Molecular and expression characterization of insulin-like signaling in development and metabolism of Aedes albopictus mosquito

doi:10.21203/rs.3.rs-2076538/v1

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Research Article

Molecular and expression characterization of insulin-like signaling in development and metabolism of Aedes albopictus mosquito

https://doi.org/10.21203/rs.3.rs-2076538/v1

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published 18 Apr, 2023

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Background

Insulin-like signaling (IS) is a conserved pathway in all metazoans. In insects, IS pathway is activated by the binding of Insulin-like peptides (ILPs) and insulin receptor (InR), and is involved in the development, reproduction, and longevity by regulating ERK/AKT cascades. A varying number of ILPs were identified in the Aedes aegypti mosquitos and other insects. Aedes Albopictus (Ae. Albopictus), a major vector for transmitting dengue fever, is fast spreading worldwide. Until now, the molecular and expression characteristics of IS pathway have not been investigated in Ae. Albopictus.

Methods

The orthologues of ILP in Ae. albopictus genome assembly were analyzed by using sequence blast. Phylogenetic analysis and molecular characterization were performed to identify the functional domains of Ae.albopictus ILPs (AalbILPs). Quantitative analysis was performed to determine the expression characteristics of ILPs, InR and ERK/AKT cascades in the lifespan, and different tissues of female adults after blood feeding. In addition, knockdown of InR was achieved by feeding larvae with E.coli-producing dsRNA to investigate the impact of IS pathway on mosquito development.

Results

We identified seven putative AalbILP genes in the Ae. Albopictus genome assembly, based on nucleotide similarity to the ILPs of Ae. Aegypti and other insects. Bioinformatics and molecular analyses suggested that the AalbILPs contain the structural motif which is conserved in the insulin superfamily. Expression levels of AalbILPs, InR and ERK/AKT varied among developmental stages and between male and female Ae. Albopictus. Quantitative analyses revealed that expression of ILP6, the putative orthologue of the IGF, was highest in midgut of female adults after blood meals. Knockdown of InR induces a significant decrease in the phosphorylation levels of ERK and AKT proteins, and results in developmental delays and smaller body sizes of Ae. Albopictus mosquito.

Conclusions

The IS pathway of Ae. Albopictus mosquito contains ILP1-7, InR, and ERK/AKT cascades, which exhibited different developmental and tissue expression characteristics. Feeding larvae with E.coli-producing InR dsRNA blocks the ERK and AKT cascades and interferes with the development of Ae. Albopictus mosquito. Our data suggest that IS pathway plays an important role in the metabolism and developmental process and could represent a target for controlling mosquito-borne diseases.

Aedes albopictus

insulin-like peptides

insulin receptor

metabolism

development

dsRNA

Insulin-like signaling (IS) is a conserved pathway in all metazoan species and plays a critical role in regulating metabolism, growth, reproduction, and lifespan [1, 2]. In insects, the IS pathway is regulated by insulin superfamily peptide hormones, such as insulin-like peptides (ILPs) and insulin growth factor peptides (IGFPs) [2–4]. Most insect ILP/IGFLP share a common structural motif called the insulin fold, containing a signal peptide and B-chain, C-chain, and A peptides [2, 5]. The number of ILPs and IGFPs varies according to different insect species [4–6]. To date, eight ILPs in the genus Drosophila [4], five to eight ILPs in mosquitoes [5, 6], and 38 ILPs in Bombyx mori [7] have been identified.

Insect ILPs display differential expression characteristics in specific tissues and developmental stages [4–6]. For instance, In eight ILPs of Drosophila (DILP1-8), the expression of DILP1,-2, -3, and − 5 are detected in the brains, while transcription of DILP4,-6, and − 7 are found in the midgut, fat body, and imaginal discs [4]. Similar spatiotemporal ILP expression patterns were also found in Ae. Aegypti mosquito [5]. Transcripts of five ILPs of Ae. Aegypti (AaegILP1,-3,-4,-7,-8) occurred predominantly in the heads (brains) of larval, pupal, and adult mosquitoes, however, transcripts of AaegILP2,-5, and − 6 were present not only in the head but also in the thorax and abdomens of all stages [5]. In Anopheles mosquitoes, some ILP transcript changes in tissues from younger females relative to patterns observed in older females [6].

The IS pathway is initiated by the binding of ILPs to the receptor InR [8, 9]. As a result of ILPs stimulation, the InR undergoes tyrosine phosphorylation [8], and employs a group of adaptor molecules, known as insulin receptor substrates (IRSs), to initiate the phosphorylation of two main protein kinases: the extracellular regulated protein kinases (ERK) and the intracellular protein kinase B (also named AKT) [1, 10, 11]. The IS pathway regulates the nutrient metabolism processes and cell cycle responses by ERK / AKT cascades to affect development and reproduction [12], as well as host-pathogen interactions[13–15]. To date, single InR was discovered in flies and mosquitoes [4, 16]. Blockade of IS pathway by knockdown of InR in several medical insects results in growth defects, metabolic abnormalities, and pathogen inhibition [17–20].

