The GPROP3 gene controls larval negative phototaxis in Aedes albopictus (Skuse) and shows structural and regulatory conservation across invasive Aedes species

Abstract

Mosquito larvae depend on light detection for predator evasion and habitat selection; however, the molecular functions underlying larval photoreception remain largely unexplored. In this paper, we describe in detail the long-wavelength-sensitive opsin GPROP3 in Aedes mosquitoes by investigating its evolutionary conservation, expression patterns, and functional role in phototactic behavior. Using phylogenetic analyses, expression profiling across different developmental stages and tissues, and functional RNAi assays, we show that GPROP3 is required for larval negative phototaxis. These studies demonstrate stage-specific expression patterns and light-dependent subcellular dynamics and identify GPROP3 as a principal photosensor necessary for larval survival behaviors. These findings advance understanding of mosquito larval sensory systems and indicate their potential for vector control strategies targeting early developmental stages.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-47356-2.

Introduction

Mosquito vision supports essential behaviors, such as host-seeking, flight navigation, and habitat selection. These visual capabilities are adapted to diverse light environments, ranging from the nocturnal habits of many mosquito species to the diurnal or crepuscular activity typical of Aedes spp. (Diptera, Culicidae)¹. This diversity in circadian activity forms the structural and functional basis for various adaptations that are reflected in evolutionary shifts across insect life stages and visual ecologies of the expression and regulation of opsins, proteins that, when bound to a chromophore (retinal), form light-sensitive visual pigments².

While vision is highly developed in the compound eyes of adult mosquitoes, the eyes of mosquito larvae are relatively more straightforward and detect primarily light-dark contrasts; this enables larvae to respond to various environmental cues in aquatic habitats³. These simplified larval visual organs, called stemmata, are located on both sides of the larval head and consist of single-lens units that sense light intensity but cannot form images. Additionally, in front of the stemmata, at the primordia of the compound eyes, there is a pair of crescent-shaped dark regions that are non-functional during the larval stages but signal the onset of adult visual system development^3,4. As the larva grows and matures into an adult, the eyes develop into a more complex adult structure, with expanded color vision and polarized-light detection.

Behavioral changes parallel this developmental progression: whereas younger larvae (2nd and 3rd instars) may exhibit little preference or even slight photophilic responses, 4th -instar larvae and pupae display increasingly pronounced negative phototaxis, congregating in dark areas⁵. However, as demonstrated by the behavioral assays in our present study, in species like Aedes albopictus, robust negative phototaxis is already fully established by the third instar. The development in sensitivity coincides with the progress of the imaginal compound eye beneath the cuticle and argues that the adult visual system begins influencing behavior well before eclosion has been reached⁵. In mosquito larvae, the rhabdom ultrastructure is highly responsive to light conditions. Rhabdoms of dark-reared specimens display dilated membrane systems with loosely arranged microvilli, whereas in light-exposed larvae, they are narrower with microvilli packed together; septate desmosomes and membrane-coated vesicles are also present⁶. These light-dependent changes support visual adaptation, increasing photosensitivity during behaviors like phototaxis and predator avoidance⁶. This avoidance behavior is robust and often stereotyped: larvae orient towards specific light signals even when this results in fatal entrapment at the bottom of the water column, prevailing over homeostatic needs such as respiration⁷.

Recent studies examined how mosquito larvae respond to light⁸, showing apparent avoidance of light in Aedes aegypti, Ae. albopictus (Diptera, Culicidae), each consistently choosing darker areas over well-lit ones. In contrast, light avoidance in Culex quinquefasciatus larvae is more context-dependent, although their adult stage displays a clear photonegative behavior⁸. Rather than a simple fear of brightness, this response involves deliberate decisions shaped by factors such as heat. When exposed to light, Ae. aegypti larvae initially exhibit negative phototaxis, orienting and moving away from the light, and then further adjust their position based on thermal conditions⁸. These thermal preferences dissipate in the absence of light; larvae exhibit higher velocities, which likely enhance foraging while reducing the risk of visual predation. Furthermore, larval phototaxis depends on wavelength: Ae. aegypti larvae are more effectively attracted to purple and yellow wavelengths but avoid, or are poorly attracted to, green and red light, a discriminatory ability likely related to unique opsin expression profiles⁸.

