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. 2022 Oct 2;25(11):105255. doi: 10.1016/j.isci.2022.105255

Neuroanatomical basis of sexual dimorphism in the mosquito brain

Jing Li 1,2,5,∗, Austin Merchant 3,5, Suyue Zhou 1, Tao Wang 1,4, Xuguo Zhou 3,7,∗∗, Chuan Zhou 1,2,6
PMCID: PMC9583127  PMID: 36277452

Summary

Female but not male mosquitoes are vectors for multiple deadly human diseases including malaria, dengue, yellow fever, and West Nile virus. However, the underlying neural substrates of sexually dimorphic behaviors remain largely unknown in mosquitoes. In this study, we found striking sexual dimorphism in brain regions in two major disease vectors, Aedes aegypti and Culex quinquefasciatus, through voxel-wise comparison of the whole brain. Female-enlarged regions include those associated with chemosensation and vision, while male-enlarged regions are linked to hearing and memory. However, some brain regions associated with vision and memory are sexually dimorphic in Ae. aegypti but not Cx. quinquefasciatus. As the first global voxel-based comparative neuroanatomical analysis of mosquito brains between sexes, this study not only sheds light on the neural substrates underlying sex-specific behaviors, but also identifies regions of interest for future research to disrupt female-specific behaviors critical to disease transmission.

Subject areas: Animals, Ethology, Behavioral neuroscience

Graphical abstract

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Highlights

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    Sexual dimorphism is demonstrated in brains of two mosquito species

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    Female enlarged brain regions are linked to chemical and visual perception

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    Male enlarged brain regions are associated with memory and auditory sensing

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    Sexual dimorphism in regional volume is different in the two species

Animals; Ethology; Behavioral neuroscience

Introduction

Sexual dimorphism is a widespread phenomenon among animals and is the result of complex combinations of natural and sexual selection. In sexually dimorphic species, males and females demonstrate differences encompassing morphological, physiological, and behavioral traits. Common examples of sexually dimorphic traits include body size, coloration, and the development of weapons or other specialized ornaments such as the antlers of male deer (Mori et al., 2017). In some animal systems, dietary divergence is observed between the sexes. Dietary divergence is often found in species where the body sizes of males and females differ significantly, with examples found among both vertebrate (Blanco-Fontao et al., 2013) and invertebrate (Pekár et al., 2011) species. In many cases, this divergence reflects simple differences in body size and gut capacity. In other cases, however, sex-specific feeding behaviors are observed that may in part be the result of differing nutritional needs between the sexes and are most likely associated with reproduction (Hierlihy et al., 2013). Overall, the existence and development of sexual dimorphism can lead to members of opposite sexes following highly divergent life histories.

Mosquitoes are medically important insects that demonstrate clear sexual dimorphism. As vectors, they transmit a number of life-threatening and debilitating diseases through their diverse host-seeking preferences and geographical distributions. Major mosquito transmitted diseases include malaria, transmitted by Anopheles spp.; Japanese encephalitis and West Nile virus, transmitted by Culex spp.; and Zika, Chikungunya, yellow fever and dengue, transmitted by Aedes spp. (Lee et al., 2018). Together, mosquito-vectored diseases represent a significant threat to public health: over half of the world’s population is at risk of contracting malaria or dengue, whereas malaria alone kills over 400,000 people annually (World Health Organization, 2020). Members of both mosquito sexes feed primarily on nectar, but females of many species must take a blood meal to either initiate vitellogenesis or increase the number of eggs produced. The act of finding and feeding on an appropriate host requires recognition of specific host cues such as CO2, body temperature, lactic acid and human odor (McMeniman et al., 2014; Zhao et al., 2022; van Breugel et al., 2015; Dekker et al., 2002). Given that blood-feeding behavior, which is responsible for mosquito-borne disease transmission to humans, is only demonstrated by females, a better understanding of the neuroanatomical basis of sexual dimorphism in mosquitoes will facilitate the development of novel and improved forms of vector control.

Previous efforts to understand the basis of mosquito sexual dimorphism have done so from a primarily genetic level. Studies in major vector species at the adult stage have revealed a significant number of genes showing sex-biased expression (Baker et al., 2011; Matthews et al., 2016), as well as the gene responsible for initiating male-specific development (Hall et al., 2015). Sex-biased gene expression has also been observed during the pupal stage, when sexual characteristics develop (Tomchaney et al., 2014). Together, these studies indicate that the transcripts of many sex-biased genes are located in the head or in the brain specifically, consistent with the role of the brain in controlling sexually dimorphic behaviors. Sexual dimorphism in brain structure has been described in a diverse array of animal species (Cachero et al., 2010; Toli et al., 2017; Spring et al., 2007), but to date only the structure of the antennal lobe (AL), which is involved in processing olfactory cues, has been shown to be sexually dimorphic in mosquitoes. Differences in both the size and number of AL glomeruli have been described in several major vector species (Ignell et al., 2005; Ye et al., 2020; Shankar and McMeniman, 2020; Riabinina et al., 2016). However, sexual dimorphism in mosquitoes is not limited to differences in olfaction, and thus it is probable that other regions of the brain differ structurally as well.

Mosquitoes display distinct, sexually dimorphic behavior between males and females, making them an optimal group in which to study the relationship between brain structure and behavior. In this study, we hypothesized that mosquito sexual dimorphism possesses a structural basis in the brain. To test this hypothesis, we selected two species, Aedes aegypti and Culex quinquefasciatus, that are of public health importance but display distinct behaviors in terms of host preference and activity period. We imaged and performed global voxel-based anatomical comparisons between the brains of males and females for each of the two species. Our results show distinct male- and female-enlarged regions throughout the brain consistent with the differing sensory requirements of each sex. This work reveals new insights into how sexually dimorphic behaviors are connected to differences in brain structure, while also indicating the probable locations of fine neural circuitry controlling male- and female-specific behaviors, which is of particular interest to mosquito control efforts.