Ae. albopictus, also named “Asian tiger mosquito”, is the most invasive mosquito species which has been introduced to North and South America, Africa, Australia, and Europe outside its original zones [21]. Ae. albopictus also transmits more than 25 arboviruses, including dengue, chikungunya, and zika virus [22]. Its fast adaptation to environments and the increasing resistance to common insecticides make the development of an efficient control strategy challenging [23]. In this study, we describe the expression characterization of the ILPs gene family from Ae. Albopictus (AalbILPs). We identified seven putative ILPs genes (Aalb ILP1–7 ) and one InR in the Ae. Albopictus genome assembly, and characterized the expression features of ILPs and InR genes, as well as ERK and AKT proteins during mosquito development and tissue of female adults following consumption of blood meals. We investigated the functional role of InR in mosquito development by feeding the larvae with dsRNA produced by E.coli expression system. Following the knockdown of InR in Ae. Albopictus larvae, we observed a significant delay of development and a reduction in the wing size of Ae. Albopictus female adults. These results provide insights into the molecular characteristic of IS pathway and its role during Ae. Albopictus development and metabolism.

Sequence alignments and phylogenetic tree analysis

Putative AalbILPs sequences from Ae. albopictus genome were investigated with the known Ae. aegypti ILPs sequences using tblastn in NCBI website(https://www.ncbi.nlm.nih.gov). ILP gene sequences of Ae. Aegypti, Anopheles gambiae (An. gambiae), and Drosophila melanogaster (D. melanogaster) were aligned with putative AalbILPs sequences using ClustalX. The phylogenetic analysis was performed in the neighbor-joining method using the MEGA X phylogenetic software package. The signal peptides (SP) were predicted processing sites by SignalP 5.0, and The amino acid alignments of ILPs were performed by using ESPript 3.0. The conserved amino acids in ILPs motif and functional domains were identified by comparison to the sequences of the insulin superfamily published before [24]. The accession numbers of ILP mRNA sequences in NCBI are shown (Additional file: Table S1).

Mosquito rearing, blood feeding, and tissues dissection

Ae. albopictus (Foshan strain) mosquitoes were maintained under the condition of 28˚C, 85% humidity with a 12-hour light/dark photoperiod. The adult mosquitoes were provided with cotton pads soaked in a 10% sucrose solution. Larvae were fed with ground yeast. Female adults were starved for 16 h before the blood feeding. The blood was donated by healthy volunteers who provided written informed consent. Mosquitoes were allowed to feed the blood meals containing 1 mM ATP by using an artificial membrane feeder at 37°C water jacket for 2h. Only fully engorged mosquitoes were used for the experiment.

Four developmental stages of mosquitoes: eggs, larvae (L1 to L4 stages), pupae, and adults༈female and male༉were collected to examine the temporal expression of IS pathway. Mosquitoes samples were prepared as described before [25]. In brief, mosquitoes were cold anesthetized, surfaced sterile in 75% ethanol, and washed twice with sterile H₂O. The different tissues of female adults including the head, thorax, fatbody, midgut, and ovary were separately dissected on ice. Around 20 individuals in each treatment were placed into 1.5 mL sterile Eppendorf tube containing 250 µL PBS buffer and homogenized in ice for 1 min. Then the tissue homogenate was centrifuged at 12,000g for 5 min at 4°C, and the supernatant was transferred to a new tube for RNA extraction.

RNA extraction and gene expression analyses

Total RNA was isolated from the tissue homogenate using TRIzol reagent (Invitrogen) and treated with DNase. The cDNA was generated from total RNA using HiScript II Q Select RT Super-Mix (Vazyme Biotech, Nanjing, China) according to the manufacturer's instructions. Approximately 500 ng of total cDNA was employed for gene expression analyses by using the quantitative PCR (qPCR) method.

ChamQ SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) was used for qPCR analysis with ABI QuantStudio 6 Pro Real-Time PCR System (Applied Biosystems, CA). The specific primers for Ae.albopictus ILP1-7 and InR genes are listed (Additional file: Table S2). The ribosomal S7 (RPS7) gene was used as the reference gene. Relative gene expression in different developmental stages was assessed by normalization to the levels of the RPS7 gene (2^−ΔCt). The comparative Ct (2^−ΔΔCt) method was used to calculate the relative expression of ILPs and InR genes in different tissues of blood-fed female adults compared to the sugar-fed adults.

Western blotting analysis

Mosquito homogenate samples were lysed in 150µL RIPA lysis buffer with phosphatase and protease inhibitors. After centrifugation of 12,000 g for 10 min, the supernatants were collected, and the protein concentrations were detected by using a BCA protein assay kit. Total protein samples were separated on 12% SDS-PAGE polyacrylamide gels and transferred to a polyvinyl difluoride (PVDF) membrane. After blocking with 5% milk in Tris-buffered solution Tween-20 (TBS-T), the PVDF membrane was labeled with primary antibodies which are separately used for detecting the total and phosphorylation ERK (1:1000), as well as total and phosphorylation AKT (1:1000) overnight at 4°C. Subsequently, secondary antibodies of HRP-conjugated goat anti-rabbit IgG (1:2000) were added and incubated with each membrane for 1h at room temperature. The signals were measured with Supersignal West Pico Chemiluminescent Reagent (Pierce, USA)for 5 min. The densitometric analysis of the Phospho- ERK and Phospho-AKT levels, as well as total ERK and total AKT levels were performed by using the ImageJ software. The levels of phosphorylated and total ERK and AKT were separately normalized to that of β-actin. The fold-change was indicated as the level of induction in each treatment compared with the control.