The Ae. aegypti larval visual system includes five distinct stemmata on each side of the head: satellite, anterior dorsal, posterior dorsal, central, and ventral. Each stemmata contains a specific number of photoreceptor cells, whose rhabdomeres converge into a central rhabdomere structure³. Two distinct classes of photoreceptors are described in Ae. aegypti larvae, based on rhodopsin expression. The primary class expresses Aaop3 (GPROP3), a long-wavelength-sensitive rhodopsin found in all stemmata. It moves from the rhabdom to the cytoplasm in response to light and returns to the rhabdom in darkness, a mechanism that appears to modulate sensitivity based on ambient light levels^3,9. Similar light-dependent redistribution, followed by degradation, was demonstrated for GPROP1/3/4 rhodopsins in adult Anopheles gambiae photoreceptors during a light-dark cycle, thereby supporting the conservation of rhodopsin trafficking as an adaptation to varying light regimes across culicid species¹⁰. A second, minor class of photoreceptors, limited to the central and satellite stemmata, expresses Aaop7 (GPROP7), a rhodopsin with a different spectral sensitivity (~ 450 nm) and minimal mobility upon light exposure³. Coexpression of diverse rhodopsins and photopigment types at the larval stage may confer basic spectral discrimination capabilities, particularly within the blue–green range^3,8. In addition, dynamic relocalization of Aaop3 is an essential adaptation to transient light environments, enhancing sensitivity to visual stimuli at any time during the circadian cycle^3,10. The features are indicative of an elaborate and transitional visual system that can support larval phototaxis and provide an early basis for the complex processing of visual information in adulthood^3,6,11.

However, despite the fundamental role of light detection in larval survival, little is known about the molecular mechanisms and photoreceptors involved. The function of rhodopsins in adult mosquitoes is well documented^10,12–14. However, there is a significant research gap regarding how specific opsins, such as GPROP3, mediate phototaxis in early developmental stages of invasive Aedes species. Addressing this gap is crucial. It enables us to understand the evolutionary and functional transition of the mosquito visual system, from simple stemmata to complex compound eyes. It may also help identify novel molecular vulnerabilities targets for control.

In the present study, we present a comparative analysis of the structural and expression profiles of the GPROP3 gene, both at temporal dimensions during development and at spatial dimensions across three larval tissues in four medically relevant mosquito species of the genus Aedes: Ae. albopictus, Ae. aegypti, Ae. koreicus, and Ae. japonicus. These invasive Aedes species are characterized by their ability to expand their range and cause significant harm to public health, economic activity, and the environment. Their continued global spread and urbanization thus render them a primary concern for the transmission of pathogens of significant public health relevance^15,16. We also report the first functional study of the GPROP3 gene in mosquitoes, conducted using RNAi in Ae. albopictus embryos and larvae, indicating a role for this gene in controlling the larval phototactic response to light.

Results

Structural characterisation of the rhodopsin GPROP3 genes in Aedes mosquitoes

To investigate the structural features and expression dynamics of the rhodopsin GPROP3 gene in Aedes mosquitoes, we adopted an integrated approach combining in silico and wet lab analyses across four species: Ae. albopictus, Ae. aegypti, Ae. koreicus, and Ae. japonicus. The Ae. aegypti GPROP3 protein sequence (AAEL006484, gene symbol: LOC5568061, 373 amino acids) was used as a query in tBLASTn searches to identify potential orthologues in other Aedes genomes available at the NCBI website (Ae. albopictus: AalbF5 - GCA_035046485.1; Ae. koreicus: Akor1v2 - GCA_034211335.2; Ae. japonicus: Ajap1v2 - GCA_034211315.2) genome¹⁷.

As observed in Ae. aegypti, the GPROP3 ortholog identified in Ae. albopictus genome (XP_019553172, chromosome 3) includes two coding exons (blue boxes) separated by a single intron (in grey), yielding a genomic span of 1.197 kb and ~ 1.492 kb, respectively (Fig. 1a, c). In contrast, the orthologs identified in Ae. koreicus (uncharacterized protein, scaffold795) and Ae. japonicus (uncharacterized protein, scaffold288) genomes show a single continuous exon of approximately 1.0 kb (Fig. 1e, g), indicating potential intron loss in these species. To verify the in silico-predicted gene structure and confirm the presence or absence of intronic sequences, we conducted PCR amplifications using exon-spanning primers on genomic DNA (gDNA) and cDNA templates from both male and female mosquitoes (Fig. 1a, c, e, g). The amplification patterns were identical in both sexes, indicating that GPROP3 undergoes the same splicing process in males and females, with no evidence of sex-specific alternative splicing.

Fig. 1.

In silico gene structure prediction and experimental validation of GPROP3 in Aedes mosquitoes. (a, c,e, g) Predicted gene models of GPROP3 in Ae. albopictus, Ae. aegypti, Ae. koreicus, and Ae. japonicus. Gene architecture was reconstructed from genome annotations and visualized using Gene Structure Display Server. Exons (blue boxes) and introns (grey lines) are shown to scale, indicating conservation or divergence of intron–exon boundaries among species. (b, d, f, h) PCR validation of the predicted gene structures using exon-spanning primers (grey arrow) on gDNA and cDNA from male and female samples. In Ae. aegypti and Ae. albopictus (Fig. 1b, d), amplification from gDNA produced a longer fragment than cDNA, consistent with the presence of an intron. In contrast, in Ae. koreicus and Ae. japonicus (c–d), PCR on gDNA and cDNA yielded identical fragment sizes, supporting the absence of intronic sequences in these species. Identical amplification products in males and females provide no evidence of sex-specific splicing across all species. The first lane on the left is the 1 kb DNA Ladder or 100 bp DNA Ladder (NEB). Primers used in the PCR amplifications for the b, d, f, and h panels are indicated by short yellow arrows.