Results

Male and female mosquito brains are anatomically different

We performed immunohistochemistry on the brains of adult male and female Aedes aegypti and Culex quinquefasciatus mosquitoes using the antibody nc82, which marks synaptic neuropils. Stained Ae. aegypti and Cx. quinquefasciatus brains were averaged into species-specific male, female, and intersex template brains and used to identify regions of the brain enlarged in either males or females of each species. Overviews of template brain generation and the volumetric differences identified are shown in Figure 1 for Ae. aegypti and Figure 2 for Cx. quinquefasciatus. We also compared randomly assigned groups containing both male and female brains for each species using the same method and the same brains to exclude any potential noise explaining the sex-specific differences we observed. Indeed, the random groups had no significantly enlarged brain regions (see Figure S1).

Figure 1.

Figure 1

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Identification of sexually dimorphic brain regions in Ae.aegypti mosquitoes

(A and B) Individual brains from male and female Ae. aegypti were averaged into a template brain for each sex. N = 10 for both sexes.

(C) An intersex template brain was generated by averaging both male and female template brains. N = 20 (10 males and 10 females).

(D and E) Regions enlarged in either male or female Ae. aegypti were identified through comparison of the intersex template brain to individual male and female brains. Heatmap indicates t-statistic values, the original 3D data have been z-projected to display maximum intensity at each pixel. N = 35 for males, N = 43 for females.

(F) Three-dimensional representation of male- and female-enlarged regions shown in D and E. Male-enlarged regions are colored blue, while female-enlarged regions are colored orange. See also Figure S1 and Data S1.

Figure 2.

Figure 2

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Identification of sexually dimorphic brain regions in Cx. quinquefasciatus mosquitoes

(A and B) Individual brains from male and female Cx. quinquefasciatus were averaged into a template brain for each sex. N = 15 for both sexes.

(C) An intersex template brain was generated by averaging both male and female template brains. N = 30 (15 males and 15 females).

(D and E) Regions enlarged in either male or female Cx. quinquefasciatus were identified through comparison of the intersex template brain to individual male and female brains. Heatmap indicates t-statistic values, the original 3D data have been z-projected to display maximum intensity at each pixel. N = 38 for males, N = 38 for females.

(F) Three-dimensional representation of male- and female-enlarged regions shown in D and E. Male-enlarged regions are colored blue, while female-enlarged regions are colored orange. See also Figure S1 and Data S1.

To further verify these differences, we manually quantified the volume of each of these enlarged regions in individual brains for the two mosquito species. A summary of regions showing sexual dimorphism in each species is shown in Table 1; in all instances, standardized nomenclature was used for brain regions (Ito et al., 2014). In addition, we compared the volumes of whole brains between each sex and species using the segmentation function in Fiji (Schindelin et al., 2012). There was no significant difference in whole brain size between sexes within each species, but male and female Cx. quinquefasciatus brains were significantly larger than their counterparts in Ae. aegypti (see Figure S2). This is consistent with the generalized linear model (GLM) analysis for sex and species effects (P-sex = 0.787, P-species = 0.003, P-sex:species = 0.426).

Table 1.

Sexually dimorphic brain regions in Aedes aegypti and Culex quinquefasciatus

Brain Region Sensory Pathway Sex Bias∗
Aedes aegypti Culex quinquefasciatus
Antennal lobe Olfactory ♀ ♀
Lateral horn Olfactory ♀ ♀
SEZ Gustatory ♀ –
SEZ_2 Gustatory ♀ ♀
Medulla Visual ♀ –
Lobula Visual ♀ –
Lobula plate Visual ♀ ♀
Fan-shaped body Visual ♀ –
Ellipsoid body Visual ♀ –
AMMC Auditory ♂ ♂
MB-Beta lobe Memorial ♂ –
MB-Gamma lobe Memorial ♂ ♂
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“∗”: Brain regions enlarged in females are denoted with “♀”, while regions enlarged in males are denoted with “♂”. Regions that were not sexually dimorphic are denoted with “-“. SEZ, subesophageal zone; AMMC, antennal motor and mechanosensory center; MB, mushroom body. Although our voxel-based comparison revealed male-specific enlargement within the lateral protocerebrum of Ae. aegypti and Cx. quinquefasciatus, it is not included in this table because we were unable to conduct volume measurements on this region to verify its enlargement.

Female-enlarged regions

Chemosensory pathway

Regions of the brain associated with olfaction were enlarged in female mosquitoes (Figures 3A–3C). Olfactory signals are processed in the antennal lobe (AL) and then relayed to neurons in the lateral horn (LH) that directly regulate innate behaviors (Martin et al., 2011; Dobritsa et al., 2003; Fishilevich and Vosshall, 2005; Silbering et al., 2011; Liang et al., 2013; Ramaekers et al., 2005). In Ae. aegypti, the AL and LH were an average of 66.8 and 79.6% larger in females than in males, respectively (Figure 3B). In Cx. quinquefasciatus, the AL and LH were an average of 73.5 and 83.8% larger in females than in males, respectively (Figure 3C).

Figure 3.

Figure 3

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Volumetric comparison of female-enlarged regions in Ae. aegypti and Cx. quinquefasciatus mosquitoes

(A) Regions associated with olfaction. AL = antennal lobe; LH = lateral horn.

(B) Comparison of AL and LH volume in male and female Ae. aegypti.

(C) Comparison of AL and LH volume in male and female Cx. quinquefasciatus.

(D) Regions associated with gustation. The volume of the subesophageal zone (SEZ) was measured, in addition to the volumes of two subregions (SEZ_1 and SEZ_2) showing especially notable levels of nc-82 staining.

(E) Comparison of SEZ and SEZ subregion volume in male and female Ae. aegypti.

(F) Comparison of SEZ and SEZ subregion volume in male and female Cx. quinquefasciatus.

(G) Neuropils of the optic lobe, which is associated with vision.

(H) Comparison of optic lobe neuropil volume in male and female Ae. aegypti.

(I) Comparison of optic lobe neuropil volume in male and female Cx. quinquefasciatus.

(J) Neuropils of the central complex. FB = fan-shaped body; EB = ellipsoid body; PB = protocerebral bridge; N = paired noduli.

(K) Comparison of central complex neuropil volume in male and female Ae. aegypti.

