Imaging and spectral analysis of autofluorescence patterns in larval head structures of mosquito vectors.

Autofluorescence (AF) in mosquitoes is currently poorly explored, despite its great potential as a marker of body structures and biological functions. Here, for the first time AF in larval heads of two mosquitoes of key public health importance, Aedes albopictus and Culex pipiens, is studied using fluorescence imaging and spectrofluorometry, similarly to a label-free histochemical approach. In generally conserved distribution patterns, AF shows differences between mouth brushes and antennae of the two species. The blue AF ascribable to resilin at the antennal bases, more extended in Cx. pipiens, suggests a potential need to support different antennal movements. The AF spectra larger in Cx. pipiens indicate a variability in material composition and properties likely relatable to mosquito biology, including diverse feeding and locomotion behaviours with implications for vector control.

Introduction

In insects, optical phenomena have received great attention since a long time, especially in Lepidoptera and Coleoptera.[1-5] Autofluorescence (AF) emission, in particular, has been mainly characterized in butterfly pigments and in resilin-enriched and chitinous body structures from different insect orders, including Diptera, providing a valuable basis for various label-free morphological, taxonomic, and functional studies.[6-13]

The optical-based investigation of the scattering properties of mosquito adults allowed to obtain promising data for sex- and species-specific detection in the field for vector surveillance. Indeed, a recently developed system based on the spectral and polarization properties of the mosquito body, integrating wing-beat frequency analysis, was shown to have the potential to optically discriminate individuals and sexes of Anopheles coluzzii Coetzee & Wilkerson, 2013 and An. arabiensis Patton, 1905.[14] On the other hand, AF has been up to now poorly investigated in Nematocera. So far, the AF signals in the antennal structural components have been characterized in relation with the ability to detect sound vibrations, according to the species- and sex-specific needs of adult midges or mosquitoes.[15,16] Moreover, we recently used both AF imaging and spectrofluorometry to characterize the fluorescing structures in the head appendages and body scales of adult males and females of the Asian tiger mosquito, Aedes albopictus (Skuse, 1894) (Diptera: Culicidae). Our AF data indicated the presence of different fluorophores, most likely resilin, chitinous compounds and melanins, providing new perspectives for studying the role of AF in the biology and behavior of the tiger mosquito, with the potential of exploiting such knowledge for in-flight mosquito detection and surveillance in the field.[17]

Ae. albopictus is considered the most abundant urban mosquito in many cities worldwide.[18] Native to Southeast Asia,[1] this species was first detected in Italy in 199020 and, given its biological plasticity and capacity to diapause,[21-23] it was able to rapidly invade various areas in Central and Northern Italian regions.[24,25] The spread in urban and suburban habitats has lead the species to share habitats with the common house mosquito, Culex pipiens Linnaeus, 1758 (Diptera: Culicidae), an ubiquitous mosquito adapted to a wide range of environments across the temperate Northern hemisphere,[26] including Italy.[2] Both Ae. albopictus and Cx. pipiens complex members play a key role in the transmission of arboviruses: Ae. albopictus is more anthropophilic than Cx. pipiens and it shows vector competence for at least 20 arboviruses, including chikungunya (CHIKV), dengue (DENV) and Zika (ZIKV) viruses.[28-30] Culex pipiens is a competent vector of arboviruses such as West Nile (WNV), Usutu (USUV) and Sindbis (SINV) viruses.[31]

The established role of Ae. albopictus and Cx. pipiens as vectors of arboviruses makes these mosquitoes key threads for public health. Consequently, expanding knowledge in their biology is essential to develop and improve approaches for their control. Despite the need of complementary interventions for adult control and of solving practical organizational and infrastructural issues,[32] larval mosquito control can be a highly effective tool to manage wild populations[33] due to the larval responsiveness to intervention measures and low mobility.[32] Moreover, targeting the larval stages contributes to reduce the proportion of adult populations being selected for insecticide resistance[34]. Although the interest in mosquito larval behavioral patterns is recently rising,[35-37] there is still a huge gap in the knowledge on the response to environmental stimuli, including nutrients and toxicants, as well as on navigation strategies and predator avoidance, required to be filled for the implementation of effective control interventions.