In Ae. albopictus, genomic PCR yielded a 1170-bp product. In contrast, RT-PCR from cDNA showed a smaller amplicon, consistent with intron splicing (Fig. 1b). A similar pattern was observed in Ae. aegypti, where the genomic amplicon measured 1090 bp, and a 72 bp shorter fragment was detected in cDNA, confirming the removal of the intron during mRNA processing (Fig. 1d). In contrast, both Ae. koreicus and Ae. japonicus shows identical amplification patterns on genomic DNA and cDNA (Fig. 1f, h), confirming the absence of an intron in the amplified regions.

Evolutionary conservation of the GPROP3 protein in Aedes mosquitoes

To investigate the evolutionary conservation and potential functional domains of the GPROP3 protein, we conducted a multiple-sequence alignment of predicted amino acid sequences from Ae. albopictus, Ae. aegypti, Ae. koreicus, and Ae. japonicus (Fig. 2). The pairwise identity scores revealed a high degree of sequence conservation between Ae. koreicus and Ae. japonicus (99.73%). Moderate similarity was observed between Ae. aegypti and both Ae. koreicus (79.19%) and Ae. japonicus (78.98%), while the sequence from Ae. albopictus showed slightly lower similarity with Ae. koreicus (77.84%) and Ae. japonicus (77.63%) (Fig. 2b). The phylogenetic tree (Fig. 2c, left) suggests distinct clades for each species, with Ae. aegypti and Ae. albopictus being closely related, followed by Ae. koreicus and Ae. japonicus. The MEME analysis (Fig. 2c, right) identified ten highly conserved motifs within the GPROP3 protein sequences of Aedes mosquitoes. Notably, the motif architecture reflects this evolutionary clustering: closely related species share nearly identical motif patterns, while minor variations are observed between more distantly related taxa. These findings indicate that evolutionary divergence within the genus Aedes has occurred under intense selective pressure to maintain key functional domains of the GPROP3 protein.

Fig. 2.

Phylogenetic analysis and conserved motif organization of the GPROP3 protein across Aedes species. (a) The protein alignment was performed using Clustal Omega and visualized with ESPript 3.0. Conserved residues are highlighted in red, and similar residues are shaded in gray. A high level of conservation is observed among the species. (b) The heatmap displays the percentage of amino acid sequence identity among Ae. albopictus, Ae. aegypti, Ae. koreicus, and Ae. japonicus. The color gradient represents the identity score, ranging from lower (green) to higher (red) conservation. (c) Phylogenetic tree and MEME-derived motif architecture of GPROP3 orthologs in Ae. albopictus (Aal), Ae. aegypti (Aae), Ae. koreicus (Akor), and Ae. japonicus (Ajap). Each color-coded box represents a conserved motif identified by MEME analysis, with identical colors corresponding to the same motif across species. The high similarity in motif composition and order suggests a strong evolutionary conservation of GPROP3 protein domains among Aedes mosquitoes. (d) The E-values indicate the statistical significance of motif conservation; values approaching zero indicate highly conserved sequences.

Furthermore, a significant degree of conservation is observed among the four sequences, particularly within regions corresponding to transmembrane domains, indicating a structurally conserved architecture (Fig. 2a). Subsequent functional annotation utilizing InterProScan identified three regions, motifs 2, 6, and 8, that align with the Family A G protein-coupled receptor-like domain, thereby supporting the hypothesis that GPROP3 is a member of the rhodopsin-like GPCR superfamily (Fig. 2d). The remaining domains did not correspond to known conserved protein families, potentially reflecting species-specific sequence divergence or the existence of uncharacterized functional elements.

Expression analysis of GPROP3 in life stage-specific Aedes mosquitoes

Gene expression patterns were then assessed by quantitative real-time RT-PCR (Fig. 3) across developmental stages (pools of embryos, larvae, pupae, and adults) and in dissected larval tissues (heads, thoraces, abdomens). GPROP3 expression was strongly observed in larvae across all species and decreased significantly in pupae and adults, suggesting a distinct or stage-specific role for the gene during larval development (Fig. 3a − d). Notably, the expression of GPROP3 is primarily localized in the stemmatal photoreceptors of Ae. aegypti larvae, particularly in the developing eyes of the larval head³. Based on this observation, the qRT-PCR analysis was restricted to the larval stage, where predominant expression was detected in the larval head (p < 0.0001), confirming the role in photoreceptive or sensory functions (Fig. 3e − h). These findings support the hypothesis that GPROP3 contributes to light-dependent behaviors during larval development, in line with the established function of rhodopsins in insect phototransduction.

