regardless of treatment with glyphosate, however the abundance and copy number of these bacteria were altered. Glyphosate treatment significantly reduced the abundance and gene copy number of Staphylococcus and increased the abundance and gene copy number of Enterobacter. This is the first report demonstrating that glyphosate can reduce the body weight H. axyridis and alter the bacterial endosymbiont community by affecting the abundance and gene copy number of dominant bacteria.

Background The worldwide pest Aphis gossypii has three-winged morphs in its life cycle, namely, winged parthenogenetic female (WPF), winged gynopara (GP), and winged male, which are all produced by a wingless parthenogenetic female (WLPF). Most studies on A. gossypii have focused on WPF, while few have investigated GP and male. The shared molecular mechanism underlying the wing differentiation in the three wing morphs of A. gossypii remains unknown. The wing differentiation of WPF was explored in a previous study. Herein, GP and male were induced indoors. The characters of the body, internal genitals, wing veins, and fecundity of GP and male were compared with those of WPF or WLPF. Compared with WLPF, the shared and separate differentially expressed genes (DEGs) were identified in these three-wing morphs. Results Newly-born nymphs reared in short photoperiod condition (8 L:16D, 18 °C) exclusively produced gynoparae (GPe) and males in adulthood successively, in which the sex ratio was GP biased. A total of 14 GPe and 9 males were produced by one mother aphid. Compared with WLPF, the three-wing morphs exhibited similar morphology and wing vein patterns but were obviously discriminated in the length of fore- and underwings, reproductive system, and fecundity. A total of 37 090 annotated unigenes were obtained from libraries constructed using the four morphs via RNA sequencing (RNA-Seq). In addition, 10 867 and 19 334 DEGs were identified in the pairwise comparison of GP versus WLPF and male versus WLPF, respectively. Compared with WLPF, the winged morphs demonstrated 2 335 shared DEGs (1 658 upregulated and 677 downregulated). The 1 658 shared upregulated DEGs were enriched in multiple signaling pathways, including insulin, FoxO, MAPK, starch and sucrose metabolism, fatty acid biosynthesis, and degradation, suggesting their key roles in the regulation of wing plasticity in the cotton aphid. Forty-four genes that spanned the range of differential expression were chosen to validate statistical analysis based on RNA-Seq through the reverse transcription quantitative real time polymerase chain reaction (RT-qPCR). The comparison concurred with the expression pattern (either up- or downregulated) and supported the accuracy and reliability of RNA-Seq. Finally, the potential roles of DEGs related to the insulin signaling pathway in wing dimorphism were discussed in the cotton aphid. Conclusions The present study established an efficiently standardized protocol for GP and male induction in cotton aphid by transferring newly-born nymphs to short photoperiod conditions (8 L:16D, 18 °C). The external morphological characters, especially wing vein patterns, were similar among WPFs, GPe, and males. However, their reproductive organs were strikingly different. Compared with WLPF, shared (2 335) and exclusively (1 470 in WLPF, 2 419 in GP, 10 774 male) expressed genes were identified in the three-wing morphs through RNA-Seq, and several signaling pathways that are potentially involved in their wing differentiation were obtained, including insulin signaling, starch and sucrose metabolism.

Spodoptera litura is a major insect with a cosmopolitan distribution and strong resistance to multiple insecticides. Determining the molecular basis and key candidate genes of the insecticide resistance of S. litura may help in managing this insect. In this study, fifth-instar S. litura larvae were subjected to transcriptome analysis at 6, 12, 24, 48, and 72 h after feeding on an LC20 dose of avermectin. The result showed that genes responding to avermectin changed dynamically with different gene counts and resistance mechanisms at the fifth instar based on a metabolic pathway map. These responses included degrading the insecticide by a series of P450 and glutathione-S-transferase enzymes starting at the 12 h time point, with subsequent increases in the number of genes involved and shifts to TOLL and immune deficiency (IMD) pathways at 48 h after feeding the insecticide. Weighted correlation network analysis (WGCNA) determined a co-expression module related to the avermectin response at 12 and 24 h (r = 0.403, p = 0.0371; r = 0.436, p = 0.023), in which a hub gene (LOC111358940) related to metalloproteinase activity was identified. In addition, Analysis of the genes in the co-expression module further revealed that eight genes encoding UDP-glucuronosyltransferases were directly associated with insecticide response in S. litura. These results provide better understanding of the avermectin response mechanism of S. litura and may be useful in developing improved control strategies for this species. SUPPLEMENTARY INFORMATION: The online version of this article (10.1007/s13205-021-02651-9) contains supplementary material, which is available to authorized users.