(L) Comparison of central complex neuropil volume in male and female Cx. quinquefasciatus. In all cases, scale bars represent 50 μm. In all scatterplots, data are shown as mean ± SEM ns, p > 0.05; ∗, p < 0.05; ∗∗, p<0.01; ∗∗∗, p<0.001; ∗∗∗∗, p<0.0001 (unpaired t-test). Statistical power values were listed below the t-statistic values of the significantly different groups. N = 10 for each group with the exception of female Ae. aegypti. LH, for which N = 12.

In addition, we measured the volume of the subesophageal zone (SEZ), the gustatory processing center in insects (Freeman and Dahanukar, 2015; Sterne et al., 2021). Two subregions showing relatively high levels of nc-82 staining were also individually measured and are referred to as SEZ_1 and SEZ_2 (Figure 3D). In Ae. aegypti, the SEZ and SEZ_2 were an average of 55.1 and 108.9% larger in females than in males, respectively (Figure 3E). In Cx. quinquefasciatus, the SEZ_2 was an average of 170.4% larger in females than in males (Figure 3F).

Visual pathway

Regions of the brain associated with visual processing were enlarged in female mosquitoes (Figures 3G–3I). In insects, visual cues are processed in the optic lobe, which consists of three distinct neuropils: The lamina, medulla, and lobula complex, which is comprised of the lobula and lobula plate (Borst et al., 2010). In Ae. aegypti, the medulla, lobula and lobula plate were an average of 37.6, 22.5 and 37.5% larger in females than in males, respectively (Figure 3H). In Cx. quinquefasciatus, the lobula plate was an average of 26.7% larger in females than in males (Figure 3I).

Visual-motor transformation occurs in the central complex, which consists of four distinct neuropils: the fan-shaped body (FB), the protocerebral bridge (PB), the paired noduli (N), and the ellipsoid body (EB) (Figure 3J) (Pfeiffer and Homberg, 2014). In Ae. aegypti, the FB and EB were an average of 31.4 and 36.1% larger in females than in males, respectively (Figure 3K). Sexual dimorphism was not found in any of the central complex neuropils in Cx. quinquefasciatus (Figure 3L).

Male-enlarged regions

In addition to male-specific expansion in the regions described in detail below, our voxel-based comparison of Ae. aegypti and Cx. quinquefasciatus brains identified large areas of male-specific expansion in a region correlating approximately with the lateral protocerebrum (Figures 1D–1F and 2D–2F), which is believed to act as an integration center for multimodal sensory inputs (Galizia, 2014; Paulk et al., 2009). This region was not included in our volume measurements because it is not clearly delineated from other brain structures, rendering it impossible to accurately assess using our methods.

Auditory pathway

Regions of the brain associated with auditory processing were enlarged in male mosquitoes (Figures 4A–4C). In insects, auditory cues are detected by the Johnston’s organs in the antennae and then processed in the antennal motor and mechanosensory center (AMMC) (Ignell et al., 2005; Kamikouchi et al., 2006; Yorozu et al., 2009; Zhou et al., 2015). In Ae. aegypti, the AMMC was 112.3% larger in males than in females (Figure 4B). In Cx. quinquefasciatus, the AMMC was an average of 278.2% larger in males than in females (Figure 4C).

Figure 4.

Figure 4

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Volumetric comparison of male-enlarged regions in Ae. aegypti and Cx. quinquefasciatus mosquitoes

(A) Regions associated with hearing. AMMC = antennal mechanosensory and motor center.

(B) Comparison of AMMC volume in male and female Ae. aegypti.

(C) Comparison of AMMC volume in male and female Cx. quinquefasciatus.

(D) Lobes of the mushroom body, which is associated with learning and memory.

(E) Comparison of mushroom body lobe volume in male and female Ae. aegypti.

(F) Comparison of mushroom body lobe volume in male and female Cx. quinquefasciatus. In all cases, scale bars represent 50 μm. In all scatterplots, data are shown as mean ± SEM ns, p > 0.05; ∗, p < 0.05; ∗∗, p<0.01; ∗∗∗, p<0.001; ∗∗∗∗, p<0.0001 (unpaired t-test). Statistical power values were listed below the t-statistic values of the significantly different groups. N = 10 for each group.

Learning and memory pathways

Regions of the brain associated with learning and memory were enlarged in male mosquitoes (Figures 4D–4F). In insects, the mushroom body (MB) is partially involved in learning and memory consolidation. The α/β, α′/β′, and γ lobes are parallel axons of three classes of Kenyon cells located in the MB (Griffith, 2014). In Ae. aegypti, the β lobe and the γ lobe were an average of 39.4 and 28.2% larger in males than in females, respectively (Figure 4E). In Cx. quinquefasciatus, the γ lobe was an average of 66.2% larger in males than in females (Figure 4F).

Ae. aegypti and Cx. quinquefasciatus brains are anatomically different

Ae. aegypti and Cx. quinquefasciatus exhibit similar differences in their head morphology between males and females (Figure 5A) but differ in behavioral traits, such as host preference and active period, prompting us to investigate whether brain structure also differed between these two species. Because Cx. quinquefasciatus whole brains of both sexes were found to be significantly larger than those of Ae. aegypti (see Figure S2), scalars of 0.85 and 0.8 were applied to male and female Cx. quinquefasciatus brains, respectively, to normalize their volumes. After normalization, the SEZ_2 of females and AMMC of males were found to be significantly larger in Cx. quinquefasciatus than in Ae. aegypti. The γ lobe of the mushroom body in males and the SEZ, medulla, lobula and lobula plate of females were significantly larger in Ae. aegypti than in Cx. quinquefasciatus (Figure 5B).

Figure 5.

Figure 5

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Comparison of Ae. aegypti and Cx. quinquefasciatus mosquitoes

(A) Images of heads of male and female Ae. aegypti and Cx. quinquefasciatus. Images illustrate differences in antennae, proboscis, and palps between sexes and species. In both Ae. aegypti and Cx. quinquefasciatus, the maxillary palps are longer and the antennae are more plumose in males than in females, whereas females have more complex proboscises than males.