Fig. 3.

Developmental and tissue-specific transcriptional profile of GPROP3 in Aedes mosquitoes. (a − d) Stage-specific expression in embryos (0–24 h), larvae (L_I−III), pupae (P), and adults (A) as determined by qRT-PCR. The x-axis indicates the developmental stage, and the y-axis shows the relative expression level normalized to the reference gene Rp49. The error bars represent the SEM (N = 10). (a) one-way ANOVA test, p-Value < 0.0001 followed by Tukey post hoc test (‘a’ vs. ‘b’ 0.0308, ‘a’ vs. ‘c’ <0.0001; ‘b’ vs. ‘c’ <0.0001). (b) one-way ANOVA test, p-Value < 0.0001) followed by Tukey post hoc test (‘a’ vs. ‘b’ 0.0014, ‘a’ vs. ‘c’ <0.0001; ‘b’ vs. ‘c’ <0.0001). (c) one-way ANOVA test, p-Value < 0.0001) followed by Tukey post hoc test (‘a’ vs. ‘b’ <0.0001). (d) one-way ANOVA test, p-Value < 0.0001) followed by Tukey post hoc test (‘a’ vs. ‘b’ 0.0002, ‘a’ vs. ‘c’ <0.0001; ‘b’ vs. ‘c’ <0.0001). (e − h) Spatial expression profile in dissected larval heads, thoraces, and abdomens. The x-axis indicates the tissue type, and the y-axis shows the relative expression level obtained by qRT-PCR. The internal reference genes were the same as above. The error bars represent the SEM (N = 10). one-way ANOVA test, p-Value < 0.0001 followed by Tukey post hoc test (‘a’ vs. ‘b’ <0.0001).

In vivo functional analysis of the GPROP3 gene in Ae. albopictus by RNAi knockdown

To study the in vivo function of the GPROP3 gene and its role in larval photoreception, Ae. albopictus was selected as a model species. RNAi knockdown of AalGPROP3 was performed using two methods (Fig. 6): microinjection of dsRNA molecules (Table 1) into one-hour-old embryos (579 embryos) and a soaking assay with dsRNA delivered to first instar larvae (Table 2) (100 larvae). As controls, 516 embryos were injected with dsRNAs targeting the exogenous Green Fluorescent Protein (GFP) gene, which is absent in the Ae. albopictus genome, and 100 larvae were soaked in the identical GFP-targeting dsRNA.

Table 1.

Number of injected embryos and relative survival rate.

Microinjection	Injected eggs (n)	Survival rate (%)	Larvae analyzed by qRT-PCR analysis (n)
dsRNA GFP	516	1.35	7
dsRNA GPROP3	579	3.97	13

Table 2.

Number of larvae exposed to dsRNA and relative survival rate.

Soaking	Larvae (n)	Survival rate (%)	Larvae analyzed by qRT-PCR analysis (n)
dsRNA GFP	100	90	25
dsRNA GPROP3	100	92	25

To evaluate the efficiency of GPROP3 silencing, the expression levels of the target gene were analyzed in third-instar larvae compared with the control group. From the microinjection experiment, a total of 13 GPROP3-interfered larvae were collected and analyzed as single individuals, whereas 7 GFP-injected control larvae were analyzed as a single pool for RNAi validation. qRT-PCR analysis revealed a statistically significant (p < 0.0001) downregulation of GPROP3 expression in the GPROP3-interfered group compared to controls (Fig. 4b). In the soaking experiment, a total of 25 GPROP3-treated and 25 GFP-treated larvae (5 pools of 5) were analyzed, confirming a significant reduction in GPROP3 expression with this method as well (Fig. 4c). These results demonstrate that both dsRNA delivery strategies—embryonic microinjection and larval soaking—were effective in reducing GPROP3 transcript levels (Fig. 4b–c).

Fig. 4.

Schematic representation of dsRNA delivery protocols in Ae. albopictus. (a) Representative images of in vitro-synthesized dsRNA. The GPROP3-specific dsRNA was 618 bp in length, while the GFP-specific control dsRNA measured 270 bp. (b) Microinjection into 1-hour-old embryos at the posterior pole using a dsRNA solution (0.5 µg/µL in injection buffer). (c) Soaking of larvae in a dsRNA solution (1000 ng) for 2 h daily over six consecutive days. (d–e) Expression levels of the Ae. albopictus GPROP3 gene in larvae subjected to RNAi at the embryonic or larval stage. Third-instar larvae (single or pooled in groups of 5) were analyzed by qRT-PCR. The x-axis shows the sample ID, and the y-axis indicates the relative expression levels. The Ae. albopictus ribosomal protein 49 gene (Aalrp49) was used as the internal reference. Error bars represent the standard error of the mean (SEM). Statistical differences between RNAi-treated groups and the control group were assessed using one-way ANOVA (p < 0.0001) followed by Dunnett’s multiple comparisons test (‘a’ vs. ‘b’, p < 0.0001).