Vector construction and dsRNA expression

Primers were designed according to the AalbInR gene(GeneBank No. XM_029853782.1), which is an orthologous gene of Ae. aegypti InR [16]. The cDNA reverse transcribed from the total RNA of Ae. albopictus female adults were used as the template for PCR amplification. The fragment of the AalbInR gene (from 3120 nt to 3530 nt in the CDS) was amplified by using PCR with primers AalbInR-F (5'-GCTCTAGAGGTTCGCCTGGTGGACATTGAG-3') and AalbInR-R (5'- CCCTCGAGATCGTGGTGCCCATCGAGTCAT-3'). The AalbInR gene with a length of 411 bp was inserted into the multiple clone sites of L4440 plasmid which were flanked with two T7 promoters in an inverted orientation by using Xba I and Xho I restriction sites. The L4440-EGFP plasmid containing a 717 bp fragment targeting EGFP was used as the control. The L4440-InR and L4440-EGFP plasmids were confirmed by sequencing. Then the recombinant plasmids transformed into E. coli HT115 (DE3) competent cells lacking RNase III by a standard transformation procedure. The positive colonies were selected by using LB agar plates (100µg/mL ampicillin) screen, PCR verification and DNA sequencing.

To produce dsRNA, a single colony of transformed bacteria was cultured in LB medium(100 µg/mL ampicillin)at 37°C with shaking at 200 rpm overnight. Then, 500 µL bacteria culture was added to 50 mL LB medium with antibiotics as above and incubated at 37°C (200 rpm) for 3 ~ 4 hours༈OD₆₀₀ = 0.6༉. Expression of dsRNA was induced by adding 0.5 mM of isopropyl-β-D-1-thiogalactopyranoside (IPTG) for another 3 hours.

Feeding mosquito larvae with E.coli expressing dsRNA

Mosquito larvae feeding experiments were carried out by using recombinant bacteria expressing dsRNA as described before[26]. In brief, age-synchronized larvae (L3 stages) were placed into 10 cm dishes containing about 10 mL deionized water. The recombinant HT115 (DE3) E. coli was induced to generate dsRNA by adding IPTG as described above. The bacteria were pelleted by centrifugation (12000 rpm) for 5 min and resuspended in sterile water. Then, cells were mixed with 1% LB-agar containing IPTG and 1 g of finely ground yeast at 42°C to keep the bacteria alive. The bacteria agar mixture was cubed into 5 mm cubes, and fed to mosquito larvae once a day. Mosquito larvae were divided into three groups: InR dsRNA(dsInR) feeding group, EGFP dsRNA (dsEGFP) feeding group and the negative control (NC) groups fed with no bacterial LB mixture. Each group contained 20 larvae. After 2 days, the bacterial diet was removed and the normal diet was added to rear the larvae for developing into the pupae and adults. The transcript level of InR and phosphorylated level of ERK and AKT protein were detected as mentioned above. The visible phenotypic changes including the body sizes, pupation and eclosion times during the experiment were recorded daily for two weeks. Each treatment was repeated at least three times.

Statistical analyses

Statistical analyses comparing gene expression were performed by using multiple comparison t test [27]. Values of gene expression were represented as mean ± SD in at least three independent experiments. All statistical analyses were performed using GraphPad Prism8 software (GraphPad Software, Inc.,La Jolla, CA): *p < 0.05, **p < 0.01, ***p < 0.005 and ****p < 0.001.

Molecular and phylogenetic characterization of AalbILP genes

Seven putative ILPs transcripts in Ae. albopictus genomic assembly were obtained by using sequence blast with known Ae. aegypti ILP1–8 genes [5]. The phylogenetic analysis was performed to discern the evolutionary relatedness of Ae. Albopictus(Aalb)ILPs with those identified in Ae.aegypti (Aaeg), An.gambiae (Aga) and D. melanogaster (Dme). We observed four clusters in the phylogenetic trees of ILPs. Aalb ILP3, -4, -5 and 7 are highly conserved with those of Ae. Aegpyti (Aaeg ILP3, -4, -5 and 7), and AagILP3, -5 and 6 in An.gambiae, which are classified into a cluster with DmeILP6, -7 and 8༈Fig. 1༉. Another cluster is formed between AaegILP8 and Aga ILP4, as well as DmeILP1-5. The third cluster strongly supports the evolutionary relationship between ILP1and ILP6 in both Aalb and Aaeg mosquitoes, as well as ILP7 in Aag mosquitoes. Interestingly, ILP2 of Aalb, Aaeg and Aga mosquito families formed an independent branch separated from the other three clusters (Fig. 1). The bootstrap analysis revealed that the lineages of ILPs are highly conserved in Aedes mosquito species, but are variant with D. melanogaster. Additionally, the phylogenetic trees also suggested the evolutionary relatedness of InR between different insect species (Additional file: Fig. S1).