Background Endosymbiotic bacteria have been reported to mediate interactions between parasitoids and their insect hosts. How parasitic wasps influence changes in host microbial communities and the relationship between them are of great importance to the study of host-parasitoid co-evolutionary and ecological interactions. However, these interactions remain largely unreported for interactions between in Aphis gossypii and Lysiphlebia japonica. Results In this study, we characterize the bacterial microbiota of L. japonica wasps at different developmental stages and monitor changes over time in the bacterial microbiota of their parasitized and non-parasitized aphid hosts, using metagenomic analysis of 16S rDNA sequencing data. Proteobacteria, Firmicutes, and Actinobacteria were the three most abundant bacterial phyla identified in L. japonica. We found that parasitism was associated with an increased abundance of Buchnera nutritional endosymbionts, but decreased abundance of Acinetobacter, Arsenophonus, Candidatus_Hamiltonella, and Pseudomonas facultative symbionts in aphid hosts. Functional analysis of enriched pathways of parasitized aphids showed significant differences in the 'transport and metabolism of carbohydrates' and 'amino acid, lipid, and coenzyme biosynthesis' pathways. Notably, the composition of symbiotic bacteria in wasp larvae was highly similar to that of their aphid hosts, especially the high abundance of Buchnera. Conclusion The results provide a conceptual framework for L. japonica interactions with A. gossypii in which the exchange of symbiotic microbes provides a means by which microbiota can potentially serve as evolutionary drivers of complex, multilevel interactions underlying the ecology and co-evolution of these hosts and parasites. This article is protected by copyright. All rights reserved.

Understanding of neonicotinoid insecticides toxicity on non-target organisms, such as bees, has indirectly promoted their soil treatment use. However, their effect on soil ecosystems haven’t fully understood. Here, based on 16S rRNA high-throughput sequencing and metagenomics, the effects of neonicotinoid insecticide thiamethoxam on bacterial communities and metabolic functions in two types of soils were studied. Thiamethoxam treatment significantly affected soil bacterial abundance, reduced microbial diversity, and changed the bacterial community structure in the short term, and the structure soon returned to a stable state. Soil type and time were important factors affecting bacterial community structure. Some plant growth-promoting rhizosphere bacteria (PGPR) including Actinobacteria were found, and their populations were reduced, while pollutant-degrading bacteria including Firmicutes were also found, and their populations were increased. Based on metagenomics analysis, thiamethoxam treatment insignificantly promoted or inhibited multiple metabolic processes, but gene abundance of some key processes significantly changed. Subtypes of 18 biodegradation genes (BDGs) and 5 pesticide degradation genes (PDGs) were identified. Thiamethoxam treatment significantly increased the abundance of BDGs and PDGs, including cytochrome P450. Potential hosts of P450 degradation genes, including the genus Rhodococcus, were discovered. Conclusions of this study will promote safety evaluation and degradation-related research on neonicotinoid insecticides in soil.