Mosquito larvae are aquatic and employ a wide range of physiological sensory systems to navigate in their habitats to find food and avoid predators.[38] As a feature common to other dipteran species, mosquito larvae have a set of anterior appendages, including the antennae that apically carry a sensory cone considered to have olfactory functions and peg organs with putative gustatory roles.[39-42] The molecular and physiological machinery underlying olfaction in mosquito larvae, accounted by the expression of olfactory, ionotropic and gustatory receptors, is increasingly being elucidated. [38,41,43-48]

Up to now, only a few studies have focused on the characterization of AF in dipteran larvae.[13] For instance, AF emission signal detected as an overall broadband or as bands at increasing wavelengths along the visible spectrum has been applied to recognize morphological structures and organs to support comparative, taxonomic, developmental and physiological studies.[49,50] The detection of AF of defined endogenous fluorophores has been specifically used in metabolic studies. For example, lifetime imaging of NAD(P)H and FAD in the larval salivary glands and fat body cells allowed to validate investigation procedures to study energy metabolism in the sperm of Drosophila melanogaster Meigen, 1830.[51] In addition, metabolic changes underlying morphogenetic processes in the fat body of developing Drosophila larvae were monitored through the AF of pteridine or kynurenine, also in parallel with the assessment of the progressive degradation of the lipid droplets detected by an anti-Stokes Raman scattering based procedure. [52,5]

As to AF of compounds associated to biomechanical functions, the bright bluish emission of resilin is a well-established feature, widely exploited for the in situ detection and localization of this protein that ensures strengthen and flexibility to organs such as wings and antennae. The bluish AF of the elastic and resilient resilin allows also its differentiation from stiffer chitinous materials, fluorescing in the yellowish-to-reddish spectral region.[9,13,54] Currently, investigations on the AF of compounds with a biomechanical function have been limited to the adult stage.[13,15,17]

In the case of mosquito larvae, behavioral differences across species have been assessed in terms of locomotion, arresting, browsing and filtering in response to physical and food-related environmental stimuli.[35] Instead, information on the structural and biomechanical properties of sensory organs and surrounding tissues that can potentially modulate sensory inputs[55] is currently lacking. Therefore, in this study we aim, for the first time, to take advantage of the above recalled AF-related material properties to investigate the larval head structures in Ae. albopictus and Cx. pipiens, with particular attention to mouth brushes and antennae, for their already recognized roles as mediators of behavioral functions.[38,5] Albeit limited to Ae. albopictus and Cx. pipiens species, the findings from our comparative study are expected to provide useful insights to favor further investigation of larval sensory behavior in these important mosquitoes, with implications for the development of approaches for the control of vectors of public health importance.

Materials and Methods

Insects

Ae. albopictus eggs were collected in Northern Italy (Seniga, BS, 45°14’35.63” N, 10°10’51.41” E) using ovitraps in the time frame July-September 2021. Eggs were allowed to hatch in autoclaved water, and kept in a climatic chamber at 25°C, with 60-75% relative humidity and a 12:12 h (light:dark) photoperiod. Cx.pipiens larvae were collected from different breeding sites in the same geographical area. All larvae were reared on fish food pellets (Goldfish Granules, Tetra GmbH, Melle, Germany) and confirmation of the identity of fourth instars was based on Rueda[57] and Romi and colleagues.[58]

Bright field and fluorescence microscopy

Individual fourth instar larvae were cold anesthetized and mounted with a drop of phosphate buffer saline (PBS) between two coverslips to allow microscopical observation of the dorsal as well as ventral side of each individual. Bright field and AF imaging was performed on 15 larvae for each of the two mosquito species by means of an Olympus BX53 fluorescence microscope (Olympus Optical Co. GmbH, Hamburg, Germany). AF was observed using an X-Cite 120 Q illumination system (120W Hg vapor short arc lamp) as the light excitation source, the UFUW optical cube mounting the 340–390 nm excitation filter, 410 nm dichromatic mirror and a 420 nm long pass filter and the Olympus objectives Plan 4x (numerical aperture, NA, 0.10), UPlanFL 10x (NA 0.30), UPlanFL 20x (NA 0.50), and UPlanFL 40x (NA 0.75). Images were recorded using an EOS 1300D Olympus camera, processed to adjust contrast and brightness with Adobe Photoshop CC 2017 v. 21.0, and assembled in panels using Adobe Illustrator CC 2017 v. 21.0. Images of AF are presented as true colors. Identity of body structures, organs and tissues has been assigned based on the available literature and, in the case of Ae. albopictus, on the comparison with Ae. aegypti.[42,57,59-66]