The conserved domain of AalbILP was analyzed by using amino acid sequences alignment with AaegILP1-8. The open reading frames (ORFs) of Aalb ILP 1–7 encode the prepropeptides with 139–178 amino acid (aa) residues. The prepropeptides of AalbILPs contain a signal peptide (SP) (11 to 32 aa), B-chain, C-peptide and A-chain (Fig. 2). AalbILP1, -2 and 5 have extensions of 20–25 amino acids in the N-terminal of B-chain, which may generate an additional bioactive peptide by the proteolytic cleavage sites in Arg and Lys residues[5]. The length of C-peptide ranges from 9 to 81 residues, and contains single or paired Arg and Lys amino acids which may be additional proteolytic cleavage sites. After proteolytic cleavage of the SP and C-peptide, the ILP peptides form the tertiary structure by linkage of the two conserved Cys residues in the B-chain and four Cys residues in the A-chain. Similar to AaegILP6, a short C peptide and C-terminal extension sequences were also found in AalbILP6, which is considered the characteristic of IGFLP [28]. Nevertheless, ILP6 of Aalb and Aaeg mosquitoes have no relatedness to DmeILP6, a putative IGFLP in D. melanogaster. Unexpectedly, no orthologues of AaegILP8 were found in Ae. Albopictus. AalbILP4 and 7 both possess an additional amino acid in the A-chain, which results in a nine amino acid spacer between the final two Cys residues instead of the usual eight—a feature not found in any other ILPs to date. Both AalbILP5 and AaegILP5 have an additional amino acid between the second and third Cys residues in the A-chain. The position of identical and similar amino acids among the peptide regions of the Aalb ILPs is given in Fig. 2, indicating that most members of the ILPs only share a few key conserved amino acids.

Expression of IS pathway genes during Ae. albopictus development

To explore the expression patterns of IS pathway genes in different developmental stages, Ae. Albopictus mosquito samples from eggs, larvae, pupae, and adults (male and female) were collected, respectively. a comprehensive survey of mRNA transcription and protein expression was conducted to investigate the temporal expression pattern of ILPs, InR and ERK/AKT.

Relative expression analyses revealed AalbILP1-7 in the embryo stage exhibited much lower expression than those of post-embryonic development stages: larva, pupa and adult. The highest expression level of AalbILP1 was observed in the larva stage, then decreased in both pupa and adult stages (Fig. 3). AalbILP2 expression was detected at the highest level in the pupa stage and exhibited no difference between the male and female adults. AalbILP3 is mainly expressed in larva and adult stages, where their levels were much higher than those in the egg and pupa stages. We observed the expression level of AalbILP4, -5, -6, and 7 were increased in the adult stage compared with those in both larva and pupa stages. Meanwhile, we detected considerably higher expression of AalbILP5 in males than those in females, while other ILPs expression showed no significant difference between the male and female adults. Similar to the ILPs, AalbInR exhibited higher expression in the postembryonic stages than that in the egg stage (Fig. 3).

Since the mRNA transcription of ILPs and InR are variants in the lifespan of Ae. albopictus, we aim to examine whether two critical protein kinases: ERK and AKT in the IS pathway also exhibit different expression patterns. As shown in Fig. 4a and b, the total ERK protein was decreased in the postembryonic stages compared to the egg stage. And the phosphorylation level of ERK protein was also decreased in the larvae, pupa, and adult stages compared with that of the egg stage (Fig. 4a and 4c). Similar to the ERK protein, the total and phosphorylated AKT protein levels were also downregulated in the larva, pupa, and adult stages compared with that of the egg stage (Fig. 4a, d, and 4e). Interestingly, we found the phosphorylated ERK level in female adults is much higher than that in male adults (Fig. 4c). In contrast, the phosphorylated AKT level in the male mosquito is much higher compared to that in the female mosquito (Fig. 4e). It suggested ERK / AKT cascades may be involved in regulating the different nutrition metabolism in male and female mosquitoes.

Impact of blood feeding on the expression of IS pathway genes

Relative expression analyses were applied to examine the tissue localization of seven ILP transcripts in female adults (Additional file: Fig. S2). In the head, the mRNA transcription of AalbILP 2–7 were detected, and ILP3 was much higher than other ILPs, which indicated that ILP3 is brain-specific. ILP6 transcript is much higher in thorax, fatbody and midgut than the other six ILPs. We observed that transcription levels of Aalb ILP2, -5, -6 and 7 are significantly higher than Aalb ILP1, -3 and 4 in the midgut. The transcripts of AalbILP2 and 6 were found to be enriched in the ovary, while ILP1 is rare in all tissues of female adults. InR transcript was high in both midgut and ovary. This revealed a differential distribution of ILP transcripts in female mosquito tissues.

Female mosquitoes need to take the blood meal for developing eggs. To explore whether expression patterns of ILPs are affected by the diet switch from sugar to blood, we collected sugar-fed and blood-fed female adults, and investigated the expression patterns of ILPs in the mosquito head and peripheral tissues. Blood feeding induced the transcription levels of AalbILP2, -6 and 7 genes upregulation, while ILP4 and 5 genes were downregulation in the head. Meanwhile, the expression levels of ILP1, -2 and 5 genes in the thorax were increased after taking the blood meals. The midgut is the main organ for blood digestion for mosquitoes. We found expression level of ILP6 is significantly increased in the blood-fed midgut compared to those of sugar-fed midgut (Fig. 5). In addition, the transcription levels of ILP3 and 4 genes were also increased after blood feeding. In the bood-fed fatbody, the ILP5 transcript was increased while ILP4 and 7 were decreased. Blood feeding inhibited the expression level of four ILPs: ILP3, -4, -5 and 7 in the ovary. The InR expression also exhibited a different pattern in the thorax, fatbody and ovary after blood feeding. These expression patterns of the ILPs genes suggest a possible role in regulating the nutrition metabolism during blood feeding.

