RESEARCH ARTICLE

Expression and light-triggered movement of rhodopsins in thelarval visual system of mosquitoesManuel Rocha, Kyle J. Kimler, Matthew T. Leming, Xiaobang Hu, Michelle A. Whaley and Joseph E. O’Tousa*

ABSTRACTDuring the larval stages, the visual system of the mosquito Aedesaegypti contains five stemmata, often referred to as larval ocelli,positioned laterally on each side of the larval head. Herewe show thatstemmata contain two photoreceptor types, distinguished by theexpression of different rhodopsins. The rhodopsin Aaop3 (GPROP3)is expressed in the majority of the larval photoreceptors. There aretwo small clusters of photoreceptors located within the satellite andcentral stemmata that express the rhodopsin Aaop7 (GPROP7)instead of Aaop3. Electroretinogram analysis of transgenic Aaop7Drosophila indicates that Aaop3 and Aaop7, both classified as long-wavelength rhodopsins, possess similar but not identical spectralproperties. Light triggers an extensive translocation of Aaop3 from thephotosensitive rhabdoms to the cytoplasmic compartment, whereaslight-driven translocation of Aaop7 is limited. The results suggest thatthese photoreceptor cell types play distinct roles in larval vision. Anadditional component of the larval visual system is the adultcompound eye, which starts to develop at the anterior face of thelarval stemmata during the 1st instar stage. The photoreceptors of thedeveloping compound eye show rhodopsin expression during the 4thlarval instar stage, consistent with indications from previous reportsthat the adult compound eye contributes to larval and pupal visualcapabilities.

KEY WORDS: Mosquito larva, Stemmata, Photoreceptors, Mosquitovision, Rhodopsin movement, Rhodopsin expression

INTRODUCTIONThe Aedes aegypti mosquito is the principal vector of thearboviruses causing yellow fever and dengue fever. This mosquitois known to use multiple sensory cues for efficient host-seekingbehavior (McMeniman et al., 2014). We sought to characterize theAedes visual system to understand how vision contributes to host-seeking and other behaviors underlying disease transmission. TheAedes genome contains ten predicted rhodopsins, encoding theG-protein-coupled receptors that initiate visual transduction (Neneet al., 2007). These were classified into five groups based onphylogenetic analysis. Three of these groups are labeled aslong-wavelength (>500 nm), short-wavelength (400–500 nm), andUV-sensitive rhodopsin (<400 nm), and two of these groups containuncharacterized family members. We previously characterized thepatterns of rhodopsin expression in the adult Aedes compound eye(Hu et al., 2009, 2011, 2012, 2014). These studies revealed featuresof the visual system in Aedes that were not predicted from study ofother invertebrate models.

One noteworthy feature is the daily cycle of rhodopsin movementfrom the cytoplasm into the photosensitive rhabdomeric membranesat dusk and then back into the cytoplasm at dawn (Hu et al., 2012).The increased rhodopsin content within the rhabdom at nightwill increase light sensitivity, which is probably a valuablephotoreceptor adaptation for acquisition of visual informationin the dim light conditions where mosquitoes are most active. Inaddition, a prevalent cause of photoreceptor degeneration bothin vertebrates (Vihtelic and Hyde, 2000; Okano et al., 2012)and invertebrates (Stark and Carlson, 1984; Meyer-Rochow et al.,2002) is exposure to bright light. For this reason, anotherbenefit of rhodopsin movement out of the rhabdoms duringdaylight may be to protect photoreceptors from bright-light-induced damage.

Here, we extend the analysis of the Aedes visual system bycharacterizing rhodopsin expression and light-triggered rhodopsinmovement during the larval stage. Mosquito development proceedsthrough four aquatic larval instars and a mobile aquatic pupal stageprior to emergence of the adult (Clements, 1999). The majorcomponents of the larval visual system are the stemmata, or larvalocelli, that mediate phototaxis and diving behaviors during the larvaland pupal periods (Kasap, 1977b).We show that there are two classesof photoreceptors within the stemmata. The major stemmatalphotoreceptor class expresses the Aaop3 rhodopsin (see Materialsandmethods for an explanation ofAedes opsin nomenclature). Aaop3exhibits extensive light-driven relocation, establishing that the light-triggered loss of∼80% rhabdom volume documented in Aedes larvalphotoreceptors (White, 1968) is associatedwith rhodopsinmovement.The minor photoreceptor class expresses the rhodopsin Aaop7. Thisrhodopsin possesses shorter wavelength sensitivity than Aaop3 andexhibits limited light-driven movement. We also show that the majoradult stage rhodopsin, Aaop1, is expressed in the developingcompound eye during the 4th larval instar. This finding is consistentwith earlier results that indicated that the compound eye maycontribute to larval and pupal vision (Kasap, 1977a).

RESULTSThe Aedes larval visual systemFrom the 1st instar, the visual system of the mosquito larva consistsof a set of five stemmata that are positioned laterally on each side ofthe head (Sato, 1953; White, 1961). Fig. 1A shows a diagramincluding the names of the stemmata. Fig. 1B–G show images of themosquito head at different developmental stages. Midway throughthe 1st instar stage, the development of the adult compound eyeinitiates within epidermal cells positioned anterior and adjacentto these stemmata. In the 3rd instar larva, two distinct regionscorresponding to the dorsal and ventral hemispheres show the initialpigmentation of the adult eye (Fig. 1C, arrowheads). Therecruitment and differentiation of adult retinal cells occur fromthese two sites, such that the two distinct pigmented eye fields arevisible in the early 4th instar larva (Fig. 1D, arrowheads). These twoReceived 24 July 2014; Accepted 26 February 2015

Department of Biological Sciences and Eck Institute for Global Health, Galvin LifeScience Building, University of Notre Dame, Notre Dame, IN 46556, USA.