Phototactic response of the AalGPROP3 knockdown larvae in Ae. albopictus

To assess whether GPROP3 silencing affected larval phototactic behavior, we analyzed the behavior of third-instar larvae under light versus shadow conditions across three groups: WT (untreated), GFP-interfered (control), and GPROP3-interfered larvae, to determine whether light-driven behavior was altered (Table 3). Pools of larvae or single larvae were placed in Petri dishes with half of the surface partially covered with black paper, or in the 3D-chamber, respectively, and the larval movements were recorded for a fixed time interval (Supplementary Video S1 and S2). The proportion of larvae in the illuminated versus shaded areas was quantified and statistically analyzed to evaluate differences in phototactic response (Fig. 5).

Table 3.

Light preference behavior of Ae. albopictus larvae following AalGPROP3 silencing by embryonic microinjection or soaking.

Treatment group	Delivery method	Larvae tested (n)	Larvae in illuminated area (n)	Larvae in shadow area (n)
WT	Injection	25	0	25
dsRNA GFP	Injection	7	0	7
dsRNA GPROP3	Injection	23	21	2
WT	Soaking	100	0	100
dsRNA GFP	Soaking	90	1	89
dsRNA GPROP3	Soaking	92	84	8

Fig. 5.

Effect of GPROP3 knockdown on Ae. albopictus larval distribution in illuminated and shadowed zones. (a) The time course of phototactic behavior in an untreated larva versus a GPROP3-silenced larva is depicted using representative sequential video frames. The assay was performed with a custom-designed, 3D-printed vertical chamber. Scale bar represents 2 cm. Control larvae migrate rapidly away from the illuminated zone toward the shaded refuge (upper-left corner), demonstrating negative phototaxis. In contrast, GPROP3-silenced larvae exhibit a clear transition to positive phototaxis, actively moving toward or remaining within the illuminated zone. (b − c) Percentage distribution of larvae choosing the illuminated versus the shadowed area following embryonic microinjection (b) and larval soaking (c). The bar charts show the behavioral choices recorded for the control group (WT, black bars; dsRNA GFP, green bars) and the GPROP3-interfered group (dsRNA GPROP3, blue bars). Significance was determined using a Chi-square test for global association and Fisher’s exact test for pairwise comparisons (**** p-Value < 0.0001).

Following AalGPROP3 RNAi treatment via embryonic microinjection, 91.3% of investigated larvae (21/23) failed to exhibit a negative phototactic response to light, remaining in the illuminated area. In contrast, all control-injected larvae (7/7) with dsRNA GFP displayed a normal negative phototactic response to light, moving within a few seconds toward the shadow area (Fig. 5b). A similar trend was observed after larval soaking: 91.3% of treated larvae (84/92) failed to activate a negative phototactic response to light, remaining in the illuminated area. In contrast, GFP controls mainly responded to light with typical negative phototactic behavior (98.9%, 89/90) (Fig. 5c). Wild-type Ae. albopictus larvae (WT) exhibited 100% negative phototactic behavior, further supporting that the light-unresponsive behavior observed in treated individuals results from GPROP3 silencing (Fig. 5b, c). These results strongly support the hypothesis that GPROP3 plays a central role in regulating larval negative phototactic behavior in Ae. albopictus.

Discussion

Mosquito larvae survival heavily relies on their capacity to detect and react to environmental cues, especially light, as it influences predator avoidance and habitat choice^8,18. The adult mosquito visual system is well-understood, but the molecular mechanisms controlling larval photoreception are less well-known¹⁹. Here, we report the detailed characterization of the long-wavelength-sensitive opsin GPROP3 in Aedes mosquitoes, demonstrating its structural conservation, larva-specific expression, and essential function in mediating negative phototaxis in Ae. albopictus.