Next, we examined ERK and AKT protein levels in female adult tissues after blood feeding. Blood meals did not influence the total ERK level in different tissue of female adults, but significantly inhibited the ERK phosphorylated levels in the ovary (Fig. 6a-c). Blood meals did not change the total AKT protein level in the different tissues of females: head, thorax, fatbody, midgut and ovary (Fig. 6a and 6d). However, we found that the phosphorylated ATK level in the midgut was significantly inhibited by blood meal, which was rarely detected in blood-fed midgut compared to the sugar-fed midgut (Fig. 6a and 6e). The results showed that blood meals regulate the IS pathway, which exhibited variety in the female midgut and ovary.

Effect of dsRNA -induced AalbInR knockdown on the mosquito development

Mosquitoes produce 5–8 ILPs and only one known InR [6, 16, 24]. To investigate the effect of IS pathway on Ae. albopictus mosquito development, we adopt the strategy of microbial-base dsRNA interference to knock down AalbInR (Fig. 7a). The transformed E. coli expressing InR dsRNA (dsInR) were confirmed by PCR (Additional file: Fig. S3), then fed to L3 larvae by oral administration, and the recombinant E. coli-producing EGFP dsRNA (dsEGFP) was taken as the dsRNA control. As shown in Fig. 7b, feeding dsInR resulted in a significant reduction in InR gene expression levels (~ 40%) compared to the dsEGFP-fed and the NC groups. Nextly, we examine the phosphorylation level of ERK and AKT proteins. Knockdown of AalbInR did not change the total protein levels for both ERK and AKT in the larvae, but phosphorylated protein levels for both ERK and AKT were significantly decreased (Fig. 7c-e). It demonstrated that knockdown of AalbInR successfully blocks the activation of ERK/AKT cascades in the IS pathway by feeding the larvae with E.coli producing dsRNAs.

To investigate the effect of AlbInR knockdown on the development of Ae. albopictus mosquito, three groups of larvae: dsInR-fed group and dsEGFP-fed group and the NC group were raised and maintained under identical conditions at all stages. The ratio of pupation and eclosion were recorded daily after feeding larvae with dsRNA to measure their developmental conditions. We observed that silence of AlbInR significantly reduces the ratio of pupation after feeding E.coli producing dsRNAs in 5 days compared with dsEGFP treatment, and the inhibition effect on the pupation can last for more than 10 days (Fig. 8a). No significant changes were found between the dsEGFP group and the NC group. Meanwhile, the eclosion times of mosquitoes after feeding AlbInR dsRNA were also delayed compared to those in the dsEGFP -fed group and the NC group (Fig. 8b).

We also evaluated the impact of AalbInR silence on the body size of larvae and adults. Body sizes of the last-instar larva, female and male adults were recorded. InR dsRNA-fed larvae exhibited a much smaller body size, while larvae in the dsEGFP-fed group exhibited a similar body size to those in the NC group (Fig. 9a and 9d). The influence on the body size could last to the adult stage (Additional file: Fig. S4). Meanwhile, the wing length of adult mosquitoes (both male and female) was measured to ascertain whether wing size is attuned with body sizes. We confirmed that the wing length of dsInR-fed group was shorter than that of dsEGFP-fed groups, and no difference between the dsEGFP-fed and NC groups (Fig. 9b, c, e and 9f). These results indicated that knockdown of InR in larvae makes the body sizes smaller when developing into adults.

Ae. Albopictus is an important vector for transmitting dengue, chikungunya, and Zika viruses, and is fast spreading worldwide now [22]. Mosquitoes need to endure metamorphosis before the female adults suck the human blood and transmit vector-borne diseases [29]. The IS pathway plays a critical role in regulating the metabolism, growth, reproduction, and development in a variety of insect species [2, 30, 31]. In this study, we characterized the molecular and expression characteristic of IS pathway genes: ILPs, InR and AKT/ERK in the lifespan and different tissues of Ae. Albopictus. Additionally, we investigated the potential role of IS pathway in mosquito development.

We identified seven putative AalbILP genes (AalbILP1-7) in the Ae. Albopictus assembly. The genome of the related mosquito Aedes aegypti contains eight ILP genes [5]. However, no gene encoding ILP8 has been identified in Ae. Albopictus species. Seven ILP genes were also found in the genome of An. stephensi, but only five unique ILPs due to two duplicated ILP genes that encode identical B and A peptides [32]. Seven AalbILPs genes were conserved in the evolutionary with ILPs from other insects, especially for Ae.aegypti ILPs (AaegILPs) [5]. Amino acid sequence alignments demonstrated that seven AalbILPs have a similar propeptide structure to the insulin superfamily including the SP, B-chain, C-chain, and A peptides, which was common in both invertebrate and vertebrate ILPs [1, 24]. Domain analyses revealed the amino acid between ILPs family members of Aedes mosquitoes is conserved key residues in necessary for appropriate processing and tertiary structure. This evolutionary conservation suggests a critical physiological role for insect ILPs [24].