*Author for correspondence ( jotousa@nd.edu)

1386

© 2015. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2015) 218, 1386-1392 doi:10.1242/jeb.111526

TheJournal

ofEx

perim

entalB

iology

areas have merged into a single eye field by the mid-4th instar larvalstage (Fig. 1E) and continue to expand anteriorly to produce thefully formed adult eye by the midpupal stage (Fig. 1F). The site ofthe larval stemmata remains visible at the posterior edge of the adulteye (Fig. 1G, arrow).

Aaop3 rhodopsin is expressed in most stemmatalphotoreceptors and exhibits light-driven movementAaop3 is a member of the long-wavelength rhodopsins. Peptidescorresponding to amino acid sequences within the N-terminal andC-terminal ends of the Aaop3 rhodopsin were used to generateAaop3 antisera. These two peptide sequences are poorly conservedwith other rhodopsin subfamily members (Fig. 2A,B, left), andthe resulting antisera were expected to uniquely recognize theAaop3 rhodopsin. The antisera stained transgenic Drosophilaexpressing Aaop3 but not wild-type or transgenic Aaop7-expressing Drosophila (Fig. 2A,B, right).In the 4th instar mosquito larval head, individual stemmata are

recognized by strong actin staining (red) within the rhabdom (R)regions (Fig. 2C). Four stemmata were identified previously(White, 1967; Brown and White, 1972) and named as the dorsalanterior (DA), dorsal posterior (DP), central (C) and ventral (V)stemmata. We identified a fifth, much smaller, stemma that waslocated dorsally to these four stemmata and named this the satellite(S) stemma (Fig. 2C). The central stemma is composed of 20–22photoreceptors. The dorsal anterior, dorsal posterior and centralstemmata each contain 9–12 photoreceptors. The satellite stemmacontains six photoreceptors. In each of the stemmata, therhabdomeres of all photoreceptors fuse into a common rhabdomstructure (Fig. 2C,E).Fig. 2C,D also shows that Aaop3 (green) is detected within all

five stemmata structures. In tissue prepared from a light-treatedlarva, Aaop3 is largely excluded from the rhabdom structures andfound dispersed within the cell body of the majority of stemmatalphotoreceptors. There are a small number of photoreceptors in the

central and the satellite stemmata lacking detectable Aaop3expression (Fig. 2C, asterisks).

The identification of Aaop3 as a stemmatal rhodopsin allowedus to test whether light exposure triggers an extensive movement ofthis rhodopsin. Indeed, in retinas of larvae fixed and dissectedin dark conditions, Aaop3 is sequestered within the rhabdoms andfound at very low levels within the photoreceptor cell bodies(Fig. 2F–H). The same region within the rhabdom of the centralstemmata is not labeled by Aaop3 (Fig. 2F, asterisk) as seen in thelight-treated retina (Fig. 2C, asterisk). The comparable views oflight-treated and dark-treated stemmata presented in Fig. 2C–E andFig. 2F–H respectively, show that there is a robust movement ofAaop3 upon transitions between light and dark conditions, withAaop3 sequestered within the rhabdomeric compartment whenlarvae are placed in dark conditions.

Aaop7 rhodopsin is expressed in a non-overlapping set ofstemmatal photoreceptorsAntibodies against Aaop7, another member of the long-wavelengthrhodopsin group, were created using the same strategy described togenerate antisera against Aaop3. Aaop7 shares limited sequenceidentitywith other long-wavelength rhodopsinswithin theN-terminalregion, making it likely that antibodies recognizing this peptidesequence are specific to Aaop7 (Fig. 3A, left). In agreement with thisexpectation, the resulting antiserum successfully labeled transgenicDrosophila retina expressing Aaop7 (Fig. 3A, middle) but failed tolabel thewild-type or Aaop3-expressing transgenicDrosophila retina(Fig. 3A, right).

To determine whether Aaop7 is expressed in the mosquito larvalstemmata, similar experiments to those already described for Aaop3were carried out with the Aaop7 antiserum. This approach revealedthat Aaop7 (green) is expressed in small groups of photoreceptorswithin the central stemmata and the satellite stemmata (Fig. 3B,arrows). The location of these groups is consistent with the view thatthe Aaop3 and Aaop7 rhodopsins are expressed in nonoverlappingsets of photoreceptors of the central and the satellite stemmata. Inmany preparations, the Aaop3 and Aaop7 groups were composed ofthree photoreceptors each in the satellite stemma, and the Aaop7group was composed of three photoreceptors in the central stemma.However, as it was not always possible to reliably count the cellbodies, some animals may not have exactly these numbers ofAaop3- and Aaop7-expressing photoreceptors.