Scanning electron microscopy

A FEG-SEM Tescan Mira3 XMU (Tescan, Brno, Czech Republic), located at the Arvedi Laboratory of CISRiC-Pavia, was used to study the external morphology of the antennae of fourth instar larvae (six individuals for each of the two mosquito species), with particular attention devoted to the antennal sensory cones. Whole larvae were collected individually and immediately fixed in 70% ethanol. The larvae were then glued to aluminium stubs with a double- sided carbon adhesive tape and coated with a thin layer of platinum in a Cressington Sputter Coater 208 HR. Observations were made at 5 kV with an SE detector at a working distance of 15 mm.

Spectrofluorimetric analysis

AF emission spectra were recorded from ten individual larvae for each of the two species under epi-illumination by means of a microspectrograph (Leitz, Wetzlar, Germany) equipped with a 100W/Hg lamp (Osram, Berlin, Germany) as the excitation source, combined with KG1-BG38 anti-thermal filters. The 366 nm bandpass interference excitation filter (Full Width at Half Intensity Maximum, FWHM = 10 nm) was used to select the 366 nm emission line of the light produced by the Hg lamp, excluding the unwanted lines at longer wavelengths, which could also affect reliable AF detection. The AF emission was collected through a 50/50 dichroic mirror and a 390 nm long pass filter, by using a 40x objective (NA 0.75). The emission signal was driven to the multichannel analyzer (Hamamatzu PMA-12 photonic model, Hamamatsu Photonics Italia Srl, Arese, Italy) by means of a fiber optic probe optically coupled to the microspectrograph exit slit. Spectra were recorded in the 400-750 nm range, covering the visible spectral interval where we expect to detect the AF emission of our samples. Data were stored on a magnetic mass memory to be then normalized to maximum peak values and assembled in graphs by means of Microsoft Excel for presentation. Normalization allowed to present the spectra with the same scale, to facilitate the comparison between their profiles and to directly evidence the relative differences in the amplitude of the emission signal at the different wavelength positions.

Results

Mosquito larval heads display multiple autofluorescence signals

Observations of the heads of fourth instar larvae under bright field microscope conditions showed morphological differences between Ae. albopictus (Figure 1 A,B,D) and Cx. pipiens (Figure 2 A,B,D). At the magnification used here to provide a general overview of the head structures, the antennae show the most evident differences between the two species. In Ae. albopictus, the antenna appears as a simple, single cylindrical segment, while in Cx. pipiens it is more elongated and complex, with a head comb carrying numerous long setae at about one third of its length, and an asymmetrically narrowing portion towards the tip carrying long sensilla. These findings are consistent with the available literature.[59,65,67] When observed under near-UV excitation light, AF signals in the heads showed an articulated distribution, in particular in the mouth structures and in the antennae (Figures 1 and 2 C,E). In both species, AF imaging showed the presence of areas with different colors and signal intensity. For example, the comparison with the bright field images evidenced an orange-reddish AF corresponding to the mouth brushes, while a brighter bluish emission allowed to detect regions/structures not directly observable under bright field conditions. These findings on the differences in AF distribution and intensity and spectral properties between the two considered mosquito species are described in detail in the next paragraphs.

Figure 1.

Morphology of the cephalic region of Ae. albopictus fourth instar larvae. A) Bright field light view of the dorsal head and thorax. Bright field (B,D) and fluorescent (C,E) light views of the dorsal (B,C) and ventral (D,E) apical portion of the head. a, antenna; apb, anteromedian palatal brush; hc, head capsule; lpb, lateral palatal brush; tm, tessellated membrane. Scale bars: 300 μm.