(B) Comparison of enlarged brain regions between Ae. aegypti and Cx. quinquefasciatus. Comparisons were made using the sex in which a brain region was enlarged; for additional clarity, labels with the “_M” suffix were compared in males, whereas those without were compared in females. For normalization, the volume of male and female Cx. quinquefasciatus brain regions were multiplied by 0.8 and 0.85 (the average volume of male and female Ae. aegypti whole brain: the average volume of male and female Cx. quinquefasciatus whole brain) respectively. AL = antennal lobe; LH = lateral horn; SEZ = subesophageal zone; FB = fan-shaped body; EB = ellipsoid body; AMMC = antennal mechanosensory and motor complex. Data are shown as mean ± SEM ns, p > 0.05; ∗, p < 0.05; ∗∗, p<0.01; ∗∗∗, p<0.001; ∗∗∗∗, p<0.0001 (Kruskal-Wallis and post-hoc Mann-Whitney U tests). Statistical power values were listed below the t-statistic values of the significantly different groups. N = 10 for each group, with the exception of Ae. aegypti. female LH, for which N = 12. See also Figure S2.

Discussion

We carried out the first full-scale voxel-based analysis of sexual dimorphism between male and female mosquito brains in two species relevant to public health, Aedes aegypti and Culex quinquefasciatus. These two blood-feeding mosquitoes show sex-specific expansion in brain regions associated with sexually dimorphic behaviors, suggesting that sex-specific development of physiological functions is correlated with regional brain expansion. The incomplete conservation of sexually dimorphic brain regions between these two species further suggests that evo-developmental factors determine which brain regions are sexually dimorphic for a given species.

Correlative relationships between differential enlargement of particular brain regions and behavioral characteristics have been documented in other animal models. In the canary, Serinus canarius, and zebra finch, Poephila guttata, brain regions involved in the acquisition and performance of song are much larger in males than in females, who do not sing (Nottebohm and Arnold, 1976). In the turnip moth, Agrotis segetum, the antennal lobe glomeruli that receive signals from ORNs that detect female sex pheromones are much larger in males than females (Hansson et al., 1992). Similarly, in Drosophila melanogaster, three antennal lobe glomeruli that receive innervation from fruGAL4 ORNs are larger in males than in females (Stockinger et al., 2005), and these ORNs are involved in detection of female sex pheromone and potentially male-male courtship behavior (Dalton et al., 2009).

In both Ae. aegypti and Cx. quinquefasciatus, females showed significant expansion in the antennal lobe (AL) and the lateral horn (LH) (Figures 3B and 3C), which are involved in olfaction. The AL is the primary olfactory center in insects and is composed of a number of glomeruli that varies among taxa and species (Stockinger et al., 2005). Each glomerulus represents the converging point for olfactory receptor neurons of a specific receptor type, such that different olfactory cues activate different patterns of glomeruli within the AL (Grabe et al., 2016). The LH is a higher order processing center of olfactory information that receives input directly from the AL, which is then transformed into a behavioral response (Schultzhaus et al., 2017; Galizia and Szyszka, 2008). Our observation of AL expansion in female mosquitoes is consistent with previous studies, which indicate that females possess more and/or larger AL glomeruli than males (Ignell et al., 2005; Ye et al., 2020; Shankar and McMeniman, 2020), potentially as a consequence of increased innervation of olfactory neurons to this region (Riabinina et al., 2016). Members of both sexes respond positively to floral odors (Jhumur et al., 2007, 2008; Basrur et al., 2020), however female mosquitoes also rely heavily on olfactory cues to locate blood-feeding hosts and oviposition sites. Odor plumes allow for long-distance host tracking and are made up of compounds emitted by hosts, primarily CO2 (Dekker and Cardé, 2011), whereas olfactory cues are used both in long-distance location and short-distance discrimination of oviposition sites (Mwingira et al., 2020). It is possible that female-specific expansion of the AL and LH is concentrated in glomeruli and LH circuits that modulate perception of host cues or participate in long- and short-range oviposition site perception.

In addition, females of both species showed significant expansion in the subesophageal zone_2 (SEZ_2) (Figures 3E and 3F). Gustatory receptor neurons (GRNs) are involved in taste perception and are located within hair-like sensilla that are generally found on an insect’s mouthparts, antennae, and at the ends of the legs. Depending on their location, these neurons project either directly or indirectly to the SEZ, which is responsible for central processing of taste (Freeman and Dahanukar, 2015). Expansion of the SEZ_2 in female mosquitoes is likely related to blood-feeding behavior. Female mosquitoes must be able to distinguish the tastes of nectar from blood, which are sweet and umami respectively, a challenge not encountered by males. In addition, the taste of blood must be palatable to females to promote feeding behavior. In Ae. aegypti, it has been demonstrated that females possess distinct populations of nectar- and blood-responsive GRNs that project to different regions of the SEZ. Blood-responsive GRNs are located in the stylet, which is the only part of the mouth in direct contact with blood during blood-feeding, whereas GRNs responding non-specifically to sugars such as glucose and fructose are located in the labium (Jové et al., 2020). Female-specific expansion of the SEZ_2 as we observed may correlate with regions innervated by stylet GRNs, consistent with these previous results.

In both Ae. aegypti and Cx. quinquefasciatus, females showed significant expansion within optic lobe neuropils (Figures 3H and 3I). The lobula plate showed expansion in females of both species. The insect eye is separated into a species-dependent number of facets, each containing photoreceptors that send signals directly to the optic lobe when activated. Among the optic lobe layers, the lobula plate plays a role in integrating local motion signals into global optic flow, facilitating insects’ optomotor response (Borst et al., 2010; Zhu, 2013). Females following an odor plume show a strong attraction to visual features that contrast with the environment. Upon approach, the presence and identity of thermal and volatile cues is used to determine whether to land on the object and initiate blood-feeding behavior (van Breugel et al., 2015). Accurate tracking and orientation toward mobile hosts likely requires enhanced perception of motion in females relative to males, which would in part explain our observed expansion of the lobula plate. Gene expression during optic lobe development is itself highly sexually dimorphic, providing a clear basis for the existence of differing visual abilities between male and female mosquitoes (Tomchaney et al., 2014).