We then examined whether Aaop7 also shows light-drivenmotility by examining the satellite and central stemmata in light- anddark-treated mosquitoes. In z-series confocal reconstructions of thesatellite stemma, Aaop7 (green) was detected only within the actin-rich (red) rhabdoms of dark-treated photoreceptors (arrow, Fig. 3C).Upon light treatment (Fig. 3D), some Aaop7 was detected withincytoplasmic vesicles (arrow) of the photoreceptor cell bodies (CB),but the majority of Aaop7 remained within the rhabdom. In a similaranalysis of the central stemma, Aaop7 was localized to the rhabdomin dark conditions (Fig. 3E), and showed limited movement to thephotoreceptor cell bodies (Fig. 3F) when treated by light. Theseresults indicate that Aaop7 exhibits less light-triggered movementthan Aaop3.

The spectral properties of Aaop7 rhodopsinComparative sequence analysis places Aaop3 and Aaop7 in thegroup of long-wavelength rhodopsins (Nene et al., 2007). Toexperimentally validate this characterization, we performed anelectroretinogram (ERG) analysis on transgenic Drosophilaexpressing these genes. Aaop3 transgenic Drosophila failed to

A B C D

E F G

SatelliteDorsal anterior

Dorsal posterior

Central

Ventral

2nd Instar Late 3rd instar Early 4th instar

Mid 4th instar Pupal Adult

Larval stemmata

Larva

Developing adult compound eye

RR

R

R

Fig. 1. Organization of the Aedes visual system during larvaldevelopment. (A) Diagram of the larval visual system showing that the larvalstemmata are composed of five photoreceptor groups, labeled from dorsal toventral as the satellite, dorsal anterior, dorsal posterior, central and ventralstemmata. The central areas colored in red represent the fused rhabdoms(R) which are composed of the rhabdomeres from individual stemmatalphotoreceptors. The adult compound eye development begins at the anteriorface of the larval stemmata and extends further in the anterior direction asdevelopment proceeds (arrows). (B–G) Microphotographs of the Aedesmosquito head showing the organization of the larval stemmata anddevelopment of the adult compound eye at larval, pupal and adult stages.Arrows in all images point to the larval stemmata. The larval stemmata persistthough the pupal stages and adult metamorphosis. Arrowheads in C andD identify the early stages of adult eye development immediately anterior to thelarval stemmata. Scale bars: 200 µm.

1387

RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 1386-1392 doi:10.1242/jeb.111526

TheJournal

ofEx

perim

entalB

iology

give an ERG response, probably because of poor Aaop3 expressionand an inability of Aaop3 to localize to Drosophila photoreceptorrhabdomeres (Fig. 2A,B). By contrast, ERG responses wereconsistently recorded from the Aaop7 transgenics. Fig. 4A showsa representative ERG trace from the Aaop7 transgenic flies, andFig. 4B shows a compilation of the data obtained from fourindividual flies. The results show that the ERG response to photon-matched stimuli was greatest at a wavelength of 450 nm. However,the Aaop7 transgenic fly retains high sensitivity to shorterwavelengths (400 and 350 nm) and does not exhibit the secondpeak response in the UV observed for Drosophila Rh1 (Fig. 4C,D)and Aaop2, the only other long-wavelength Aedes opsin that hasbeen analyzed in this way (Hu et al., 2011).

Rhodopsin expression in the developing adult eyeThe mosquito adult compound eye develops from a placodeimmediately anterior to the larval stemmata. This developmentalprocess is already in progress during the 1st instar larva, and the 1stommatidial units are fully differentiated by the late 3rd instar(White, 1961). New ommatidial units continue to be added until the

pupa is 24 h old (Sato, 1953). A larval whole-mount preparationallowed us to investigate the timing of rhodopsin expression in thedeveloping compound eye.

Fig. 5A shows the retinal region of a 4th instar larva. Theexpansion of the developing adult eye in the anterior direction ismarked by the line of three arrows. Aaop7-expressing ommatidia(green) are present near the anterior edge of the developingcompound eye (Fig. 5A, asterisk). In this preparation, we were notable to determine whether Aaop7 is restricted to a particular class ofphotoreceptors within the developing ommatidium. However, wealso detected Aaop7 at lower levels in mature ommatidia borderingthe larval stemmata (Fig. 5B). In these mature ommatidia, Aaop7expression is restricted to the R8 photoreceptor positioned centrallywithin the fused rhabdom structure. Aaop7 is also detected withinthe axonal projections from photoreceptors of the developingcompound eye and larval stemmata (arrows, Fig. 5C).

Aaop1 is the major rhodopsin of the adult compound eyebecause it is expressed in all R1–R6 photoreceptors and mostR8 photoreceptors (Hu et al., 2012). We made use of a larvalwhole-mount preparation to determine the onset of Aaop1