Figure 2.

Morphology of the cephalic region of Cx. pipiens fourth instar larvae. A) Bright field light view of the dorsal head and thorax. Bright field (B,D) and fluorescent (C,E) light views of the dorsal (B,C) and ventral (D,E) apical portion of the head. a, antenna; apb, anteromedian palatal brush; hc, head capsule; lpb, lateral palatal brush; MxBo, maxillary body; tm, tessellated membrane. Scale bars: 300 μm.

Autofluorescence imaging of mouth structures

In both species, strong orange-reddish AF signals arise from the mouth brushes in the larval mouthparts (Figures 1 and 2 C,E, Figure 3, Figure 4). Mouth brushes comprise both lateral palatal brush (lpb), anteromedian palatal brushes (apb) and mandibular brushes.[56] Although the mouth parts are located ventrally (Figures 1 and 2 D,E, Figure 3G, Figure 4 E,F), portions of the labrum extends anteriorly and their AF can be observed also dorsally (Figures 1 and 2 B,C, Figure 3 A-F, Figure 4 A,B).

Figure 3.

Morphology of the mouth brushes region in the head of Ae. albopictus fourth instar larvae. Fluorescent light views of the apical portion of the head (A, dorsal view), and of the lateral palatal brushes (B). Bright field (C) and fluorescent (D) light dorsal views of the tessellated membrane region. Higher magnification of the anteromedian palatal brush, lateral palatal brush, and tessellated membrane structure in bright field (E) and fluorescent (F) light dorsal views. Fluorescent light view of the ventral labial region (G). apb, anteromedian palatal brush; epla, post-epipharyngeal lobe area; la, labial area; lpa, lateral plate of clypeus area; lpb, lateral palatal brush; mdo, fenestra for articulation of mandible and maxilla; MxBr, maxillary brushes; ppl, postpalatal lobe; sma, submaxillary apodeme; tm, tessellated membrane. Scale bars: A,C,D,G) 150 μm; B,E,F) 75 μm.

Figure 4.

Morphology of the mouth brushes region in the head of Cx. pipiens fourth instar larvae. Fluorescent light view of the apical portion of the head (A, dorsal). Higher magnification fluorescent light view of the tessellated membrane, cross-bars region, anteromedian palatal brush, and lateral palatal brush (B, dorsal). Bright field (C) and fluorescent (D) light views of dissected lateral palatal brushes and cross-bars region. Fluorescent light view of the ventral labial region (E). Higher magnification fluorescent light view of the ventral maxillary bodies area (F). a, antenna; apb, anteromedian palatal brush; cl, clypeus; crb, cross-bars; DMxS, dorsal maxillary suture; lb, labial plate; lpb, lateral palatal brush; MxBo, maxillary body; MxP, maxillary palps; pc, preclypeus; pcs, preclypeal spines; tm, tessellated membrane. Scale bars: A,E) 300 μm; B,C,D) 75 μm; F) 150 μm.

In both species, blue AF areas are also visible, either from the dorsal and ventral view (Figures 1 to 4). The most remarkable signal rises from the palatal tessellated membrane (tm), which is particularly visible dorsally (Figure 3 A,D,F; Figure 4 A,B,F). The tessellated membrane is a bossed membranous surface located between the median labral plate and the anteromedian palatal and lateral palatal penicular areas.[60] In Ae. albopictus, the blue AF evidences two tessellated membranes, separated by the projecting palatum (Figure 3 A,D,F), similar to what has already been reported in Ae. Aegypti.[59] As compared with Ae. albopictus, in Cx. pipiens the blue AF in the tessellated membrane area appears to be more extended (Figure 4 A,B), in agreement with previously published morphological studies in members of the Culex genus.[65,68]

The ventral side of the head shows additional fluorescing structures in both Ae. albopictus (Figure 3G) and Cx. pipiens (Figure 4 E,F). These include the mandible and maxillary brushes, which display a marked orange-reddish AF. Also, blue AF signals can be seen as brilliant thin lines, such as those visible in correspondence of the dorsal maxillary sutures (DMxS) in Cx. pipiens (Figure 4 E,F).

