Role In Insect Reproduction Research Articles

MiRNAs targeted transcription factors HaGATAa/b to mediate the post-mating switch in Helicoverpa armigera female reproductive behavior.

The process of mating induces significant shifts in female reproductive behavior across various species, with the postmating behavioral switch playing a crucial role in insect reproduction. Previous studies have demonstrated the regulatory role of GATA transcription factors in vitellogenin transcription and egg formation in insects, while miRNAs have been implicated in modulating GATA expression and insect reproductive processes. Nevertheless, the precise regulatory mechanism underlying the interaction between miRNAs and GATA transcription factors in the postmating behavioral switch remains largely unexplored. In this study, we identified two key GATA transcription factors, HaGATAa and HaGATAb, as central players in orchestrating the postmating behavior of H. armigera using transcriptomics and RNAi technologies. HaGATAa was found to act upstream of HaGATAb, regulating its expression. Furthermore, we observed a postmating increase in miR-282 levels in females, targeting HaGATAa to regulate egg-laying capacity. Conversely, the decreased expression of miR-2 following mating functioned as a negative feedback regulator, influencing the expression of HaGATAb and thus impacting the postmating behavior of female individuals. Our results revealed a signal-mediated feedback regulatory mechanism that sustains female postmating behavior in H. armigera. These findings not only establish a strong basis for understanding the postmating behavior mechanisms in female moths, but also offer valuable insights for identifying potential targets for pest control strategies. © 2024 Society of Chemical Industry.

Knockdown of the salivary protein gene NlG14 caused displacement of the lateral oviduct secreted components and inhibited ovulation in Nilaparvata lugens.

Saliva plays important roles in insect feeding, but its roles in insect reproduction were rarely reported. Here we reported that the knockdown of a salivary gland-specific gene NlG14 disrupted the reproduction through inhibiting the ovulation of the brown planthopper (BPH), Nilaparvata lugens (Stål), one of the most devastating rice pests in Asia. NlG14 knockdown caused the displacement of the lateral oviduct secreted components (LOSC), leading to the ovulation disorder and the accumulation of mature eggs in the ovary. The RNAi-treated females laid much less eggs than their control counterparts, though they had the similar oviposition behavior on rice stems as controls. NlG14 protein was not secreted into the hemolymph, indicating an indirect effect of NlG14 knockdown on BPH reproduction. NlG14 knockdown caused the malformation of A-follicle of the principal gland and affected the underlying endocrine mechanism of salivary glands. NlG14 reduction might promote the secretion of insulin-like peptides NlILP1 and NlILP3 from the brain, which up-regulated the expression of Nllaminin gene and then caused the abnormal contraction of lateral oviduct muscle. Another explanation was NlG14 reduction disrupted the ecdysone biosynthesis and action through the insulin-PI3K-Akt signaling in ovary. Altogether, this study indicated that the salivary gland specific protein NlG14 indirectly mediated BPH ovulation process, which established a connexon in function between insect salivary gland and ovary.

Molecular Characterization of Vitellogenin and Its Receptor in Spodoptera frugiperda (J. E. Smith, 1797), and Their Function in Reproduction of Female.

The fall armyworm Spodoptera frugiperda is a highly polyphagous invasive pest. The strong reproductive capacity is an important factor in the rapid colonization and expansion of S. frugiperda. Vitellogenin (Vg) and vitellogenin receptor (VgR) play important roles in insect reproduction. As the precursor of vitellin (Vn), Vg provides essential nutrition for embryonic development, and VgR mediates the uptake of Vg by oocytes. In this context, we cloned and characterized these two genes of S. frugiperda (SfVg and SfVgR) and evaluated their expression profiles in different developmental stages and tissues. The RNA interference experiment was used to investigate their function in vitellogenesis. The ORF values of SfVg and SfVgR were 5250 and 5445 bp, encoding 1749 and 1815 amino acid residues, respectively. The qRT-PCR results revealed that both SfVg and SfVgR were highly expressed in female adults; SfVg was specifically expressed in the fat body, whereas SfVgR was highly expressed in the ovary. In addition, the depletion of either SfVg or SfVgR hindered oocyte maturation and ovarian development, leading to a significant decrease in fecundity. The present study reveals the importance of SfVg and SfVgR in the vitellogenesis of S. frugiperda, laying a theoretical foundation for the development of pollution-free pest control strategies with SfVg and SfVgR as new targets.