A Aaop3 N-term

B

C D

E

F G

H

Aaop3 C-term

Light Dark

Aaop3 transgenic

Aaop3 transgenic Aaop7 transgenic

Aaop7 transgenic

Wild type

Wild type

Actin Actin Actin

Actin Actin Actin

Actin Actin ActinActin

Developing adult eye Developing

adult eye

Fig. 2. Aaop3 rhodopsin is expressed and undergoes light-inducedmovement in larval stemmata. (A) The sequence alignment of the N-terminal (N-term)region of Aaop3 with other long-wavelength rhodopsins (Aaop1, Aaop2, Aaop4, Aaop5 and Aaop7) is shown at left. The images on the right show thatimmunostaining with antiserum directed against this Aaop3 sequence produces a higher signal (green) in the retina of Drosophila expressing the Aaop3transgene (middle) than in the retina of wild-type and Aaop7 transgenic Drosophila (left, right). In all micrographs, actin is stained by phalloidin and is labeled red.(B) Left, the sequence of the C-terminal region (C-term) of Aaop3 is aligned with other long-wavelength rhodopsins. The images on the right are an examination ofthree Drosophila strains showing specificity of the Aaop3 antiserum directed against this C-term sequence. A higher signal (green) is observed in the retinaof Drosophila expressing the Aaop3 transgene (middle) than in controls (left, right). (C–E) Aaop3 N-term antiserum (Aaop3N) detects Aaop3 within the cell body(CB) regions of the five larval stemmata (S, satellite; DA, dorsal anterior; DP, dorsal posterior; C, central and V, ventral) of light-adapted mosquitoes. Therhabdoms (labeled R in both the central stemma and one ommatidium of the developing adult eye) are stained for actin (red). No Aaop3 staining is seen in the adulteye. Aaop3 staining is also absent in the small areas of satellite and central stemmata marked by asterisks. D and E show Aaop3 and actin staining separately tohighlight the lack of Aaop3 colocalization with the rhabdoms in the light-adapted animal. (F–H) The Aaop3N antiserum detects Aaop3 in dark-treated larvaewithin the actin-rich rhabdoms. Panel F is labeled as described for C, except that the orientation of the image in F does not permit identification of the satelliteregion lacking Aaop3 expression. Scale bars: 20 µm (A,B), 5 µm (C,F).

1388

RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 1386-1392 doi:10.1242/jeb.111526

TheJournal

ofEx

perim

entalB

iology

rhodopsin expression in the developing compound eye. In 4thinstar larva, Aaop1 expression is readily detected in theposterior mature ommatidia of the developing compound eye(Fig. 5D). The magnified image (Fig. 5E) shows Aaop1expression within the fused rhabdom and also within the cellbodies surrounding the fused rhabdom (arrow). Given that theR1–R6 photoreceptors are positioned in this peripheral location(Hu et al., 2012), these results indicate that the initiation ofAaop1 expression within the R1–R6 photoreceptors occursduring the 4th instar larval stage.

DISCUSSIONIn this report, we characterized the expression pattern and light-triggered movement of rhodopsins in Aedes larval photoreceptors.These photoreceptors are positioned laterallyon the head andorganized

into five stemmata. The majority of stemmatal photoreceptor cellsexpress the long-wavelength Aaop3 rhodopsin. In a lighted environ-ment, Aaop3 localizes to cytoplasmic vesicles in the photoreceptor cellbody, whereas under dark conditions, Aaop3 moves into the rhabdom.This extensive light–dark translocation of a rhodopsin was previouslyobserved for Aaop1 in adult Aedesmosquitoes and it is proposed to bean adaptation mechanism for maximizing the light sensitivity range ofphotoreceptors (Hu et al., 2012).

The pioneering research of White (1967) used the Aedes larvalvisual system to identify a daily cycle in which rhabdoms are lost atdawn and regenerated at dusk. Membrane loss during the dawntransition reduces the rhabdom surface area by 50–70%. Thisreduction is accompanied by the formation of large numbers ofcytoplasmic multivesicular bodies (White, 1968). Subsequently,similar light-triggered loss of rhabdoms or rhabdomeres wasdocumented in adult photoreceptors of other invertebrates,including crabs (Nässel and Waterman, 1979), spiders (Blestet al., 1978) and tipilid flies (Williams and Blest, 1980). More recentstudies revealed an extensive redistribution of rhodopsin duringthe membrane cycling process. In the horseshoe crab Limuluspolyphemus, the 10% loss of membrane volume at dawn (Sacunaset al., 2002) is accompanied by a 50% loss of rhodopsin content(Katti et al., 2010). The adult Aedes photoreceptors exhibit greaterchanges at dawn, where a 50% loss of membrane volume (Brammeret al., 1978) is accompanied by an almost 100% loss of rhodopsincontent (Hu et al., 2012). Thus, it is likely that rhodopsin movementis the trigger for membrane renewal and that this process occurs todifferent extents in different species. The identification of Aaop3as the larval stemmatal rhodopsin involved in the extensive

A

BC D

E F

Aaop7 N-term

Satellite

Central

Aaop7 transgenic Aaop3 transgenic

Wild type

Actin Actin

ActinActin Actin

Fig. 3. Aaop7 is expressed in a small number of satellite and centralstemmatal photoreceptors. (A) The sequence alignment of the N-terminalregion of Aaop7 with other long-wavelength rhodopsins (Aaop1, Aaop2,Aaop3, Aaop4 and Aaop5) is shown at left. The images on the right show thatimmunostaining with antiserum directed against this Aaop7 sequenceproduces a higher signal (green) in the retina of Drosophila expressing theAaop7 transgene (middle) than in the retina of wild-type and Aaop3 transgenicDrosophila (right). In all micrographs, actin is stained by phalloidin and islabeled red. (B) In mosquito larvae, the Aaop7 antiserum detects Aaop7expression in a small number of photoreceptors (arrows) within the satelliteand central stemmata (S, satellite; DA, dorsal anterior; DP, dorsal posterior;C, central and V, ventral). (C,D) In dark-treated photoreceptors (C), Aaop7(green, arrow) is found within the satellite rhabdom (R). Following lighttreatment (D), Aaop7 (green) is detected within both the rhabdom andcytoplasmic vesicles within the cell bodies (arrow). (E,F) Dark-treatedphotoreceptors of the central stemma (E) shows Aaop7 location in therhabdom (R), but not the cell bodies (CB) whereas light treatment (F) results insomemovement of Aaop7 from the rhabdom to the cell bodies (CB, arrow in F).Scale bars: 20 µm (A,B), 5 µm (C–F).