The in silico and molecular analyses show that GPROP3 is highly conserved among Aedes species at the protein level, including within the transmembrane domains typical of G protein-coupled receptors (GPCRs)²⁰. This high degree of conservation thus indicates strong selective pressure to maintain spectral sensitivity and signaling capability in this rhodopsin, which are critical for survival in an aquatic environment. However, we found a clear genomic split: whereas in Ae. aegypti and Ae. albopictus, an intron is present within the GPROP3 gene, which has not been present in Ae. koreicus and Ae. japonicus. This phylogenetic split aligns with the separate clades identified in our evolutionary analysis and thus indicates lineage-specific genomic simplification in Ae. japonicus and Ae. koreicus group. Despite these genomic differences, protein architecture appears resilient and underpins the gene’s functional relevance. Expression profiling demonstrated that GPROP3 is mainly transcribed during the larval stage and is highly repressed in pupae and adults. While the Aedes genome encodes other long-wavelength-sensitive rhodopsins, such as AalGPROP4 and AalGPROP5, which contribute to the broader spectral sensitivity of the species²¹, the severe decline of GPROP3 transcripts in adulthood suggests a highly specialized role for this specific paralog in immature stages. This is in contrast to other mosquito rhodopsins, such as Aaop1, which is crucial for adult behaviors and is subject to a sophisticated diurnal trafficking machinery to sustain light sensitivity^12,22. The severe decline of GPROP3 transcripts in adulthood suggests functional takeover by the adult visual system during metamorphosis, paralleling the situation in Drosophila, where different photoreceptor subsystems operate at various life stages^12,23. The functional significance of rhodopsins in mosquito behavior is extensively documented in adults, where they modulate complex tasks such as host-seeking, flight orientation, and circadian entrainment. For instance, RNAi knockdown of AalbOpsin1 in Ae. albopictus adults abolish negative phototaxis, confirming its dominant role in the compound eye¹³. Similarly, in Anopheles gambiae, perturbations of AgOp1 disrupt light-dependent rhodopsin trafficking, reducing nocturnal visual sensitivity and affecting crepuscular flight¹⁰.

In contrast, GPROP3 expression is highly enriched within the larval head, in a way that would be entirely consistent with localization in the stemmata. These observations are in larvae, thereby supporting the behavior underlying larval orientation to light gradients that may be advantageous for predator avoidance or thermoregulation^8,24. This contrasts with extra-ocular photoreception mechanisms in other Dipterans; for example, in Drosophila melanogaster larvae, high-intensity light avoidance is mediated by class IV dendritic arborization neurons tiling the body wall, while Bolwig organs handle dim light²³. Predominant expression in Aedes larvae heads suggests a primary reliance on the ocular stemmata system rather than diffuse dermal photoreceptors to drive phototactic behavior and thus an evolutionary divergence in how these larvae perceive danger signals. These findings are in line with the broader context set by recent studies emphasizing the importance of larval sensory systems to the development and survival of mosquitoes^25,26.

Within the stemmata, GPROP3 undergoes light-dependent intracellular translocation from rhabdomeres to the cytoplasm, a dynamic redistribution that is proposed to fine-tune photoreceptor sensitivity and modulate phototactic behavior by enhancing larval responsiveness to environmental fluctuations^3,27. This mechanism mirrors the rhodopsin trafficking (e.g., GPROP1/3/4) observed in adult An. gambiae photoreceptors during light-dark cycles, suggesting a striking conservation of the molecular machinery for light adaptation across culicid life stages¹⁰.

Thus, silencing of GPROP3 by embryonic microinjection or larval soaking RNAi disrupts this regulatory mechanism, resulting in a near-total loss of natural negative phototaxis. While control larvae rapidly migrated to shadowed areas, GPROP3-silenced larvae became insensitive to light and remained in the illuminated areas. Importantly, this severe behavioral shift strongly indicates a lack of functional redundancy between GPROP3 and other larval opsins, such as GPROP7. Although GPROP7 is known to be expressed in a minor subset of stemmatal photoreceptors³, its presence is clearly insufficient to compensate for the loss of GPROP3. This confirms that GPROP3 operates as the indispensable primary photosensor driving the light-avoidance escape response in Aedes larvae. These results are in line with functional studies in agricultural pests such as the melon fly Zeugodacus cucurbitae (Diptera, Tephritidae), in which silencing of long-wavelength-sensitive opsins resulted in a significant reduction of phototactic efficiency under green light²⁸. However, in contrast to the melon fly, in which opsin expression fluctuates under light stress, GPROP3 is a primary sensor; without it, larvae are “blind” to the light gradient and unable to carry out the escape response.

Altogether, our data confirm prior evidence that soaking-mediated dsRNA delivery can be an efficient method for reaching mosquito larvae^29–31, as it is successful for environmental RNAi in other insects and aquatic animals^32,33. Moreover, the sensitivity of larval phototaxis behavior underscores the biological relevance of our target gene and suggests that perturbing light responses could affect larval distribution within the water column and potentially increase vulnerability to predators and stressors^34,35. Future research will need to more precisely define the spectral sensitivities of GPROP3 and determine whether it influences additional light responses, such as circadian rhythm entrainment and thermal avoidance.

In summary, this research enhances the understanding of mosquito sensory biology during the immature stages. It provides a foundation for exploring the larval visual system as a potential target for developing innovative vector control strategies within the broader evolutionary context of insect opsin expression across development³⁶. By enhancing our understanding and developing better RNAi techniques, studies on these pathways could eventually lead to new methods for controlling mosquito populations based on their specific behaviors.