Insects undergo distinct developmental stages and these stages possess very distinct nutrient acquisition and metabolism conditions [1, 2]. In Drosophila, ILP genes play an important role in regulating feeding preference and metabolism [2]. Transcriptional profiles of AalbILP1-7 revealed low expression levels for all AalbILP in the egg stage, which is consistent with the expression pattern of their ortholog ILPs in Ae. aegypti mosquito [5]. It suggested that ILP genes may not be constitutively active in embryonic development [24]. As developing into the larva stage, all seven AalbILPs expressions are increased, indicating that the ILPs are involved in supporting the metabolic needs during development [17]. Meanwhile, we observed the expression level of four AalbILPs (ILP3, -5, -6 and7) genes were higher in male adults, while ILP2 expression was much higher in female adults, especially in the ovary tissues. Male mosquitoes solely obtain carbohydrates from their diets; however, female mosquitoes need to change their diet from carbohydrate-rich nectar to vertebrate blood for ovary development[33, 34]. The different expression of ILPs between male and female mosquitoes may reflect their distinct functions in regulating nutrient acquisition [24].

Blood feeding is an important physiological activity for female mosquitoes [34]. Blood meals trigger ILPs released from specific cells or organs into circulation to digest protein and lipids, as well as stimulate egg maturation [32, 34, 35]. Insect nervous system is considered the primary source of ILPs [1, 4]. Blood feeding induces AalbILP2, -3, -6 and 7 mRNAs expression up-regulated in the female brain. ILP3 is the most abundant ILP in the brain of Ae. Albopictus. The release of ILP3 from the brain of Ae. aegypti after blood feeding regulate the enzymes that digest the blood meal and yolk protein expression by the fat body to stimulate the proliferation of hemocytes [34]. Insect midgut and fatbody are also considered important sources of ILPs [5, 32, 36]. We observed AalbILP2 and ILP6 expression were up-regulated in the blood-fed midgut of female adults. In Drosophila, DILP2 is the most closely related to mammalian insulin, and DILP6 is characterized as the putative IGF-like ILP [4]. The insect fatbody is an analog of the vertebrate liver and adipose tissue [12, 36]. Over-expression of DILP6 in the adult fat body extends lifespan and increases longevity-associated metabolic phenotypes [36]. However, fat body DILP6 expression represses other DILPs production and secretion in the brain [36]. In Ae. aegypti, blood-feeding boosts serotonin concentration and elevates the serotonin receptor Aa5HT2B transcript level, which modulates the expression of ILP2, -4, -5 and 6 in the fatbody [30].

IS pathway is activated by the binding of ILPs and InR, and promotes the phosphorylation level of ERK and AKT proteins [37]. Although variant number of ILPs have been identified in mosquitoes, only single known InR was reported for a long time in both Aedes and Culex species [16, 19]. The InR from Ae. aegypti and Drosophila are similar to mammalian InR/IGFRs [16, 37]. Complete removal of InR in Drosophila throughout development causes early larval lethality [38]. The deficiency of InR reduces lipid synthesis and reproductive function in the insect Rhodnius prolixus, a vector of Chagas disease [18]. We found that InR expression is increased in the post-embryonic development stages. Application of bacteria expressing dsRNA to knockdown InR in the larvae stage led to the delay of development and much smaller body size in the adults, which may be related to the blockade of ERK and AKT cascades. Similarly, RNAi against the IS pathway genes of Maruca vitrata can interfere with larval growth, leading to small pupae or significant larval mortality [39]. It indicated that the bacteria expressing dsRNA specific to IS components can be used to develop dsRNA insecticide [40].

Here we present expression analyses of the AalbILPs gene family and the functional study of InR in mosquito development. Our study confirmed IS pathway genes: ILP1-7, InR, and ERK/AKT in Ae. Albopictus genome assembly. Sequence homology suggested conservation between AalbILP and other insects' ILPs, and the presence of insulin family domains supports ILPs as a factor in regulating the development and metabolism of Ae. Albopictus. Expression characteristics revealed complexities of IS pathway genes between stages and sexes and suggest that IS pathway of female adult mosquito is regulated by blood feeding. In addition, it demonstrated that transformed E. coli expressing dsRNA specific to AalbInR has significant inhibition activity by oral application, which is the development of novel control tools for Ae. Albopictus mosquitoes.

Ae. Albopictus

Aedes albopictus

Ae. Aegypti

Aedes Aegypti

An. Gambiae

Anopheles gambiae

D. melanogaster

Drosophila melanogaster

Bombyx mori

Insulin-like signaling

ILPs

Insulin-like peptides

InR

insulin receptor

IGFPs

insulin growth factor peptides

IRSs

insulin receptor substrates

ERK

extracellular regulated protein kinases༛AKT:protein kinase B༛ SP:the signal peptides

RT-qPCR

real-time quantitative PCR

IPTG

isopropyl-β-D-1-thiogalactopyranoside

T-ERK

Total ERK

T-AKT

Total AKT

P-ERK

phosphorylated-ERK

P-AKT

phosphorylated-AKT.