A B

Aaop7

600 550 500 450 400 350 600

100

130

100

50

0

50

0

600

550

500

450

400

350

300

600

550

500

450

400

350

300

600 550 500 450

Wavelength (nm)

400 350 600

C D

Rh1

5 s

5 s

1 mV

1 mV

450

nm

resp

onse

(%)

Fig. 4. ERG spectral analysis of transgenicDrosophila expressing Aaop7rhodopsin. (A) An ERG trace obtained from an Aaop7 transgenic Drosophila.Light pulses of 1 s duration with an equalized photon count (approximately20 μE m−2 s−1) for 600 nm, 550 nm, 500 nm, 450 nm, 400 nm, 350 nm and600 nm were administered at 16 s intervals. (B) Mean responses (error barsare s.e.m., N=4) were calculated for each light stimulus relative to the peakresponse to the 450 nm stimulus. (C) An ERG trace for Drosophila expressingRh1 using the same paradigm as in A. (D)Mean responses for Rh1-expressinganimals (N=3) calculated as in B. The Drosophila genotypes were constructedsuch that Aaop7 (A,B) or Rh1 (C,D) was the only rhodopsin contributing tothe light response. The complete genotypes were: Aaop7: w norpA/Y;<pRh1-Aaop7> cn bw/cn bw; <pRh1-norpA> ninaEI17/ninaEI17 and Rh1: wnorpA/Y; cn bw; <pRh1-norpA> ninaEI17/+.

1389

RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 1386-1392 doi:10.1242/jeb.111526

TheJournal

ofEx

perim

entalB

iology

light-driven membrane renewal makes the Aedes larva an excellentexperimental system to further analyze this process.Two separate groups of three photoreceptors within the central

and satellite stemmata express the Aaop7 rhodopsin instead ofAaop3. Direct ERG recordings of the Aedes stemmata establishedthat its peak spectral sensitivity is at 520 nm (Seldin et al., 1972).Microspectrophotometric analysis further showed that therhodopsins of the stemmata together have an absorbance spectralpeak at 515 nm (Brown and White, 1972). Both values areconsistent with what would be expected upon expression of Aaop3and Aaop7, as sequence comparisons classify these as long-wavelength rhodopsins (Nene et al., 2007). We attempted toindividually assess the properties of Aaop7 and Aaop3 byexpressing each of these rhodopsins in transgenic Drosophila.We found that Aaop7 showed a peak sensitivity near 450 nm.However, the major stemmatal rhodopsin Aaop3 was notsufficiently expressed in transgenic Drosophila to be analyzed inthis fashion. We reasoned that because Aaop3 is the major larvalrhodopsin, the stemmatal 520 nm peak sensitivity observed in theearlier studies would largely be the result of the Aaop3 spectralproperties. Aaop7, then, is shifted towards shorter wavelengths.Therefore, the presence of two classes of photoreceptorsexpressing either Aaop3 or Aaop7 might provide some colordiscrimination within the blue–green region of the visible lightspectrum.The existence of two classes of Aedes stemmatal photoreceptors

distinguished by rhodopsin expression is similar to the situation in

Drosophila. In the Drosophila larval visual system, known asBolwig’s organ, one class consists of three or four photoreceptors thatarise fromprimaryprecursor ‘foundercells’ (Suzuki andSaigo, 2000).These photoreceptors express the rhodopsin Rh5 (Sprecher et al.,2007). During embryogenesis, these founder cells recruit secondaryprecursors that develop into a second class of photoreceptorsexpressing the rhodopsin Rh6 (Sprecher et al., 2007). In Aedes, it ispossible that there is a conserved developmental pathway in whichAaop7 precursor cells recruit the Aaop3-expressing photoreceptorcells. However, Aedes developmental processes ultimately result infive stemmatal units of different sizes and only two of these containAaop7-expressing cells. These considerations indicate that thedevelopmental steps specifying the mosquito larval eye are morecomplex than the Drosophila larval eye.

The adult compound eye rudiment is first detected during the 1stinstar larval stage. It is evident as a thickening of the epidermis,referred to as the optical placode, immediatelyanterior to the stemmata(Sato, 1953; White, 1961). Photoreceptors are born during a wave ofdifferentiation starting at the posterior edge, closest to the larvalstemmata, and then moving anteriorly. As a result, the posteriorregions contain more mature ommatidial units than the anteriorregions. The process appears similar to the extensively studiedmorphogenesis of the adultDrosophila eye (Treisman, 2013). Duringommatidial differentiation, Aaop7 is briefly expressed in ommatidialunits at the anterior edge of developing compound eye. Earlier in thediscussion, we hypothesized that the Aaop7-expressing cells may bethe founder cells for construction of stemmatal units. The transientexpression of Aaop7 in adult ommatidial units could also play adevelopmental role. In this regard, it is noteworthy thatAaop7 appearsto localize to the axons of both founder stemmatal cells and founderR8 cells of the developing adult ommatidia. The axons of thesephotoreceptors track together into the optic lobe, raising a possibilitythat Aaop7 expression plays a role in axonal pathfinding or relateddevelopmental process.