Several central complex neuropils also showed female-specific expansion in Ae. aegypti (Figure 3K). The central complex is located at the center of the insect brain and acts as an integration center that combines and transforms multimodal sensory inputs from both sides of the brain into context-dependent behavioral outputs (Pfeiffer and Homberg, 2014). In Ae. aegypti, the ellipsoid body (EB) and fan-shaped body (FB) were enlarged in females relative to males. The central complex as a whole is believed to act as the brain’s navigation center (Honkanen et al., 2019), and the EB and FB play discrete, established roles in visual pattern recognition and spatial orientation (Liu et al., 2006; Pan et al., 2009). As described above, host-seeking females must orient toward and follow odor plumes, identify potential hosts, and track the movement of mobile hosts. It is possible that to respond to these unique navigational and visual challenges, females of some mosquito species show expansion within the central complex. It should be noted, however, that the central complex, specifically the EB and FB, are involved in the regulation of other complex behaviors, such as nociception and sleep homeostasis (Hu et al., 2018; Qian et al., 2017), and that it is impossible to determine the utility of female-specific expansion in these regions without further investigation.

In both Ae. aegypti and Cx. quinquefasciatus, males showed significant expansion in the AMMC (Figures 4B and 4C). The AMMC receives and processes sensory input from the Johnston’s organs located in the antennae and influences the insect’s perception of sound, wind, and gravity (Ignell et al., 2005). The male-specific expansion that we observed is likely related to sound processing. Male mosquitoes possess plumose antennae (Figure 5A) and show extreme sensitivity to the distinctive sound produced by the wingbeats of females (Gibson and Russell, 2006). In many mosquito species, including Ae. aegypti and Cx. quinquefasciatus, males periodically form large, transient mating swarms. Females enter the swarm solely to mate and leave after copulation. Thus, swarming males must be able to listen for and distinguish the wingbeats of individual females from those of potentially thousands of males (Fawaz et al., 2014). Expansion of sound-processing subregions of the AMMC would feasibly contribute to the enhanced hearing ability required to successfully locate females during mating swarms.

In both Ae. aegypti and Cx. quinquefasciatus, males showed significant expansion in the γ lobe of the mushroom body (MB), whereas male-specific expansion of the β lobe of the MB was observed in Ae. aegypti (Figures 4E and 4F). The MB plays a role in associative learning and memory, particularly when olfaction is involved, although not exclusively (Heisenberg, 2003; Akalal et al., 2006). Given the complex role of the MB, further study is necessary to determine the utility of MB enlargement in males.

Brain regions showing sexual dimorphism were generally consistent between Ae. aegypti and Cx. quinquefasciatus, indicating that these differences are conserved across mosquito species. However, several regions that showed sexual dimorphism in Ae. aegypti did not show it in Cx. quinquefasciatus. These regions were the β lobe of the mushroom body, the medulla, the lobula, the ellipsoid body (EB), the fan-shaped body (FB) and the subesophageal zone (SEZ). The utility of male-specific expansion of the β lobe requires further investigation but may be the result of species-specific differences in mating and courtship behavior.

The medulla, lobula, EB, and FB are all likely involved in visual identification and navigation toward hosts by females. Expansion of these regions in Ae. aegypti but not Cx. quinquefasciatus could be the result of differences in host-seeking behavior between these species. In terms of host preference, Ae. aegypti females overwhelmingly prefer to feed on humans, whereas Cx. quinquefasciatus females appear to either prefer avian hosts or to act as opportunistic feeders on avian and mammal hosts (Takken and Verhulst, 2013; Fitzpatrick et al., 2019). Differences in activity patterns are also observed between these two species. Ae. aegypti is a day-biting mosquito and is most active in the morning and afternoon (Taylor and Jones, 1969), whereas Cx. quinquefasciatus is a night-biting mosquito and is most active after dusk and throughout the night (Suom et al., 2010; Gowda and Vijayan, 1993). Given this difference, it is possible that vision is more integral to Ae. aegypti host-seeking than to that of Cx. quinquefasciatus. Ae. aegypti females are able to perceive low levels of light, but if kept in total darkness cannot engage in host-seeking behavior, underlying the importance of vision in host-seeking for this species (Kawada et al., 2005). The importance of vision in Ae. aegypti host-seeking is further underscored by observations in urban areas, where sources of artificial light are abundant and Ae. aegypti biting activity is commonly recorded outside of daylight hours (Chadee and Martinez, 2000; Smith et al., 2018). Vision may play a diminished role in Cx. quinquefasciatus host-seeking, with increased emphasis placed on the use of chemical and thermal cues, although studies in this regard are lacking. Our comparison of the volumes of female Ae. aegypti and Cx. quinquefasciatus brain regions provides further support for this idea (Figure 5B). Although Cx. quinquefasciatus females had significantly larger whole brain volume than Ae. aegypti females, the normalized volumes of the medullas, lobulas and lobula plates of Ae. aegypti females were significantly larger than those of Cx. quinquefasciatus females. A similar relationship between photoperiodism and brain investment has been observed in hawk moths. Diurnal species show increased investment into the optic lobes and regions of the brain that process vision, whereas nocturnal species demonstrate increased investment into the antennal lobes and regions that process olfaction (Stöckl et al., 2016), providing a clear connection between brain anatomy and behavior.

In this study, we have identified key regions of the mosquito brain exhibiting sexual dimorphism in two major vector species, Ae. aegypti and Cx. quinquefasciatus (Figure 6). Our results align with expectations regarding the functions of these regions and reflect the dimorphic behavior of male and female mosquitoes. Males congregate in large mating swarms and use their strong sense of hearing to locate solitary females entering the swarm, whereas females require a blood meal to reproduce, which introduces unique and potentially species-specific challenges associated with finding and feeding on hosts. Altogether, our work correlates sexually dimorphic behavior with brain structure, which carries with it strong implications for our understanding of the neuroanatomical basis of mosquito sexual dimorphism, as well as for the fields of vector control and disease management.

Figure 6.

Figure 6

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Anatomical dimorphisms in mosquito brain suggest sexually dimorphic behaviors

This schematic drawing visualizes general anatomical dimorphism in the sexually dimorphic mosquito brain and their theoretical behavioral correlates, which suggest future research directions. All images used in this figure were generated by the authors.