Autofluorescence distribution is not homogeneous along the antenna

Differences between the distribution of the AF signals of Ae. albopictus and Cx. pipiens are particularly visible in the antennae, either at the basis, along the scape, in the distal antennal sensory cone (asc), and in the sensilla. In Ae. albopictus, the blue AF observable at the bases of the antennae rises from both the dorsal (Figure 5 A,B) and ventral (Figure 5 C,D) side of the antacoria (ant). The antacoria is an unmelanized antenna-bearing membrane covering the antennal socket and connecting the antenna with the antennal prominence (apr)[59,60,69] (Figure 5A) at the anterolateral lobe of the cranium in culicid larvae. In Cx. pipiens, the antacoria does not appear to fluoresce, while blue AF is detected in an ample portion of the cranium ventrally to the basis of the antennae, which corresponds to the ventral head sclerites (Figure 4E, Figure 5 F,H,J). The blue AF is detected also in the antennal ridge (ar) (Figure 5 F,H), which is described in the literature as the thickened rim around the outer margin of the antennal socket.[60]

Figure 5.

Antennal basis of mosquito larvae. Bright field (A,C) and fluorescent (B,D) light views of the antennal prominence in Ae. albopictus (A,B, dorsal; C,D, ventral), evidencing the blue-fluorescent antacoria. Bright field (E,G) and fluorescent light (F,H) dorsal views of the Cx. pipiens antennal prominence; images taken from two focusing planes (E,F,G,H). Bright field (I) and fluorescent (J) light views of the ventral antennal prominence in Cx. pipiens. a, antenna; ant, antacoria; apr, antennal prominence; ar, antennal ridge; hc, head capsule; lpb, lateral palatal brush; MxBo, maxillary body; sc, scape. Scale bars: 100 μm.

Differences in AF distribution occur also along the antenna, especially in the antennal sensory cone. The antennal sensory cone has been previously described in Cx. pipiens,[61] but not yet in Ae. albopictus. Therefore, we decided to complement with SEM analyses (Figure 6) our bright field and AF-based studies (Figure 7), to provide a first description of this structure in the tiger mosquito.

Figure 6.

Scanning electron micrograph of the antenna of Ae. albopictus fourth instar larvae. A) Antennal scape with apical sensory structures. B, C) Detailed view of the antennal sensory cone region at increasing magnifications. ah, antennal hair; asc, antennal sensory cone; bPeg, basiconic-like peg organ; lcs, long chaetoid sensillum; lts, long trichoid sensillum; sPeg, sinusoidal peg organ. Scale bars: A) 50 μm; B) 20 μm; C) 10 μm.

Figure 7.

Antennal sensory organs in Ae. albopictus fourth instar larvae. Bright field (A,C,E) and fluorescent (B,D,F) light views of the antennal tip. In B), the inset shows the fluorescent light view of the apical portion of the antenna superimposed to the bright field image. ah, antennal hair; asc, antennal sensory cone; bPeg, basiconic-like peg organ; lts, long trichoid sensillum; so, antennal hair socket; sPeg, sinusoidal peg organ. Scale bars: 50 μm.

In Ae. albopictus, the antenna is a single tubular segment, with an antennal hair (ah) in the middle and a terminal sensilla-carrying membranous region (Figures 6, 7). The structures we observed at the antennal tip are similar to the sensory appendages already characterized in Aedes species and especially in Ae. aegypti.[42,61,66,70] This species is among the most widely studied mosquitoes due to its relevance as arbovirus vector[71-73] and the ease of laboratory rearing, making it to be often considered as a model organism, also for studies on Ae. albopictus.[74,75] On the basis of the information available in Ae. aegypti,[70] we described the antennal tip of Ae. albopictus as comprising a large cone and smaller sensilla, including a basiconic-like, thin sinusoidal peg organ (sPeg) emerging from the wall of the antennal sensory cone without a basal socket. For the peg organ of Ae. aegypti, a potential role as osmoreceptor was suggested by Zacharuk and Blue.[66] Our observations in Ae. albopictus revealed also the presence of longer chaetoid and trichoid sensilla (lcs and lts, respectively) (Figures 6 and 7). These have been identified according to the similarity with structures described in other Aedes species,[61] although electrophysiological analyses would be essential to assign a definitive sensory function in the larva of the tiger mosquito. The AF imaging showed a blue emission rising from the antennal sensory cone of Ae. albopictus, especially evident at the basis of the central blunt-pointed cone hair located on top of the cuticular cylinder (Figure 7 B,D). Lower AF signals have been also observed at the basis of the antennal sensory cone cylinder, in the socket of the long trichoid sensillum, and in the socket of the antennal hair located on the scape (Figure 7 B,D,F).