In the Aedes compound eye, differentiated photoreceptors arepresent in the most-posterior ommatidia by the middle of the 4thinstar larva. The anterior edge of the eye reaches maturity at the24-h-old pupal stage (Sato, 1953). Axonal projections from theadult photoreceptors during this same time period are accompaniedby development and organization of the lamina and medulla opticlobe neuropil (Mysore et al., 2014). In the swimming larva and pupaof Aedes and other mosquitoes, a shadow moving across the visualfield triggers an avoidance response consisting of a downwardmovement from the surface of the water (Kasap, 1978). Todistinguish between the role of the larval stemmata and the adultcompound eye in this behavior, Kasap (1977a) used black paint tocover the stemmata, the compound eye or both. In Aedes 4th instarlarvae and pupae, only painting over both the stemmata and thecompound eye resulted in statistically significant defects in thelight-triggered behavior. These results indicate that photoreceptorsof the compound eye may begin to augment the light-sensingcapabilities of the larval stemmata during the 4th instar stage. Ourobservation that Aaop1 is already expressed in the R1–R6photoreceptors at this time is consistent with this view.

MATERIALS AND METHODSMosquito, Drosophila strains and gene nomenclatureThe Liverpool strain of Aedes aegypti was used. Aedes aegypti rhodopsinsreferred to as Aaop1, Aaop3 and Aaop7 correspond to GPROP1(AAEL006498), GPROP3 (AAEL006484) and GPROP7 (AAEL007389)of the Aedes aegypti genome project (Nene et al., 2007, https://www.vectorbase.org). To create the Aaop3- and Aaop7-expressing Drosophila

A B

D E

C

Developing adult eye

Larvalstemmata

Developing adult eye

Developing adult eye

Larvalstemmata

Larv

al s

tem

mat

aLa

rval

stem

mat

a

Actin

Actin

Actin

Actin Actin

Fig. 5. Rhodopsin expression in the developing compound eye. (A) Theadult eye develops in an anterior direction (three arrows) from the larvalstemmata, and regularly spaced individual ommatidia are identified by actin-rich rhabdoms (red). In the 4th larval instar, Aaop7 is expressed inphotoreceptors along the anterior border of the developing compound eye(green, one ommatidium is marked by asterisk) in addition to its expression inthe larval satellite and central stemmata. (B) In mature ommatidia near thelarval stemmata, Aaop7 is detected only within the central region of the fusedrhabdom, corresponding to the R8 photoreceptor cell. (C) Aaop7 is alsodetected in the axon tracts of photoreceptor cells along the anterior border ofthe developing compound eye (short arrows), as well as in axon tracts of thestemmatal Aaop7 photoreceptors (long arrow). (D) In the 4th larval instar,Aaop1 is detected in the more mature ommatidium located in the posteriorregion of the developing compound eye (arrow). (E) A magnified view of thisregion of the compound eye reveals that Aaop1 is localized to both therhabdom and cytoplasmic vesicles within the cell bodies of peripheral R1–R6photoreceptor cells (arrow). Scale bars: 20 µm (A,B,D), 5 µm (B,E).

1390

RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 1386-1392 doi:10.1242/jeb.111526

TheJournal

ofEx

perim

entalB

iology

transgenic lines, their open reading frames were retrieved from genomicDNA by PCR, P-element transformation plasmids were created, andgermline transformation was carried out as previously described for othermosquito rhodopsins (Hu et al., 2009, 2011).

Immunostaining of whole-mounted larval stemmataLarval heads were removed and fixed for 4–6 h at 4°C in 2%paraformaldehyde (Electron Microscopy Sciences, stock 16% solution) inphosphate-buffered saline (PBS). Collection of heads in the dark wasperformed under dim red light using a 650 nm long-pass filter. Heads werethen transferred to PBS and kept at 4°C for up to 5 days. Larval stemmatawere dissected in PBS and incubated in primary antibody [anti-Aaop1, anti-Aaop3 C-terminus, anti-Aaop3 N-terminus, or anti-Aaop7 N-terminusantisera, generated as described previously (Hu et al., 2011)] diluted 1:100in BNT (1× PBS, 0.1% BSA, 0.1% Tween-20 and 250 mmol l−1 NaCl)overnight at 4°C. After three 10 min washes in PBS, the stemmata wereincubated in secondary antibody (Alexa-Fluor-488-conjugated goat anti-rabbit IgG, 1:400 in BNT) and Alexa-Fluor-594-conjugated phalloidin(1:40 in BNT) for 4 h at room temperature. After three 10 min washes inPBS, stemmata were mounted in 10 µl of Vectashield (Vector Laboratories,Burlingame, CA). Tape strips mounted on glass ring slides were used to holdthe coverslip above the stemmatal tissue to prevent damage to the tissue.Stemmata were visualized with a Nikon A1R confocal microscope, andbrightness and contrast on the resulting photomicrographs were uniformlyadjusted using Photoshop CS5 software.