Background: Three wing morphs exists in the life cycle of the worldwide pest Aphis gossypii, i.e., wing parthenogenetic female (WPF), gynopara (GP) and male, which were produced mostly by crowding and host quality, photoperiod, loss of X chromosome, respectively. However, the shared molecular mechanism underlying their wing differentiation remains an enigma. Here we firstly induced gynoparae and males indoors and compared the characters of these wing morphs in body, internal genitals and fecundity. Then we identified the shared and separate differentially expressed genes (DEGs) and signaling pathways potentially involved in the wing morphs regulation in WPF, GP and male compared to wingless parthenogenetic female (WLPF). Results: Newly-born nymphs reared in short photoperiod condition exclusively produce gynoparae and males in adulthood successively, in which the sex ratio is gynoparae biased. Compared with WLPF, three wing morphs have similar morphology in bodies but is obviously discriminated in the reproductive system and fecundity. Built upon our previous study, 37 090 annotated unigenes were obtained from libraries constructed by the four morphs above through RNA-sequencing, in which 10 867, 19 334 DEGs were identified in pairwise comparison of GP vs. WLPF, Male vs. WLPF, respectively. Furthermore, 2 335 shared DEGs including 1 658 up- and 677 downregulated were obtained in these wing morphs compared to WLPF. The 1 658 shared up-regulated DEGs were enriched in multiple signaling pathways including insulin, FoxO, MAPK, strarch and sucrose metabolism, fatty acid biosynthesis and degradation which hint their key roles in the regulation of wing plasticity in cotton aphid. Gene expression levels were validated by using Pearson’s correlation (r) and potential roles of 15 DEGs related to the insulin signaling pathway in cotton wing dimorphism were discussed. Conclusions: The results of this study establish a solid foundation for deciphering molecular mechanisms underlying the switch between wingless and wing morphs in the cotton aphid and provide valuable resources for future research on the host-alternating aphid species.

Odorant-binding proteins (OBPs) play a significant role in the olfactory signal transduction of insects and help them locate hosts, oviposition sites, and mating partners. The sap-sucking insect, Aphis gossypii Glover (Homoptera: Aphididae), is a destructive cosmopolitan pest and yet the molecular mechanisms by which A. gossypii perceives pheromones and host volatiles remain unknown. In this study, we identified and characterized 6 OBPs, using the A. gossypii RNA-seq transcriptome dataset previously constructed in our laboratory. Real-time PCR indicated specific expression patterns of the 6 genes, which had different levels of expression based on development stage, tissue, morph, and life cycle. In addition, binding specificities of the 6 proteins investigated using the ligand-binding assays showed that all 6 OBPs exhibited high binding affinities towards Phlorizin dehydrate while AgosOBP3 and AgosOBP4 had strong affinity to beta-ionone and AgosOBP8 displayed higher binding affinities for Nerolidol and Cis-3-hexenyl acetate compared to other OBPs.

Lipids play an important role in energy storage, membrane structure stabilization and signaling. Parasitoids are excellent models to study lipidomics because a majority of them do not accumulate during their free-living life-stage. Studies on parasitoids have mostly focused on the changes in the lipids and gene transcripts in hosts and little attention has been devoted to lipidomics and transcriptomics changes in parasitoids. In this study, a relative quantitative analysis of lipids and their gene transcripts in 3-days-old Lysiphlebia japonica larva (3 days after spawning) and pupae were performed using liquid chromatography, mass spectrometry and RNA-seq. Thirty-three glycerolipids and 250 glycerophospholipids were identified in this study; all triglycerides and the vast majority of phospholipids accumulated in the pupal stage. This was accompanied by differentially regulated lipid uptake and remolding. Furthermore, our data showed that gene transcription was up-regulated in key nutrient metabolic pathways involved in lipid synthesis in 3-days-old larvae. Finally, our data suggests that larva and pupa of L. japonica may lack the ability for fatty acids synthesis. A comprehensive, quantitative, and expandable resource was provided for further studies of metabolic regulation and molecular mechanisms underlying parasitic response to hosts defense.

Lysiphlebia japonica (Ashmead) is a predominant parasitoid of cotton-melon aphids in the fields of northern China with a proven ability to effectively control cotton aphid populations in early summer. For accurate normalization of gene expression in L. japonica using quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR), reference genes with stable gene expression patterns are essential. However, no appropriate reference genes is L. japonica have been investigated to date. In the present study, 12 selected housekeeping genes from L. japonica were cloned. We evaluated the stability of these genes under various experimental treatments by RT-qPCR using four independent (geNorm, NormFinder, BestKeeper and Delta Ct) and one comparative (RefFinder) algorithm. We identified genes showing the most stable levels of expression: DIMT, 18S rRNA, and RPL13 during different stages; AK, RPL13, and TBP among sexes; EF1A, PPI, and RPL27 in different tissues, and EF1A, RPL13, and PPI in adults fed on different diets. Moreover, the expression profile of a target gene (odorant receptor 1, OR1) studied during the developmental stages confirms the reliability of the chosen selected reference genes. This study provides for the first time a comprehensive list of suitable reference genes for gene expression studies in L. japonica and will benefit subsequent genomics and functional genomics research on this natural enemy.