Chromatin-Remodelling ATPases ISWI and BRM Are Essential for Reproduction in the Destructive Pest Tuta absoluta.

The tomato leaf miner (Tuta absoluta) is one of the top 20 plant pests worldwide. We cloned and identified the chromatin-remodelling ATPase genes ISWI and BRM by RACE and bioinformatic analysis, respectively; used RT-qPCR to examine their expression patterns during different life cycle stages; and elucidated their roles in insect reproduction using double-stranded RNA injections. The full-length cDNA of TaISWI was 3428 bp and it encoded a 1025-aa polypeptide. The partial-length cDNA of TaBRM was 3457 bp and it encoded a 1030-aa polypeptide. TaISWI and TaBRM were upregulated at the egg stage. Injection of TaISWI or TaBRM dsRNA at the late pupa stage significantly inhibited adult ovary development and reduced fecundity, hatchability, and longevity in the adult females. To the best of our knowledge, the present study was the first to perform molecular characterisations of two chromatin-remodelling ATPase genes and clarify their roles in T. absoluta fecundity. Chromatin-remodelling ATPases are potential RNAi targets for the control of T. absoluta and other insect pests. The present study was also the first to demonstrate the feasibility of reproductive inhibitory RNAi as a putative approach for the suppression of T. absoluta and other Lepidopteran insect populations.

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET–TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

Molecular characterization and enzyme inhibition studies of NADP+- farnesol dehydrogenase from diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae).

Juvenile hormone III (JH III) plays an important role in insect reproduction, development, and behavior. The second branch of JH III production includes oxidation of farnesol to farnesal by farnesol dehydrogenase. This study reported the identification and characterization of Plutella xylostella farnesol dehydrogenase (PxFoLDH). Our results showed that PxFoLDH belongs to the short-chain dehydrogenase/reductase superfamily, consisting of a single domain with a structurally conserved Rossman fold, an NAD(P) (H)-binding region and a structurally diverse C-terminal region. The purified enzyme displayed maximum activity at 55$\ $°C with pH 9.5 and was stable in the temperature below 70$\ ^\circ $C. PxFoLDH was determined to be a monomer with a relative molecular weight of 27 kDa and highly specific for trans, trans-farnesol, and NADP+.Among analog inhibitors tested, farnesyl acetate was the most effective inhibitor with the lowestKivalue of 0.02 µm. Our findings showed this purified enzyme may represent as NADP+-farnesol dehydrogenase.

Neutral Ceramidase Is Required for the Reproduction of Brown Planthopper, Nilaparvata lugens (Stål).

Ceramides are bioactive sphingolipids that have been implicated in insect development; however, their role in insect reproduction remains poorly understood. Here, we report the pivotal role of neutral ceramidase (NCER) in the female reproduction of the brown planthopper (BPH), Nilaparvata lugens (Stål), a significant pest in rice cultivation in Asia. LC-MS/MS demonstrated that, among different developmental stages of BPH, the levels of ceramides were highest in 1st instar nymphs and lowest in adults. The transcription of NCER was negatively correlated with the levels of ceramides at different developmental stages of BPH, in that the transcript levels of NCER were the highest, whereas ceramides levels were the lowest in BPH adults. Knocking down NCER through RNA interference (RNAi) increased the levels of ceramides in BPH females and ovaries, which resulted in a delay in oocyte maturation, a reduction in oviposition and egg hatching rate, as well as the production of vulnerable offspring. Transmission electron microscopy (TEM) analysis and TdT-mediated dUTP Nick-End Labeling (TUNEL) assays showed mitochondrial deficiency and apoptosis in NCER-deficient oocytes. Taken together, these results suggest that NCER plays a crucial role in female reproduction in BPH, likely by regulating the levels of ceramides.

