Visual processing in Anopheles gambiae mosquito larvae

Abstract

Stemmata are larval eyes found in holometabolous insects, unlike compound eyes which give far greater resolution. Aedes aegypti mosquito larvae possess both the larval and adult eyes in the late L3 instar. Both eyes are predicted to express different opsins, which has been shown to influence phototaxis behaviour. Light avoidance behaviour could be further explained by optic lobe size, which controls many visual behaviours in insects. My aim is to characterise the developmental morphology of both larval and adult eyes, opsin expression, optic lobes and spectral sensitivity, of this unique visual system and investigate how these factors mediate light avoidance behaviour.
To investigate spectral sensitivity across larval development, I quantified opsin expression and used electroretinogram recordings. I show that Opsin6 (Op6), predicted to be a green-sensitive opsin, has higher expression than all other opsins in larval instars L1 and early L3. In contrast, Opsin1, Opsin3 (green-sensitive), and Opsin9 (blue-sensitive) gradually increase in expression in the late L4 stage and the adults. In line with the opsin data, L4 larvae showed a strong electroretinogram (ERG) response to ultraviolet (UV), blue, and green light. I also show for the first time that Anopheles gambiae adults have three peaks of spectral sensitivity to ultraviolet (UV), blue, and green light.
The results of the opsin and electrophysiology data led me to hypothesise that larvae will respond to green and blue light. To test my hypothesis, I conducted behavioural experiments that measured light avoidance of Anopheles gambiae larvae to different wavelengths of light. I found that late L3/L4 instars indeed avoid green, red, white, and blue light. In contrast, I found L2/early L3 larvae had less of a response to all stimuli. Given that early instars (L2/early L3) are not predicted to express the blue-sensitive opsin, Opsin9, it is reasonable to conclude
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that the adult eye is functional during the L4 stage. The significant increase in light avoidance behaviour may be further explained by the brain anatomy data collected in the synchrotron, which we used to measure optic lobe size. I found that the optic lobes do not form until the late L3 larval instar, which could explain why late L3/L4 larval instars show stronger light avoidance.
The emergence of the functional adult eye, which expresses multiple opsins alongside the larval eye, makes the larvae highly sensitive to different wavelengths of light. I show that lens morphology differs between ommatidia formed in the larva and fully formed ommatidia in the adult stages. I also tracked the development of the larval eye. I found it shrinks significantly and is retracted behind the adult eye in day 10–adult mosquitoes. The results here suggest that both the larval eye and adult eye are functional during the larval stage