Gut microbiota has been suggested as an important component of host defense. However, microbiota in other parts of the body have rarely been characterized. In our present work, we give a detailed view of the body of Spodoptera exigua larvae, the midgut with intestinal contents (MG), midgut excluding intestinal contents (PM), intestinal contents (IC) and remaining parts excluding the MG (RM), with the objective of revealing the complexity of microorganisms and comparing the biodiversity of the S. exigua larvae-associated microbiota. Our results provide the first evidence that third and fifth larvae samples of S. exigua were dominated by members of the phyla Firmicutes and Proteobacteria. In addition, there were no obvious differences in relative abundances of microbiota among MG, PM and RM at the phylum level. MG3 and MG5 (nearly RM5) harbored the richest microbial community, with much higher abundance of Halomonas, Pseudomonas and Methylobacterium, respectively. Intestinal contents contained the lowest diversity and highest abundance of Enterococcus. Knowledge of the bacteria in a major herbivorous insect, such as S. exigua, can facilitate the acquisition of special biological resources for agricultural pest control.

An amendment to this paper has been published and can be accessed via the original article.

Common varieties of genetically modified (GM) cotton increasingly display insect-resistant properties via expression of bacterial-derived toxins from Bacillus thuringiensis (Bt). This necessitates a deeper understanding of the possible effects of these crops on non-target insects. The mirid bug Apolygus lucorum is a major pest in cotton production in China, however, the effect of GM cotton on this non-target species is currently virtually unknown. This insect is exposed to these transgenic plants by consuming genetically modified (GM) leaves. In this study, laboratory experiments were conducted to assess the toxicity of CCRI41 and CCRI45, (genetically modified cotton varieties which express the toxins Cry1Ac and CpTI (Cowpea Trypsin Inhibitor)) on nymphs and adults of A. lucorum. There was no detectable increase in mortality after A. lucorum fed on GM cotton leaves for 20 days. While we detected trace amounts of Cry1Ac proteins in both A. lucorum nymphs and adults (10 ng/g fresh weight), the expression of genes related to detoxification did not detectably differ from those feeding on non-GM cotton. Our binding assays did not show Cry1Ac binding to receptors on the midgut brush border membrane from either A. lucorum nymphs or adults. Our findings collectively indicate that feeding on leaves of the GM cotton varieties CCRI41 and CCRI45 have few toxic effects on A. lucorum.

Reference genes have been utilized in estimating gene expression levels using quantitative reverse transcriptase-quantitative polymerase chain reaction (qRT-PCR) analysis. Aphidius gifuensis Ashmaed is one of the most widely used biological control agents for aphids. The biological properties of this species have been studied in detail, and current investigations are focused on elucidating the regulatory mechanisms in its host However, the appropriate reference genes for target gene expression studies have not been identified. In this study, the expression profiles of 12 candidate reference genes were evaluated under different experimental conditions(development stage, sex, tissue type, diet) by using dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt. In addition, RefFinder was used to rank the overall stability of the candidate genes. Finally, we recommend three optimal reference genes for the normalization of qRT-PCR data in the presence of specific variables, which include ACTB, RPL13, and PPI for different developmental stages; RPS18, ACTB, and RPL13 for sexes; RPL13, PRII3, and RPS18 in different tissue types; and RPL13, RPL27, and ACTB in diverse diets. The present study has identified optimal reference genes that could be used in estimating the expression levels of specific genes under these conditions following the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines, which would facilitate in advancements in functional genomics research on A. gifuensis.