Immunofluorescence of cryosectionsWhite-eyed Drosophila heads were removed and placed in fixative (4%paraformaldehyde with 5% sucrose) overnight at 4°C. The heads were thenwashed three times for 10 min each time in a solution of 5% sucrose in 1×PBSand incubated overnight at 4°C.Theywere then incubated in30%sucrose in1×PBS solution overnight at 4°C, and then placed in 30% sucrose in 1× PBS andTissue Freezing Medium (TFM, Triangle Biomedical Sciences, Cincinnati,OH, USA) mixed 1:1. The heads were embedded in TFM and sectioned at10 µm at −27°C. The sections were washed in 1× PBS for 20 min andincubated in blocking buffer (1× PBS, 5% normal goat serum, 0.3% Triton X-100 and 1% dimethylsulfoxide) for 1 h at room temperature. Sections wereincubated overnight at 4°C in primary antibody against Aaop1, Aaop3 orAaop7 diluted 1:100 in blocking buffer. After three 10 min washes in 1× PBSwith 0.1% Tween-20, the sections were incubated in secondary antibody(Alexa-Fluor-488-conjugated goat anti-rabbit IgG, 1:400 in BNT) and Alexa-Fluor-594-conjugated phalloidin (1:40 in BNT) for 1 h at room temperature.After three 10 min washes in 1× PBS with 0.1% Tween-20 and one wash inPBS for 5 min, sections were mounted in Vectashield (Molecular Probes).Cryosectionswere visualizedusing aLeicaDM5000 fluorescencemicroscope.The photomicrographs were uniformly processed with Photoshop CS5 forbrightness and contrast.

Spectral analysis by ERG recordingsAaop3 and Aaop7 transgenic Drosophila were placed in geneticbackgrounds to create white-eyed flies with only the Aaop7 or theDrosophilaRh1 rhodopsin expressed in the R1–R6 photoreceptors and withno responses from the R7 and R8 photoreceptors. Therefore only the activityof Aaop7 or the Rh1 control rhodopsin was represented in the ERG response(Ahmad et al., 2006). The Aaop7- and the Rh1-expressing Drosophilawerethen subjected to ERG analysis as described previously (Hu et al., 2014).Narrow band pass and neutral density filters (Oriel, Stratford, CT) were usedto attenuate a 1000 W tungsten light source (Oriel, Irvine, CA) so that theflies could be exposed to light stimuli of comparable photon content(20 μE m−2 s−1) at 600 nm, 550 nm, 500 nm, 450 nm, 400 nm and 350 nmwavelength.

AcknowledgementsConfocal microscopy was conducted within the Notre Dame Integrated ImagingFacility.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsM.R., X.H., M.A.W. and J.E.O. developed the concepts or approach. M.R., K.J.K.,M.T.L., X.H., M.A.W. and J.E.O. performed experiments or data analysis. M.R.,K.J.K., M.T.L., X.H., M.A.W. and J.E.O. prepared or edited the manuscript prior tosubmission.

FundingThe work supported by the National Institutes of Health [grant numberR01EY06808]. Deposited in PMC for release after 12 months.

ReferencesAhmad, S. T., Natochin, M., Barren, B., Artemyev, N. O. and O’Tousa, J. E.

(2006). Heterologous expression of bovine rhodopsin inDrosophila photoreceptorcells. Invest. Ophthalmol. Vis. Sci. 47, 3722-3728.

Blest, A. D., Kao, L. and Powell, K. (1978). Photoreceptor membranebreakdown in the spider Dinopis: the fate of rhabdomere products. CellTissue Res. 195, 425-444.

Brammer, J. D., Stein, P. J. and Anderson, R. A. (1978). Effect of light and darkadaptation upon the rhabdom in the compound eye of the mosquito. J. Exp. Zool.206, 151-156.

Brown, P. K. and White, R. H. (1972). Rhodopsin of the larval mosquito. J. Gen.Physiol. 59, 401-414.

Clements, A. N. (1999). The Biology of Mosquitoes Vol 2-Sensory Reception andBehaviour. Wallingford, UK: CABI Publishing.

Hu, X., England, J. H., Lani, A. C., Tung, J. J., Ward, N. J., Adams, S. M., Barber,K. A., Whaley, M. A. and O’Tousa, J. E. (2009). Patterned rhodopsin expressionin R7 photoreceptors of mosquito retina: implications for species-specificbehavior. J. Comp. Neurol. 516, 334-342.

Hu, X., Whaley, M. A., Stein, M. M., Mitchell, B. E. and O’Tousa, J. E. (2011).Coexpression of spectrally distinct rhodopsins inAedes aegyptiR7 photoreceptors.PLoS ONE 6, e23121.

Hu, X., Leming, M. T., Metoxen, A. J., Whaley, M. A. and O’Tousa, J. E. (2012).Light-mediated control of rhodopsin movement in mosquito photoreceptors.J. Neurosci. 32, 13661-13667.

Hu, X., Leming, M. T., Whaley, M. A. and O’Tousa, J. E. (2014). Rhodopsincoexpression in UV photoreceptors of Aedes aegypti and Anopheles gambiaemosquitoes. J. Exp. Biol. 217, 1003-1008.