In Cx. pipiens, the antenna appears as an elongated segment with an asymmetrically narrowing at the tip (Figures 8, 9). The antennal tip is characterized by the presence of three long chaetoid sensilla, one long and one short trichoid sensilla, besides the antennal sensory cone, as shown by our bright field, fluorescent light and SEM observations (Figures 8 B,C and 9C). The antennal sensory cone is observable in bright field as a central pyramidal hair with a rather transparent blunt tip (Figure 8B), which instead is clearly visible in fluorescent light conditions due to its bright bluish AF emission (Figure 8C), similar to what observed in Ae. albopictus (Figure 7 B,D). As evident from both optical microscopy (Figure 8B) and SEM analysis (Figure 9C), the antennal sensory cone is surrounded by long chaetoid sensilla with one short trichoid sensillum (sts). One long trichoid sensillum with a thickened base and a rounded tip is located laterally to the antennal sensory cone (Figures 8B and 9C). The antenna carries also a long lateral hair comb (hco) that includes numerous solitary hairs and trichoid sensilla different in size (Figures 8A and 9 A,C). In Cx. pipiens, besides the AF emission observed in the sensory cone, a bluish AF is also visible from the whole surface of the long trichoid sensillum located in the sensory area at the antennal tip, as well as from its socket and from the rim of the apical portion of the antenna (Figure 8C). The short chaetoid sensilla distributed along the basal portion of the antennal scape, shown by SEM analysis (Figure 9 A,B), can be observed also in bright field (Figure 8A) and AF conditions (Figure 8A, inlet) as dark, non-fluorescent structures.

Figure 8.

Antennal sensory organs in Cx. pipiens fourth instar larvae. A) Bright field light view of the basal portion of the antenna showing the chaetoid sensilla. The inset shows the fluorescence of the antennal basal region at a higher magnification, with the black, non-fluorescent chaetoid sensilla. Bright field (B) and fluorescent (C) light views of the antennal tip with sensory structures. a, antenna; asc, antennal sensory cone; cs, chaetoid sensilla; hc, head capsule; hco, head comb; lcs, long chaetoid sensilla; lts, long trichoid sensillum; rim, rim of the apical portion of the antenna; se, setae; so, socket of long trichoid sensillum; sts, short trichoid sensillum. Scale bars: 100 μm.

Figure 9.

Scanning electron micrograph of the antenna of Cx. pipiens fourth instar larvae. A) Whole antenna. B) Basal portion of the antennal scape. C) Detailed view of the antennal sensory cone region. a, antenna; asc, antennal sensory cone; cs, chaetoid sensilla; hc, head capsule; hco, hair comb; lcs, long chaetoid sensilla; lts, long trichoid sensillum. Scale bars: A) 200 μm; B,C) 20 μm.