Materials and methods

Insects

Wild-type Ae. albopictus strain used in this study was provided by Centro Agricoltura Ambiente “Giorgio Nicoli” Srl (CAA) and has been maintained in the Department of Biology- Federico II University (Naples) and at the Insect Pest Control Laboratory (IPCL) in Seibersdorf (Austria). The insects were reared under standard laboratory conditions (27 ± 1 °C, 80% relative humidity, and a 14/10 h day/night photoperiod³⁷. The samples of Ae. aegypti, Ae. koreicus, and Ae. japonicus were kindly provided by Prof. Fabrizio Lombardo (University of Rome La Sapienza), Prof. Sara Epis (University of Milan “La Statale”), and Dr. Fabrizio Montarsi (IZSVe, Istituto Zooprofilattico Sperimentale delle Venezie), respectively.

Isolation of nucleic acids

Genomic DNA was extracted from male and female adult mosquitoes (N = 3) using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Total RNA was isolated using the RNeasy Micro Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. The samples included: embryos (E0-24 h), larvae (L I-III), pupae (P), and adult males and females (M and F) (N = 10 per stage); different larval tissues: head (H), thorax (T), and abdomen (A) (N = 10 per tissue); larvae treated with dsRNA (L III) (N = 21, 25, or 1). DNA and RNA quantification were performed using NanoDrop 2000/2000c Spectrophotometers (Thermo Scientific, Wilmington, NC, USA). First-strand cDNA synthesis was conducted using the LunaScript RT SuperMix Kit (NEB, Ipswich, MA, USA) following the manufacturer’s instructions.

PCR, RT-PCR, and RT-qPCR

PCRs and RT-PCRs were performed using the LongAmp Taq DNA Polymerase (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions. Appropriate annealing temperatures and cycle numbers were adjusted empirically for each primer pair. The primer list is reported in Supplementary Table S1. The primer pair targeting the rp49 gene was used as a positive control³⁸. Gene expression levels during development and in larval tissues were studied using a QuantStudio 5 Real-Time PCR System (Thermo Scientific, Wilmington, NC, USA). RT-qPCR analysis of RNAi samples was performed using the CFX96 Touch Deep Well system (Bio-Rad, California, USA), following established protocols³⁷. Primer sequences for qRT-PCR are detailed in Supplementary Table S2. Gene expression levels were quantified using the 2^−ΔΔCt method³⁹, with rp49 as the internal control.

Sequence and phylogenetic analyses

Homology searches for GPROP3 proteins and genes were performed using the NCBI BLAST tool (http://www.ncbi.nlm.nih.gov/BLAST/) with the BLASTp and tBLASTn algorithms. Exon-intron structure diagrams were generated using the Gene Structure Display Server (https://gsds.gao-lab.org/). Multiple sequence alignment of all GPROP3 sequences was performed using Clustal Omega⁴⁰ and visualized with ESPript 3.0, which enabled annotation of conserved residues and visualization of secondary-structure predictions⁴¹. Phylogenetic analysis of the sequences was done by Molecular Evolutionary Genetic Analysis (MEGA) software (version 11.0.13)⁴², with bootstrap values derived from 1000 replicates. The number is shown as a percentage for each node. The branch lengths were drawn to the indicated scale. Analysis of conserved motifs was performed using the online MEME (Multiple Expectation Maximization for Motif Elicitation) tool version 5.5.7⁴³, with minimum and maximum motif widths of 6 and 30 residues, respectively, and a maximum of 10 motifs; all other parameters were set to their defaults.

Preparation of Double-Stranded RNA (dsRNA) by In Vitro Transcription

Specific primers containing T7 promoter sequences were designed for the synthesis of dsRNA targeting AalGPROP3 (AalGPROP3_T7+/AalGPROP3_T7-) and GFP (eGFP_T7+/eGFP_T7-) genes (Supplementary Table S3). PCR products were purified using the Monarch DNA Gel Extraction Kit (NEB, Ipswich, MA, USA), and dsRNAs were synthesized using the MEGAscript RNAi T7 kit (Invitrogen, Waltham, MA, USA) according to the manufacturer’s recommendation.

Delivery of dsRNA in Ae. albopictus

The dsRNAs were injected into 1-hour-old Ae. albopictus embryos at the posterior pole using a mix composed of 1 µg/µl of dsRNA (dsRNA_GPROP3 or dsRNA_GFP) and injection buffer (5 mM KCl, 0.1 mM sodium phosphate, pH 6.8) using the XenoWorks Digital Microinjector (Sutter Instrument Co., Novato, CA), as described in³⁷. This experiment was conducted at the Insect Pest Control Laboratory (IPCL) in Seibersdorf (Austria) (Fig. 6a). In addition, the dsRNAs were delivered using a larval soaking assay. Ae. albopictus larvae (1st to 3rd instar) were immersed in in vitro-transcribed dsRNA (1000 ng total) dissolved in dechlorinated tap water for 2 h per day over six consecutive days. After each exposure, larvae were returned to their feeding trays (Fig. 6b).