Funding

This work was supported by the National Natural Science Foundation of China (No.82172305) and the Natural Science Foundation of Zhejiang Province（No.LY21H190004） for financial support. The funders played no role in the study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

Authors’ contributions

WL, SL: Conceived and designed the study, reviewed the manuscript; and acquired the funding. YD, XL, JD, ZL, RG, TY, YZ and SC carried out the experiments. YD performed statistical analyses. WL, YD wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Taguchi A, White MF. Insulin-like signaling, nutrient homeostasis, and life span. Annual review of physiology. 2008;70:191–212.
Semaniuk UV, Gospodaryov DV, Feden'ko KM, Yurkevych IS, Vaiserman AM, Storey KB, et al. Insulin-Like Peptides Regulate Feeding Preference and Metabolism in Drosophila. Front Physiol. 2018;9:1083.
Fujinaga D, Shiomi K, Yagi Y, Kataoka H, Mizoguchi A. An insulin-like growth factor-like peptide promotes ovarian development in the silkmoth Bombyx mori. Scientific reports. 2019;9 1:18446.
Gronke S, Clarke DF, Broughton S, Andrews TD, Partridge L. Molecular evolution and functional characterization of Drosophila insulin-like peptides. PLoS genetics. 2010;6 2:e1000857.
Riehle MA, Fan Y, Cao C, Brown MR. Molecular characterization of insulin-like peptides in the yellow fever mosquito, Aedes aegypti: Expression, cellular localization, and phylogeny. Peptides. 2006;27 11:2547–60.
Marquez AG, Pietri JE, Smithers HM, Nuss A, Antonova Y, Drexler AL, et al. Insulin-like peptides in the mosquito Anopheles stephensi: Identification and expression in response to diet and infection with Plasmodium falciparum. General and Comparative Endocrinology. 2011;173 2:303–12.
Nassel DR, Vanden Broeck J. Insulin/IGF signaling in Drosophila and other insects: factors that regulate production, release and post-release action of the insulin-like peptides. Cellular and molecular life sciences: CMLS. 2016;73 2:271–90.
Chowanski S, Walkowiak-Nowicka K, Winkiel M, Marciniak P, Urbanski A, Pacholska-Bogalska J. Insulin-Like Peptides and Cross-Talk With Other Factors in the Regulation of Insect Metabolism. Front Physiol. 2021;12:701203.
Luckhart S, Riehle MA. The insulin signaling cascade from nematodes to mammals: insights into innate immunity of Anopheles mosquitoes to malaria parasite infection. Developmental and comparative immunology. 2007;31 7:647–56.
Slack C, Alic N, Foley A, Cabecinha M, Hoddinott MP, Partridge L. The Ras-Erk-ETS-Signaling Pathway Is a Drug Target for Longevity. Cell. 2015;162 1:72–83.
Valzania L, Mattee MT, Strand MR, Brown MR. Blood feeding activates the vitellogenic stage of oogenesis in the mosquito Aedes aegypti through inhibition of glycogen synthase kinase 3 by the insulin and TOR pathways. Developmental biology. 2019;454 1:85–95.
Hun LV, Luckhart S, Riehle MA. Increased Akt signaling in the fat body of Anopheles stephensi extends lifespan and increases lifetime fecundity through modulation of insulin-like peptides. Journal of insect physiology. 2019;118:103932.
Liu WQ, Chen SQ, Bai HQ, Wei QM, Zhang SN, Chen C, et al. The Ras/ERK signaling pathway couples antimicrobial peptides to mediate resistance to dengue virus inAedesmosquitoes. PLoS neglected tropical diseases. 2020;14 8.
Ragab A, Buechling T, Gesellchen V, Spirohn K, Boettcher AL, Boutros M. Drosophila Ras/MAPK signalling regulates innate immune responses in immune and intestinal stem cells. EMBO J. 2011;30 6:1123–36.
Patel RK, Hardy RW. Role for the phosphatidylinositol 3-kinase-Akt-TOR pathway during sindbis virus replication in arthropods. Journal of virology. 2012;86 7:3595–604.
Riehle MA, Brown MR. Insulin receptor expression during development and a reproductive cycle in the ovary of the mosquito Aedes aegypti. Cell Tissue Res. 2002;308 3:409–20.
Zhang H, Liu J, Li CR, Momen B, Kohanski RA, Pick L. Deletion of Drosophila insulin-like peptides causes growth defects and metabolic abnormalities. Proceedings of the National Academy of Sciences of the United States of America. 2009;106 46:19617–22.
Silva-Oliveira G, De Paula IF, Medina JM, Alves-Bezerra M, Gondim KC. Insulin receptor deficiency reduces lipid synthesis and reproductive function in the insect Rhodnius prolixus. Biochim Biophys Acta Mol Cell Biol Lipids. 2021;1866 2:158851.
Nuss AB, Brown MR, Murty US, Gulia-Nuss M. Insulin receptor knockdown blocks filarial parasite development and alters egg production in the southern house mosquito, Culex quinquefasciatus. PLoS neglected tropical diseases. 2018;12 4:e0006413.
Haqshenas G, Terradas G, Paradkar PN, Duchemin JB, McGraw EA, Doerig C. A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells. Cell Rep. 2019;26 3:529 – 35 e3.