DNA methylation suppresses chitin degradation and promotes the wing development by inhibiting Bmara-mediated chitinase expression in the silkworm, Bombyx mori

BackgroundDNA methylation, as an essential epigenetic modification found in mammals and plants, has been implicated to play an important role in insect reproduction. However, the functional role and the regulatory mechanism of DNA methylation during insect organ or tissue development are far from being clear.ResultsHere, we found that DNA methylation inhibitor (5-aza-dC) treatment in newly molted pupae decreased the chitin content of pupal wing discs and adult wings and resulted in wing deformity of Bombyx mori. Transcriptome analysis revealed that the up-regulation of chitinase 10 (BmCHT10) gene might be related to the decrease of chitin content induced by 5-aza-dC treatment. Further, the luciferase activity assays demonstrated that DNA methylation suppressed the promoter activity of BmCHT10 by down-regulating the transcription factor, homeobox protein araucan (Bmara). Electrophoretic mobility shift assay, DNA pull-down and chromatin immunoprecipitation demonstrated that Bmara directly bound to the BmCHT10 promoter. Therefore, DNA methylation is involved in keeping the structural integrity of the silkworm wings from unwanted chitin degradation, as a consequence, it promotes the wing development of B. mori.ConclusionsThis study reveals that DNA methylation plays an important role in the wing development of B. mori. Our results support that the indirect transcriptional repression of a chitin degradation-related gene BmCHT10 by DNA methylation is necessary to keep the proper wing development in B. mori.

N-glycosylation Site Analysis Reveals Sex-related Differences in Protein N-glycosylation in the Rice Brown Planthopper (Nilaparvata lugens)

Glycosylation is a common modification of proteins and critical for a wide range of biological processes. Differences in protein glycosylation between sexes have already been observed in humans, nematodes and trematodes, and have recently also been reported in the rice pest insect Nilaparvata lugens Although protein N-glycosylation in insects is nowadays of high interest because of its potential for exploitation in pest control strategies, the functionality of differential N-glycosylation between sexes is yet unknown. In this study, therefore, the occurrence and role of sex-related protein N-glycosylation in insects were examined. A comprehensive investigation of the N-glycosylation sites from the adult stages of N. lugens was conducted, allowing a qualitative and quantitative comparison between sexes at the glycopeptide level. N-glycopeptide enrichment via lectin capturing using the high mannose/paucimannose-binding lectin Concanavalin A, or the Rhizoctonia solani agglutinin which interacts with complex N-glycans, resulted in the identification of over 1300 N-glycosylation sites derived from over 600 glycoproteins. Comparison of these N-glycopeptides revealed striking differences in protein N-glycosylation between sexes. Male- and female-specific N-glycosylation sites were identified, and some of these sex-specific N-glycosylation sites were shown to be derived from proteins with a putative role in insect reproduction. In addition, differential glycan composition between males and females was observed for proteins shared across sexes. Both lectin blotting experiments as well as transcript expression analyses with complete insects and insect tissues confirmed the observed differences in N-glycosylation of proteins between sexes. In conclusion, this study provides further evidence for protein N-glycosylation to be sex-related in insects. Furthermore, original data on N-glycosylation sites of N. lugens adults are presented, providing novel insights into planthopper's biology and information for future biological pest control strategies.

Molecular characterization of the Krüppel-homolog 1 and its role in ovarian development in Sogatella furcifera (Hemiptera: Delphacidae).

Juvenile hormone (JH) plays a pivotal role in insect reproduction. The Krüppel-homolog 1 (Kr-h1) is a JH-inducible zinc finger transcription factor that has also been found to play a role in insect reproduction, however, its function varies across species. In this study, we cloned SfKr-h1 from Sogatella furcifera and investigated its role in ovarian development. The open reading frame of SfKr-h1 is 1 800bp encoding 599 amino acids. The putative amino acid sequence of SfKr-h1 contains eight putative C2H2-type zinc finger domains and is highly homologous with the Kr-h1s of other hemipteran species. Expression of SfKr-h1 peaked 96h after adult emergence and was highest in the ovary. RNA interference (RNAi) knockdown of SfKr-h1 substantially reduced the transcription of SfVg, and arrested ovarian development. These results suggest that SfKr-h1 is critical for normal ovarian development in S. furcifera.