Kasap, M. (1977a). Response of blinded larvae and pupae of mosquitoes to visualstimuli. Bull. Fac. Sci. Hacettepe Univ. 6, 17-25.

Kasap, M. (1977b). Black and white background color preference of the larvae andpupae of mosquitoes. Bull. Fac. Sci. Hacettepe Univ. 6, 27-34.

Kasap, M. (1978). Response of the larvae and pupae of Aedes aegypti, Anophelesstepephansi and Culex pipens to a moving shadow- 1. Commun. Fac. Sci. Univ.Ankara Ser. C Zool. 3, 17-32.

Katti, C., Kempler, K., Porter, M. L., Legg, A., Gonzalez, R., Garcia-Rivera, E.,Dugger, D. and Battelle, B.-A. (2010). Opsin co-expression in Limulusphotoreceptors: differential regulation by light and a circadian clock. J. Exp.Biol. 213, 2589-2601.

McMeniman, C. J., Corfas, R. A., Matthews, B.J., Ritchie, S. A. and Vosshall,L. B. (2014). Multimodal integration of carbon dioxide and other sensory cuesdrives mosquito attraction to humans. Cell 156, 1060-1071.

Meyer-Rochow, V. B., Kashiwagi, T. and Eguchi, E. (2002). Selectivephotoreceptor damage in four species of insects induced by experimentalexposures to UV-irradiation. Micron 33, 23-31.

Mysore, K., Flannery, E., Leming, M. T., Tomchaney, M., Shi, L., Sun, L.,O’Tousa, J. E., Severson, D. W. and Duman-Scheel, M. (2014). Role ofsemaphorin-1a in the developing visual system of the disease vector mosquitoAedes aegypti. Dev. Dyn. 243, 1457-1469.

Nassel, D. R. and Waterman, T. H. (1979). Massive diurnally modulatedphotoreceptor membrane turnover in crab light and dark adaptation. J. Comp.Physiol. A 131, 205-216.

Nene, V., Wortman, J. R., Lawson, D., Haas, B., Kodira, C., Tu, Z., Loftus, B., Xi,Z., Megy, K., Grabherr, M. et al. (2007). Genome sequence of Aedes aegypti, amajor arbovirus vector. Science 316, 1718-1723.

Okano, K., Maeda, A., Chen, Y., Chauhan, V., Tang, J., Palczewska, G., Sakai, T.,Tsuneoka, H., Palczewski, K. and Maeda, T. (2012). Retinal cone and rodphotoreceptor cells exhibit differential susceptibility to light-induced damage.J. Neurochem. 121, 146-156.

Sacunas, R. B., Papuga, M. O., Malone, M. A., Pearson, A. C., Jr, Marjanovic, M.,Stroope, D. G., Weiner, W. W., Chamberlain, S. C. and Battelle, B.-A. (2002).Multiple mechanisms of rhabdom shedding in the lateral eye of Limuluspolyphemus. J. Comp. Neurol. 449, 26-42.

Sato, S. (1953). Structure and development of the compound eye ofAedes (Finlaya)japonicus Theobald. Sci. Rep. Tohoku Univ. Ser 4 20, 33-44.

Seldin, E. B., White, R. H. and Brown, P. K. (1972). Spectral sensitivity of larvalmosquito ocelli. J. Gen. Physiol. 59, 415-420.

Sprecher, S. G., Pichaud, F. and Desplan, C. (2007). Adult and larvalphotoreceptors use different mechanisms to specify the same rhodopsin fates.Genes Dev. 21, 2182-2195.

1391

RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 1386-1392 doi:10.1242/jeb.111526

TheJournal

ofEx

perim

entalB

iology

Stark, W. S. and Carlson, S. D. (1984). Blue and ultraviolet light induced damage tothe Drosophila retina: ultrastructure. Curr. Eye Res. 3, 1441-1454.

Suzuki, T. and Saigo, K. (2000). Transcriptional regulation of atonal required forDrosophila larval eye development by concerted action of eyes absent, sine oculisand hedgehog signaling independent of fused kinase and cubitus interruptus.Development 127, 1531-1540.

Treisman, J. E. (2013). Retinal differentiation in Drosophila. Wiley Interdiscip. Rev.Dev. Biol. 2, 545-557.

Vihtelic, T. S. and Hyde, D. R. (2000). Light-induced rod and cone cell death andregeneration in theadult albinozebrafish (Danio rerio) retina.JNeurobiol.44, 289-307.

White, R. H. (1961). Analysis of the development of the compound eye in themosquito, Aedes aegypti. J. Exp. Zool. 148, 223-239.

White, R. H. (1967). The effect of light and light deprivation upon the ultrastructure ofthe larval mosquito eye. II. The rhabdom. J. Exp. Zool. 166, 405-425.

White, R. H. (1968). The effect of light and light deprivation upon the ultrastructure ofthe larval mosquito eye. III. Multivesicular bodies and protein uptake. J. Exp. Zool.169, 261-277.

Williams, D. S. and Blest, A. D. (1980). Extracellular shedding of photoreceptormembrane in the open rhabdom of a tipulid fly. Cell Tissue Res. 205,423-438.

1392

RESEARCH ARTICLE The Journal of Experimental Biology (2015) 218, 1386-1392 doi:10.1242/jeb.111526

TheJournal

ofEx

perim

entalB

iology