Fig. 6.

Experimental methodologies for gene silencing via RNAi. (a) Injection strategy: Mosquito eggs are collected and aligned horizontally (1) to facilitate dsRNA microinjection using a micromanipulator (2). Following treatment, eggs are kept at a controlled temperature (28 °C) (3), then the eggs are immersed in ddH₂O to hatch (4). (b) Soaking strategy: Larvae are placed in multi-well plates containing an aqueous solution of synthesized dsRNA. The insect demonstrates the internalization of dsRNA molecules from the external environment into the larval cells.

Behavioral Assays in Ae. albopictus

The behavioral experiments, named “Light Test”, were performed under the standard environmental conditions of the insectary. To assess whether GPROP3-interfered larvae exhibited different light-response behaviors, we employed a custom-designed, 3D-printed vertical assay chamber fabricated from Polylactic Acid (PLA) (Shenzhen Esun Industrial Co., Ltd., Shenzhen, China), a biodegradable thermoplastic from renewable resources⁴⁴, on an Anycubic Kobra 2 Max (Shenzhen Anycubic Technology Co., Ltd.) (Fig. 7a) (Supplementary Data S1). This dual-reservoir apparatus allows simultaneous, separate testing of both control (Reservoir A) and treated (Reservoir B) larvae (Fig. 7b). The device has three functional zones: a bottom holding reservoir, a central “Illuminated area”, and an upper ‘Shadow area’. A grid gate separates the reservoir from the testing arena. A light source was placed uniformly within the chamber, thereby illuminating the target zone. At the same time, the upper part was obscured with a 3D-printed blackout cap to simulate a dark refuge. In the assay, single larvae were placed in the reservoirs, and their vertical migration and orientation to the light/shadow gradient were monitored and recorded. We selected 3rd -instar larvae rather than 4th -instar larvae to avoid confounding variables associated with pre-pupal physiological and behavioral transitions.

Fig. 7.

Schematic representation of the custom 3D-printed apparatus designed for larval phototaxis assays. (a) Individual modular 3D-printed components: Component A, the dual-chamber reservoir for simultaneous testing of control and treated groups; Component B, the blackout cap used to generate the shadow zone; and Component C, the gate that separates the illuminated and shaded zones while allowing larval passage. (b) Diagram of the assembled experimental setup. The device is positioned over a light source to create a distinct “Illuminated area” and a “Shadow area”. The dual-reservoir design allows for the concurrent assessment of control larvae and RNAi-treated larvae, under identical environmental conditions.

Statistical analysis

Data are presented as mean ± standard error (SE) from at least three independent biological replicates. Student’s t-test was used to analyze the statistical differences between the two groups. Larval distribution data were analyzed via contingency tables, using Pearson’s Chi-square for global association and Fisher’s exact test for pairwise comparisons between treatments and controls. For comparison among multiple samples, one-way analysis of variance (ANOVA) was combined with Tukey’s or Dunnett’s post hoc test (P < 0.05). All statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software, San Diego, USA). Unless otherwise specified, detailed information on statistical analyses is provided in the corresponding figure legends.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

MS conceived the study. MS and MV conceived and designed the experiments. MV, PDL, KN, and AEO performed the experiments. RP performed the 3D modeling and printing of the behavioral assay chamber. MV and MS drafted the manuscript, which was critically revised by PDL, RP, KN, and KB. KB and MS supervised the project. All authors read and approved the final version of the manuscript.

Funding

This work was supported by the EU within the NextGeneration EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT) to MS. This study was also financially supported by the Insect Pest Control Subprogramme of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture and the United States State Department in the frame of the “Enhance Agency’s Capacity to Provide Support to Member States to Control Aedes Mosquitoes as Vectors of Human Pathogens, Particularly Zika Virus, Using Integrated Vector Management Approaches with a Sterile Insect Technique Component” project.

Data availability

All data generated or analysed during this study are included in this published article and its Supplementary Information files. The genome assemblies analyzed in this study are available in the NCBI repository under the following accession numbers: GCA_035046485.1 (Ae. albopictus, AalbF5), GCA_034211335.2 (Ae. koreicus, Akor1v2), and GCA_034211315.2 (Ae. japonicus, Ajap1v2). The specific gene sequences analyzed are available under accession numbers AAEL006484 (Ae. aegypti) and XP_019553172 (Ae. albopictus). The 3D-printing files (.stl) for the behavioral assay chamber are available as Supplementary Material.

Declarations

Competing interests

The authors declare no competing interests.