Studies on the Epiphallus and spermatheca in some species of the genus Acrotylus (Acridoidea: Orthoptera) of Pakistan

The study of Acridoidea is one of the significant work for the aim of insects systematic process. This Order Orthoptera comprised of insects with paurometabolus type metamorphosis,including grasshoppers,Crickts and locusts.During present work five species and subspecies of the genus Acrotylus were observed namely Acrotylus humbertianus (Saussure), A. insubricus insubricus (Scopoli) A. patruelis(Herrich-Schaffer) A. longipes longipes (Charpentier) and A. longipes subfasciatus . The spermatheca is an organ playing a key role in insect reproduction, it allowing the storage of spermatozoa and making them available when it needed, More ever there is information about the genitalia of these grasshoppers .Where as some information is present in the literature on morphology of species of the genus Acrotylus (Fieber).At present my studies expose the diagnostic characters of epiphallus and Spermatheca that have useful value for the purpose of accurate identification. Epiphallus with moderately wide bridge, narrow ancorae and bilobate lophi. Spermatheca is a coiled duct of ectodermal origion. It is of varying sizes and shapes. The spermatheca well dilates to form a sac like structure, for storing the spermatozoa which enter during copulation. Where as this study will provide the beneficial support for new research workers in future.

Cell-free expression, purification and characterization of Drosophila melanogaster odorant receptor OR42a and its co-receptor

Olfactory receptors (OR), a group of classic membrane proteins, plays a vital role in insect reproduction and acclimatization. Deciphering the molecular mechanism of insect olfaction could enhance pest control and environmental protection. Studies on ORs have faced a major bottleneck due to the notoriously strong hydrophobicity of ORs, which results in difficult expression in heterologous cell systems. Here, we demonstrated that insect ORs could be functionally produced using the E. coli cell-free protein synthesis system (CFPS), in which the highest yield of total ORs is 350 μg per 1 ml reaction. We tested the effects of detergent types and concentrations on soluble expression of ORs. The ORs showed a classic α-helical infrared spectrum. Quartz crystal microbalance (QCM) was used to demonstrate that ORs fold correctly and respond to their ligands. This is the first report that insect OR42a could be functionally produced in vitro. This approach may facilitate the development of biomimetic olfactory biosensors and may also be utilized for drug positioning and development, environmental protection and agriculture.

Amelogenin domain‐containing NlChP38 is necessary for normal ovulation in the brown planthopper

The brown planthopper (BPH), Nilaparvata lugens, is a major threat to rice production. The eggshell plays an important role in insect reproduction. The constituents and formation process of BPH eggshells remains largely unknown. Here, we report a novel eggshell-associated protein, NlChP38, containing an amelogenin domain, that is essential for normal ovulation in the BPH. NlChP38 is specifically expressed in the follicular cells from egg chambers at both RNA and protein levels. RNA interference of NlChP38 resulted in oocytes with loose and thin eggshell structure and caused ovulation difficulties. Immunofluorescence localization showed NlChP38 is deposited between follicular cells and oocytes during late choriogenesis. These results indicate that NlChP38 plays an important role in eggshell formation and could be a potential target for RNA interference control of the BPH.

Juvenile hormone signaling in insect oogenesis.

Juvenile hormone (JH) plays a crucial role in insect reproduction, but its molecular mode of action only became clear within the last decade. We here review recent findings revealing the intricate crosstalk between JH and ecdysone signaling with nutrient sensing pathways in Drosophila melanogaster, Aedes aegypti, Tribolium castaneum and Locusta migratoria. The finding for a critical role of ecdysis triggering hormone (ETH) in both molting and ooogenesis now also highlights the importance of an integrated view of development and reproduction. Furthermore, insights from non-model insects, especially so social Hymenoptera and termites, where JH function gradually becomes decoupled from reproduction and plays a role in division of labor, emphasize the need to consider life cycle and life history strategies when studying insect reproductive physiology.

Lipophorin receptor regulates Nilaparvata lugens fecundity by promoting lipid accumulation and vitellogenin biosynthesis