Limitations of the study

In this study, we have made voxel-wise comparisons of male and female brains in two major mosquito disease vectors, Aedes aegypti and Culex quinquefasciatus. Furthermore, we established a correlative relationship between anatomical dimorphism in brain structure and dimorphic behavior. However, these relationships are speculative and functional study is necessary to establish causal links between dimorphic brain regions and behaviors. Although we were able to manually label and quantify the volumes of major sensory regions, such as the optic and antennal lobes, some regions, most notably the lateral protocerebrum, could not be measured in this fashion because of difficulty in distinguishing their boundaries. Therefore, although our deformation-based morphometry analysis identified these regions as sexually dimorphic, this result could not be verified. More work is needed to study the mechanisms underlying regional expansion and find the neurons regulating host-seeking behavior in the sexually dimorphic brain regions that we have identified.

STAR★Methods

Key resources table

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
Mouse monoclonal anti-Bruchpilot antibody (nc82) Developmental
Studies Hybridoma
Bank		 Cat#nc82;
RRID: AB_2314866
Goat anti-mouse, Alexa Fluor 546 Thermo Fisher
Scientific		 Cat# A-11030;
RRID: AB_2534089
Chemicals, peptides, and recombinant proteins
Paraformaldehyde (PFA) Electron Microscopy
Sciences		 Cat# 15713
DPX Mountant Sigma-Aldrich Cat# 44581
Normal goat serum Sigma-Aldrich Cat# G9023
Deposited data
Confocal data for the generation of template brains This paper https://doi.org/10.5061/dryad.51c59zwbq
Experimental models: Organisms/strains
Aedes aegypti Zou Lab, Institute of Zoology, Chinese Academy of Sciences N/A
Culex quinquefasciatus Cui Lab, Institute of Zoology, Chinese Academy of Sciences N/A
Software and algorithms
Fiji Schindelin et al., 2012 https://fiji.sc
Computational Morphometry Toolkit (CMTK) Rohlfing and Maurer, 2003; Rohlfing, 2012; Jefferis et al., 2007; Ostrovsky et al., 2013 https://www.nitrc.org/frs/?group_id=212
MATLAB 2016a MathWorks RRID: SCR_001622
GraphPad Prism 7 GraphPad Software RRID: SCR_002798
Adobe Illustrator CS6 16.0.0 Adobe Inc. RRID: SCR_010279
Amira 2019.1 Thermo Fisher Scientific RRID: SCR_007353
Python 2.7.18 Python Programming Language RRID: SCR_008394
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Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Xuguo “Joe” Zhou (xuguozhou@uky.edu).

Materials availability

This study did not generate new unique reagents.

Experimental model and subject details

Aedes aegypti

Mosquitoes belonging to the Ae.aegypti Rockefeller strain were raised as described previously (Hays and Raikhel, 1990). Rearing of adults and larvae was conducted at 27°C and 70% relative humidity under a 12:12 L:D cycle. Larvae were raised in non-crowded conditions (200 larvae per 30 × 25 × 7 cm basin containing 1.5 L distilled water) and fed on standard diet (equal parts rodent diet, lactalbumin, and brewer’s yeast). Food was added when necessary and water was kept relatively clear and odor-free. Following pupation, pupae were placed in a plastic cage (dimensions: 5.5 cm diameter, 3 cm depth) to await eclosion. Each cage contained the pupae from a total of four basins. Adult mosquitoes were continuously fed water and 10% sucrose (w/v) solution by wick in a 30 × 20 × 15 cm box. All procedures for vertebrate animal use were approved by the Animal Care and Use Committees of the Chinese Academy of Sciences (IOZ-IACUC-2021-164).

Culex quinquefasciatus

Cx.quinquefasciatus mosquitoes used in this study were collected from Haikou city, Hainan province in 2013 and have been maintained in the laboratory until use (Shen et al., 2022). A total of 200 adult mosquitoes were collected in a 30 × 30 × 30 cm fabric box provisioned with cotton balls saturated with 10% sucrose (w/v) solution. Other rearing procedures were identical to those described for Ae. aegypti. All procedures for vertebrate animal use were approved by the Animal Care and Use Committees of Chinese Academy of Sciences (IOZ-IACUC-2021-164).

Method details

Fixation and immunochemistry

For the brains used to generate the template brains and register to the intersex template brain in Cx. quinquefasciatus, fixation and immunochemistry were carried out as described (Riabinina et al., 2016; Jové et al., 2020; Zhao et al., 2022), with some modification. Four to six days after eclosion, adult male and female mosquitoes were anesthetized on ice for 5 min and their heads were carefully removed from the body with sharp forceps. Heads were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) diluted in 0.1 M Millonig’s phosphate buffer (pH 7.4) mixed with 0.25% Triton X-100 in a 1.5 mL tube and incubated for 1.5 h at 4°C. After washing the fixed heads in cold PBS (PBS) three times, brains were dissected and placed in 0.5 mL protein LoBind tubes (Eppendorf) filled with cold PBS. The brains were washed with PBS containing 0.25% Triton X-100 (PBT) for approximately 2 h (4 × 5min + 3 × 10min + 3 × 20 min) at room temperature (RT) and permeabilized with 4% Triton X-100 and 2% normal goat serum (NGS) (Sigma) in PBS at 4°C for two days. After washing with PBT for approximately 1 h (2 × 5min + 2 × 10min + 2 × 20 min) at RT, brains were incubated for two days at 4°C with 400 μL of the primary antibody anti-nc82 (mouse, 1:50; DSHB), which was diluted in PBT with 2% NGS. After washing with PBT for approximately 2 h (3 × 5min + 3 × 10min + 3 × 20 min) at RT, brains were incubated for two days at 4°C with 400 μL of the secondary antibody Alexa Fluor goat anti-mouse 546 (1:500; Life technologies), which was diluted in PBT with 2% NGS. Brains were then washed with PBT for approximately 2 h (3 × 5min + 3 × 10min + 3 × 20 min) and fixed in 4% paraformaldehyde diluted with PBS for 4 h at RT. After washing with 0.3% PBT for 1 h (3 × 20 min) at RT, brains were mounted onto a poly-L-lysine (PLL)-coated coverslip immersed in PBS. The mounted brains were then dehydrated in graded ethanol (30%, 50%, 70%, 95%, 100%, 100%, 100%; 5 min per step) and cleared in xylene (100%, 100%, 100%, 5 min per step). Several drops of DPX (Sigma) were added onto the PLL-coverslip to cover the brains. The coverslip was inverted and gently placed on a slide with two coverslips used as spacers. The slide was ventilated in a fume hood for at least 2 days prior to imaging.