Su X, Guo Y, Deng J, Xu J, Zhou G, Zhou T, et al. Fast emerging insecticide resistance in Aedes albopictus in Guangzhou, China: Alarm to the dengue epidemic. PLoS neglected tropical diseases. 2019;13 9:e0007665.
Benelli G, Wilke ABB, Beier JC. Aedes albopictus (Asian Tiger Mosquito). Trends in parasitology. 2020;36 11:942–3.
Liu H, Liu L, Cheng P, Yang L, Chen J, Lu Y, et al. Bionomics and insecticide resistance of Aedes albopictus in Shandong, a high latitude and high-risk dengue transmission area in China. Parasites & vectors. 2020;13 1:11.
Sharma A, Nuss AB, Gulia-Nuss M. Insulin-Like Peptide Signaling in Mosquitoes: The Road Behind and the Road Ahead. Frontiers in endocrinology. 2019;10:166.
Liu WQ, Chen SQ, Bai HQ, Wei QM, Zhang SN, Chen C, et al. The Ras/ERK signaling pathway couples antimicrobial peptides to mediate resistance to dengue virus in Aedes mosquitoes. PLoS neglected tropical diseases. 2020;14 8:e0008660.
Whyard S, Erdelyan CN, Partridge AL, Singh AD, Beebe NW, Capina R. Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs. Parasites & vectors. 2015;8:96.
Chotiwan N, Brito-Sierra CA, Ramirez G, Lian E, Grabowski JM, Graham B, et al. Expression of fatty acid synthase genes and their role in development and arboviral infection of Aedes aegypti. Parasites & vectors. 2022;15 1:233.
Fujinaga D, Kohmura Y, Okamoto N, Kataoka H, Mizoguchi A. Insulin-like growth factor (IGF)-like peptide and 20-hydroxyecdysone regulate the growth and development of the male genital disk through different mechanisms in the silkmoth, Bombyx mori. Insect biochemistry and molecular biology. 2017;87:35–44.
Li XP, Lin D, Zhang Y, Chen SQ, Bai HQ, Zhang SN, et al. Expression and characterization of anticoagulant activity of salivary protein alALP from Asian tiger mosquito Aedes albopictus. Trop Biomed. 2020;37 1:116–26.
Ling L, Raikhel AS. Serotonin signaling regulates insulin-like peptides for growth, reproduction, and metabolism in the disease vector Aedes aegypti. Proceedings of the National Academy of Sciences of the United States of America. 2018;115 42:E9822-E31.
Defferrari MS, Orchard I, Lange AB. An Insulin-Like Growth Factor in Rhodnius prolixus Is Involved in Post-feeding Nutrient Balance and Growth. Frontiers in neuroscience. 2016;10:566.
Marquez AG, Pietri JE, Smithers HM, Nuss A, Antonova Y, Drexler AL, et al. Insulin-like peptides in the mosquito Anopheles stephensi: identification and expression in response to diet and infection with Plasmodium falciparum. Gen Comp Endocrinol. 2011;173 2:303–12.
Pakpour N, Akman-Anderson L, Vodovotz Y, Luckhart S. The effects of ingested mammalian blood factors on vector arthropod immunity and physiology. Microbes and infection. 2013;15 3:243–54.
Schneider DS, Castillo J, Brown MR, Strand MR. Blood Feeding and Insulin-like Peptide 3 Stimulate Proliferation of Hemocytes in the Mosquito Aedes aegypti. PLoS pathogens. 2011;7 10:e1002274.
Castillo J, Brown MR, Strand MR. Blood feeding and insulin-like peptide 3 stimulate proliferation of hemocytes in the mosquito Aedes aegypti. PLoS pathogens. 2011;7 10:e1002274.
Okamoto N, Yamanaka N, Yagi Y, Nishida Y, Kataoka H, O'Connor MB, et al. A Fat Body-Derived IGF-like Peptide Regulates Postfeeding Growth in Drosophila. Developmental Cell. 2009;17 6:885–91.
Garofalo RS. Genetic analysis of insulin signaling in Drosophila. Trends Endocrinol Metab. 2002;13 4:156–62.
Texada MJ, Lassen M, Pedersen LH, Koyama T, Malita A, Rewitz K. Insulin signaling couples growth and early maturation to cholesterol intake in Drosophila. Current biology: CB. 2022;32 7:1548-62 e6.
Al Baki A, Jung JK, Kim Y. Alteration of insulin signaling to control insect pest by using transformed bacteria expressing dsRNA. Pest Manag Sci. 2020;76 3:1020–30.
Ding BY, Shang F, Zhang Q, Xiong Y, Yang Q, Niu JZ, et al. Silencing of Two Insulin Receptor Genes Disrupts Nymph-Adult Transition of Alate Brown Citrus Aphid. Int J Mol Sci. 2017;18 2.

No competing interests reported.

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Molecular and expression characterization of insulin-like signaling in development and metabolism of Aedes albopictus mosquito

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Journal Publication

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Abstract

Background

Methods

Results

Conclusions

Figures

Background

Methods

Sequence alignments and phylogenetic tree analysis

Mosquito rearing, blood feeding, and tissues dissection

RNA extraction and gene expression analyses

Western blotting analysis

Vector construction and dsRNA expression

Statistical analyses

Results

Molecular and phylogenetic characterization of AalbILP genes

Impact of blood feeding on the expression of IS pathway genes

Effect of dsRNA -induced AalbInR knockdown on the mosquito development

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1