Insect lipophorin receptor (LpR) belongs to the low-density lipoprotein receptor (LDLR) superfamily and plays an essential role in fecundity by mediating the incorporation of lipophorin into developing oocytes. Here we report the identification and characterization of a full-length cDNA encoding a putative LpR from the brown planthopper, Nilaparvata lugens. The deduced amino acid sequence of NlLpR possesses the conserved structural motifs of LDLR family members, and displays a high degree of similarity with sequences from other insect LpRs. NlLpR is transcribed throughout oogenesis with its maximum level on day 7 after adult female emergence. NlLpR is highly expressed in the fat body and ovary, with relative low levels in the head, epidermis and midgut. Knockdown of NlLpR using double-stranded RNA (dsRNA) led to decreased triacylglycerol (TAG) content, retarded development of ovaries and decreased fecundity. Further functional analyses revealed that NlLpR works through nutritional signaling pathway-dependent activation of S6 kinase to regulate vitellogenin (Vg) biosynthesis during vitellogenesis and oocyte development. Disrupting of ecdysone receptor (EcR) expression and 20-hydroxyecdysone (20E) topical application demonstrated that NlLpR is regulated by ecdysone at transcript level. These results suggest that LpR is essential for Vg synthesis in the fat body and lipid uptake by developing oocytes, thus playing a critical role in insect reproduction.

Dynamic Notch Signaling Specifies Each Cell Fate in Drosophila Spermathecal Lineage.

Spermathecae are glandular organs in the insect female reproductive tract that play essential roles in insect reproduction; however, the molecular mechanism involved in their development is largely unknown. Drosophila spermathecae consist of class-III secretory units, in which each secretory cell (SC) discharges its products to the central lumen through an end-apparatus and a canal. Secretory unit formation in Drosophila spermathecae utilizes a fixed cell lineage, in which each secretory unit precursor (SUP) divides to produce one pIIb cell and one pIIa cell. The former differentiates into an apical cell (AC), whereas the latter divides again to produce an SC and a basal cell (BC). It is unclear how each cell acquires its identity and contributes to secretory unit formation. Here, we demonstrate that Notch signaling is required and sufficient for the specification of lumen epithelial precursors (LEPs; vs. SUPs), pIIb (vs. pIIa), and SCs (vs. BCs) sequentially. To our surprise, Notch activation in LEPs and SCs apparently utilizes different ligand mechanisms. In addition, Notch signaling both suppresses and activates transcription factors Hindsight (Hnt) and Cut during spermathecal lineage specification, supporting the notion that Notch signaling can have opposite biological outcomes in different cellular environments. Furthermore, LEP-derived epithelial cells (ECs) and ACs show distinct cellular morphology and are essential for securing secretory units to the epithelial lumen. Our work demonstrates, for the first time, the dynamic role of Notch signaling in binary cell fate determination in Drosophila spermathecae and the role of ECs and ACs in secretory unit formation.

Endocrine disruption of sexual selection by an estrogenic herbicide in the mealworm beetle (Tenebrio molitor)

The role that endocrine disruption could play in sexual selection remains relatively untested, and although estrogens occur in insects, little information exists about their biological role in insect reproduction. Atrazine is a commonly applied herbicide that mimics estrogen in vertebrates. Tenebrio molitor were raised from egg to adult under a gradation of environmentally relevant atrazine exposures and a non-treated control. Atrazine was delivered in the drinking water ad libitum. Female T. molitor were provided with a choice between unrelated males raised under three levels of atrazine exposures. Female preference for males demonstrated a non-monotonic inverted U-shaped response to atrazine exposure. There was no significant difference between the control and the high exposure to atrazine. Excluding the control, female preference increased as exposure concentration increased. These results have important repercussions for nonlethal effects of endocrine disruption on populations, their capacity to interfere with sexual selection, and the role of estrogen in pheromone communication among insects.

Farnesyl Phosphatase, a Corpora allata Enzyme Involved in Juvenile Hormone Biosynthesis in Aedes aegypti