Given that it was easier to handle Ae. aegypti heads than Cx. quinquefasciatus heads, the fixation and immunochemistry for the brains used to generate the template brains and register to the intersex template brain in Ae. aegypti were carried out as described previously (Zhou et al., 2014; Wang et al., 2020). Specifically, brains were dissected from anesthetized mosquitoes directly, followed by fixation with 2% paraformaldehyde diluted with PBS for 55min at RT. After washing with PBT (all washing PBT contained 0.3% Triton X-100) for 1 h (3 × 20 min) at RT, brains were permeabilized with 0.3% PBT containing 5% NGS at RT for 1 h. Brains were then incubated for 24 h at 4°C with 200 μL of the primary antibody anti-nc82 (1:40), which was diluted in 0.3% PBT with 5% NGS. After washing with PBT for 1 h (3 × 20 min) at RT, brains were incubated for 24 h at 4°C with 100 μL of the secondary antibody Alexa Fluor goat anti-mouse 546 (1:500), which was diluted in 0.3% PBT with 5% NGS. Other steps were the same as those used for Cx. quinquefasciatus brains.

Image acquisition

For the Cx. quinquefasciatus brains used for manual volume quantification and all Ae. aegypti brains, confocal stacks were acquired using a Zeiss LSM 710 confocal microscope. Whole brains were imaged at 1024 × 1024 pixel resolution at 1 μm intervals using a Plan-Apochromat 20x/0.8 M27 objective. Mushroom body lobes were imaged at 1024 × 1024 pixel resolution at 1μm intervals using an EC Plan-Neofluar 40x/1.30 oil DIC M27 objective. For the Cx. quinquefasciatus brains used to generate template brains, confocal stacks were acquired using a LEICA DM6 CS confocal microscope. Whole brains were imaged at 1024 × 1024 pixel resolution at 1 μm intervals using an HC PL APO CS2 20x/0.75 DRY objective. All images were taken at 8-bit color depth.

Template brain generation

Template brain generation was carried out as described previously using the registration software Computational Morphometry Toolkit (CMTK; http://www.nitrc.org/projects/cmtk/) (Cachero et al., 2010; Jefferis et al., 2007; Rohlfing and Maurer, 2003; Rohlfing, 2012; Ostrovsky et al., 2013). Raw confocal images were pre-processed by rotating them so that each was oriented in the same direction, then exported as NRRD files with Fiji (https://fiji.sc) (Schindelin et al., 2012) using plugins for reading and writing registration output (http://teem.sourceforge.net/nrrd/format.html). We constructed shape-averaged templates corresponding to the brains of males and females of the two species through nonrigid registration. For Ae. aegypti, 10 male and 10 female brains were used. For Cx.quinquefasciatus, 15 male and 15 female brains were used. Brains used for template generation were selected on the basis of symmetry and uniformity of shape. Because there were large differences in morphology between male and female brains in both species, we computed a new template by shape-averaging the male and female templates. We refer to this as an intersex template.

Deformation-based morphometry

Deformation-based morphometry (DBM) is a nonrigid registration method that has been used to study human and Drosophila brain structure (Ashburner et al., 1998; Rohlfing et al., 2006; Cachero et al., 2010; Jefferis et al., 2007). The aim of this approach is to systematically locate differences in the amount of expansion or contraction in each point of a sample brain compared to a template. The amount of deformation, quantified by the volume change, at any particular grid point is evaluated by the Jacobian determinant. The Jacobian determinant will be 1 when there is no relative size difference between a sample brain and the template at that point, >1 when the sample brain is larger than the template brain at that point, and <1 when the template brain is larger than the sample brain at that point. In order to compare the volume of brain regions across the sexes, we calculated the Jacobian determinant at a voxel-wise scale for each male and female brain through registration to the corresponding intersex template brain using CMTK’s registration, warp, and reformatx functions. For each species, approximately 40 individual brains of each sex were registered to the intersex template brain to calculate systematic volume expansion or contraction (Ae. aegypti male, n = 35; Ae. aegypti female, n = 43; Cx. quinquefasciatus male, n = 38; Cx. quinquefasciatus female, n = 38). Brains were excluded from this analysis if they were damaged or otherwise asymmetric.

Before t-test analysis, the raw Jacobian determinants were normalized by dividing by the median Jacobian determinant for each brain to remove the overall difference in size between individual brains and the intersex template brain. The median Jacobian determinant is an approximation of the relative brain size compared to the intersex template brain. The normalized Jacobian determinants of all individual brains were used to perform a per-voxel t-test between sexes and identify male- and female-enlarged brain regions using the ttest function of CMTK.

Manual quantification of brain regions

Regions identified as showing male or female enlargement relative to the intersex template brain were manually labeled in each individual Ae. aegypti and Cx. quinquefasciatus brain using the “Brush Tool” and “Segmentation Editor” plugins in Fiji. 10 brains were quantified per sex and species, respectively, excepting the LH in female Ae. aegypti, which was measured in 12 brains. We took the D. melanogaster (https://v2.virtualflybrain.org/) and Ae. aegypti (https://www.mosquitobrains.org/) brain structure as references, and boundaries between regions were clearly delineated as a result of nc82 staining. We quantified the raw confocal data of brains that were stained using the fixation procedure described above for Ae. Aegypti brains for the two species. In Ae. aegypti, labeling was performed by two individuals. Both individuals labeled ALs and AMMCs. SEZs, OLs, MBs, FBs, EBs, PBs, Ns and LHs in both male and female brains were labeled by a single individual, who labeled all of the listed regions. In Cx. quinquefasciatus, labeling of all regions in both males and females was performed by a single individual, who had also participated in labeling for Ae. aegypti. Following labeling, the volume of each region was calculated using the “3D Manager” plugin in Fiji.