BackgroundThe juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The late steps of JH III biosynthesis in the mosquito Aedes aegypti involve the hydrolysis of farnesyl pyrophosphate (FPP) to farnesol (FOL), which is then successively oxidized to farnesal and farnesoic acid, methylated to form methyl farnesoate and finally transformed to JH III by a P450 epoxidase. The only recognized FPP phosphatase (FPPase) expressed in the corpora allata (CA) of an insect was recently described in Drosophila melanogaster (DmFPPase). In the present study we sought to molecularly and biochemically characterize the FPP phosphatase responsible for the transformation of FPP into FOL in the CA of A. aegypti.MethodsA search for orthologs of the DmFPPase in Aedes aegypti led to the identification of 3 putative FPPase paralogs expressed in the CA of the mosquito (AaFPPases-1, -2, and -3). The activities of recombinant AaFPPases were tested against general phosphatase substrates and isoprenoid pyrophosphates. Using a newly developed assay utilizing fluorescent tags, we analyzed AaFPPase activities in CA of sugar and blood-fed females. Double-stranded RNA (dsRNA) was used to evaluate the effect of reduction of AaFPPase mRNAs on JH biosynthesis.Conclusions AaFPPase-1 and AaFPPase-2 are members of the NagD family of the Class IIA C2 cap-containing haloalkanoic acid dehalogenase (HAD) super family and efficiently hydrolyzed FPP into FOL. AaFPPase activities were different in CA of sugar and blood-fed females. Injection of dsRNAs resulted in a significant reduction of AaFPPase-1 and AaFPPase-2 mRNAs, but only reduction of AaFPPase-1 caused a significant decrease of JH biosynthesis. These results suggest that AaFPPase-1 is predominantly involved in the catalysis of FPP into FOL in the CA of A. aegypti.

Aldehyde dehydrogenase 3 converts farnesal into farnesoic acid in the corpora allata of mosquitoes

The juvenile hormones (JHs) play a central role in insect reproduction, development and behavior. Interrupting JH biosynthesis has long been considered a promising strategy for the development of target-specific insecticides. Using a combination of RNAi, in vivo and in vitro studies we characterized the last unknown biosynthetic enzyme of the JH pathway, a fatty aldehyde dehydrogenase (AaALDH3) that oxidizes farnesal into farnesoic acid (FA) in the corpora allata (CA) of mosquitoes. The AaALDH3 is structurally and functionally a NAD+-dependent class 3 ALDH showing tissue- and developmental-stage-specific splice variants. Members of the ALDH3 family play critical roles in the development of cancer and Sjögren–Larsson syndrome in humans, but have not been studies in groups other than mammals. Using a newly developed assay utilizing fluorescent tags, we demonstrated that AaALDH3 activity, as well as the concentrations of farnesol, farnesal and FA were different in CA of sugar and blood-fed females. In CA of blood-fed females the low catalytic activity of AaALDH3 limited the flux of precursors and caused a remarkable increase in the pool of farnesal with a decrease in FA and JH synthesis. The accumulation of the potentially toxic farnesal stimulated the activity of a reductase that converted farnesal back into farnesol, resulting in farnesol leaking out of the CA. Our studies indicated AaALDH3 plays a key role in the regulation of JH synthesis in blood-fed females and mosquitoes seem to have developed a “trade-off” system to balance the key role of farnesal as a JH precursor with its potential toxicity.

A Quantitative Assay for the Juvenile Hormones and Their Precursors Using Fluorescent Tags

BackgroundThe juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The lipophilic nature of JHs and their precursors, in conjunction with their low concentration in tissues and susceptibility to degradation had made their quantification difficult. A variety of methods exist for JH quantification but few can quantify on the femtomole range. Currently applied methods are expensive and time consuming. In the present study we sought to develop a novel method for accurate detection and quantification of JHs and their precursors.MethodsA sensitive and robust method was developed to quantify the precursor, farnesoic acid (FA) and juvenile hormone III (JH III) in biological samples. The assay is based on the derivatization of analytes with fluorescent tags, with subsequent analysis by reverse phase high performance liquid chromatography coupled to a fluorescent detector (HPLC-FD). The carboxyl group of FA was derivatized with 4-Acetamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH). Tagging the epoxide group of JH III required a two-step reaction: the opening of the epoxide ring with sodium sulfide and derivatization with the fluorescent tag 4-(N,N-Dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methylamino)-2,1,3-benzoxadiazole (DBD-COCl).ConclusionsThe method developed in the present study showed high sensitivity, accuracy and reproducibility. Linear responses were obtained over the range of 10–20 to 1000 fmols. Recovery efficiencies were over 90% for JH III and 98% for FA with excellent reproducibility.SignificanceThe proposed method is applicable when sensitive detection and accurate quantification of limited amount of sample is needed. Examples include corpora allata, hemolymph and whole body of female adult Aedes aegypti and whole body Drosophila melanogaster. A variety of additional functional groups can be targeted to add fluorescent tags to the remaining JH III precursors.