Image processing and visualization

The t-test heatmaps generated by DBM analysis were displayed using the corresponding intersex template brain as the background using the Matplotlib imshow function in MATLAB 2016a. Visualization of voxel-wise male- and female-enlarged brain regions and the manually quantified brain regions was carried out in Amira 2019.1 (Thermo Fisher Scientific) (Stalling et al., 2005). The Isosurface function was used to display voxel-wise male- and female-enlarged brain regions. A cutoff value of ±2.5 was used as the t-value threshold. To visualize each of the manually quantified brain regions, the segmentation editor function was used.

External head photography

Three different adult individuals from each species and each sex were anesthetized with diethyl ether and fixed on a piece of plasticine. Within 4 h of anesthetization, the head of each individual was photographed from a top-down view using an Olympus DSX1000 digital microscope and a DSX10-SXLOB Plan 3x/0.09 objective (Olympus, Japan). Out of the three images captured for each species/sex combination, the clearest was chosen for later use.

Quantification and Statistical analysis

Statistical analysis of volume comparisons between sexes and species was conducted using GraphPad Prism 7. We identified significant differences between pairwise groups using unpaired t-tests and between multiple groups using a Kruskal-Wallis ANOVA test followed by a post-hoc Mann-Whitney U test to correct for multiple comparisons where applicable. Differences were considered significant if p ≤ 0.05. We then conducted power statistics analysis for each comparison with significant differences. For comparisons between the whole brain volumes of males and females in the two species, a generalized linear model (GLM) of gamma family and log link function was used to test the interactions between species for each sex, between sexes for each species, and the interaction between species and sex. p values were calculated with wald test. Differences were considered significant if p ≤ 0.05. The power analysis and the GLM analysis were conducted with Python. All associated code is included in the supplemental files (see Data S2).

Acknowledgments

We are grateful to Zhen Zou and Feng Cui for providing Aedes aegypti and Culex quinquefasciatus colonies. We thank Zhengzhong Huang for imaging mosquitoes, Zhilei Zhao for sharing the protocol and suggestions about the dissection and staining. We also thank Haonan Zhou, Kai Shi, Mao Wang, Siping Li and Yuting Zhang for their assistance in maintaining mosquitoes. Special thank goes to Chao Ning for sharing insights in data analysis, and Jue Xie and Yucheng Sun for commenting on an earlier draft. This work was supported by the State Key Laboratory of Integrated Management of Pest, Insects, and Rodents, IOZ, CAS to CZ and XGZ (Nos. Y952824103 and Y929731103). AM’s summer research in China (2018 and 2019, respectively) was supported in part by the National Natural Science Foundation of China to XGZ (No. 31828009), and a Confucius Institute Student Study in China Travel Grant, University of Kentucky, to AM. Contributions by XGZ and AM were supported in part by the USDA National Institute of Food and Agriculture, Hatch projects KY008071 and KY008090, to XGZ.

Author contributions

C.Z., J.L., and X.G.Z. designed the study; J.L., A.M., S.Y.Z., and T.W. conceived the experiments; J.L. and A.M. drafted the manuscript; X.G.Z. revised and finalized the manuscript. All authors read and approved the final version of the manuscript.

Declaration of interests

The authors declare no competing interests.

Inclusion and diversity

We support inclusive, diverse, and equitable conduct of research.

Published: November 18, 2022

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2022.105255.

Contributor Information

Jing Li, Email: lijing@szbl.ac.cn.

Xuguo Zhou, Email: xuguozhou@uky.edu.

Supplemental information

Document S1. Figures S1

and S2

mmc1.pdf (192.8KB, pdf)
Data S1. Representative brain images used for generating the male and female templates, related to Figures 1 and 2

For A. aegypti, confocal stacks were acquired using a Zeiss LSM 710 confocal microscope. Whole brains were imaged at 1024 × 1024 pixel resolution at 1 μm intervals using a Plan-Apochromat 20x/0.8 M27 objective. For C. quinquefasciatus, confocal stacks were acquired using a LEICA DM6 CS confocal microscope. Whole brains were imaged at 1024 × 1024 pixel resolution at 1 μm intervals using an HC PL APO CS2 20x/0.75 DRY objective. All images were taken at 8-bit color depth. Raw confocal images were pre-processed by rotating them so that each was oriented in the same direction

mmc2.zip (30.9MB, zip)
Data S2. Code for power, related to Figures 3, 4, and 5

Code used to run the raw data to generate Figures 3B, 3C, 3E, 3F, 3H, 3I, 3K, 3L, 4B, 4C, 4E, 4F, and 5B.

mmc3.txt (2.1KB, txt)

Data and code availability

  • •

    Confocal data used in the generation of template brains have been deposited at Dryad and are publicly available as of the date of publication. The DOI is listed in the key resources table.

  • •

    All original code is available in this article’s supplemental information.

  • •

    Any additional information required to reanalyze the data reported in this article is available from the lead contact upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1

and S2

mmc1.pdf (192.8KB, pdf)
Data S1. Representative brain images used for generating the male and female templates, related to Figures 1 and 2

For A. aegypti, confocal stacks were acquired using a Zeiss LSM 710 confocal microscope. Whole brains were imaged at 1024 × 1024 pixel resolution at 1 μm intervals using a Plan-Apochromat 20x/0.8 M27 objective. For C. quinquefasciatus, confocal stacks were acquired using a LEICA DM6 CS confocal microscope. Whole brains were imaged at 1024 × 1024 pixel resolution at 1 μm intervals using an HC PL APO CS2 20x/0.75 DRY objective. All images were taken at 8-bit color depth. Raw confocal images were pre-processed by rotating them so that each was oriented in the same direction

mmc2.zip (30.9MB, zip)
Data S2. Code for power, related to Figures 3, 4, and 5

Code used to run the raw data to generate Figures 3B, 3C, 3E, 3F, 3H, 3I, 3K, 3L, 4B, 4C, 4E, 4F, and 5B.

mmc3.txt (2.1KB, txt)

Data Availability Statement

  • •

    Confocal data used in the generation of template brains have been deposited at Dryad and are publicly available as of the date of publication. The DOI is listed in the key resources table.

  • •

    All original code is available in this article’s supplemental information.

  • •

    Any additional information required to reanalyze the data reported in this article is available from the lead contact upon request.

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