Spectrofluorometric analysis

The AF spectra recorded from the fluorescing structures of Ae. albopictus antennae cover the 390-580 nm interval, with an increasing position of the wavelength of the maximum peak from about 445 nm, to 455 and 460 nm respectively for antacoria, sensory cone and scape (Figure 10A, Table 1). The AF spectra recorded from Cx. pipiens antennae cover a similar spectral interval. In this case, however, spectra show maximum peak positions at about 455 nm for both the cranial antennal basis and the antennal sensory cone, and at about 470 nm for the scape (Figure 10B, Table 1). In addition, as compared to Ae. albopictus (Figure 10A), AF spectra recorded in Cx. pipiens (Figure 10B) appear to be wider. The AF spectra recorded from the tessellated membrane have a maximum peak position at about 415 nm in Ae. albopictus and at about 425 nm in Cx. pipiens, which also shows spectra with a wider emission profile and a slight shift toward longer wavelengths. Similar differences between Ae. albopictus and Cx. pipiens can be observed also for the AF spectra collected from the mouth brushes (Figure 10C, Table 1). In these structures, the AF occurs in the 400-680 nm interval, and the maximum peak position is found at about 460-490 nm for Ae. albopictus, and in the 470-530 region for Cx. pipiens.

Table 1.

Maximum peak position and full width at half intensity maximum (FWHM) of AF spectra recorded from fluorescing structures of Ae. albopictus and Cx. pipiens larvae.

Mosquito species	Structure	Peak maximum°	FWHM°
Ae. albopictus	Antacoria	440-450 nm	110-115 nm
	Antennal sensory cone	450-460 nm	105-110 nm
	Antennal scape	440-475 nm	120-130 nm
	Tessellated membrane	410-420 nm	65-75 nm
	Mouth brushes#	460-490 nm	120-140 nm
Cx. pipiens	Cranial antennal basis	445–465 nm	125-135 nm
	Antennal sensory cone	450–465 nm	120-130 nm
	Antennal scape	460–490 nm	130-140 nm
	Tessellated membrane	420-440 nm	100-120 nm
	Mouth brushes#	470-530 nm	150-160 nm

°Peak emission and FWHM may vary depending both on the conditions of measurement applied; #peak emission and FWHM may also vary depending both on the combined presence of different biomolecules and fluorophores.

Discussion

This study reports the first characterization of the AF signals in head structures of larvae of the mosquitoes Ae. albopictus and Cx. pipiens. Within the morphological traits characterizing the head appendages of the two species, our data revealed interesting differences as to both the distribution of AF signals and colors in the microscope images, and the spectral shape profiles. These findings indicated the presence of various fluorescing biomolecules, in turn relatable to the different biological and behavioral features of the two species.

In this respect, it is worth to recall what is generally observed in insects, the cuticle of which, for example, can produce AF emission ranging from the blue-green to deep-red wavelengths, depending on the presence of multiple components with different AF emission properties.[7,9,15,76,77] Well-sclerotized chitinous exoskeletal structures are usually autofluorescent in red, the tough-flexible cuticle in yellow-green, and the relatively flexible material containing a high proportion of resilin in light blue.[9]

Our images show both in Ae. albopictus and Cx. pipiens larvae a strong blue AF emission from a hairless cuticle area, dorsally continuous with the hair plaque, corresponding to the tessellated membrane. The tessellated membrane was originally described by Christophers,[59] and it is considered a structure with particular stretchability.[63] This property is in full accordance with the enriched presence of resilin, ensuring elasticity to the tessellated membrane as well as to the cross bars that allow the mouth hairs to move.[63,78] These studies, along with the literature reporting on the resilin spectral profiles,[79,80] allow to interpret our imaging and spectral data on the bluish emission of the tessellated membrane as indicative of the presence of resilin. Our AF microscope images reveal also a more extended tessellated membrane in Cx. pipiens than in Ae. albopictus. This finding is in agreement with the engagement of the tessellated membrane in supporting the movement of the long filaments of the lateral palatal brushes.[56] Indeed, in Aedes the tessellated membrane should withstand minor efforts than in Cx. pipiens, since it carries simpler, relative shorter, thin and soft lateral labral brush hairs, which likely do not contribute in the creation of the feeding current, as suggested by the literature.[63] In addition, it is to note that the spectral profile we recorded in Ae. albopictus is narrower than that obtained from Cx. pipiens, suggesting differences in the composition of the tessellated membrane between the two species. These changes can be related to the involvement of the resilin-enriched tessellated membrane in withstanding different efforts to provide movability as well as soft and elastic support to the mouth brushes for feeding. In this regard, it is worth recalling that the differences in the fluorescing signal may depend on changes in the resilin polymeric molecular features, as well as on the coexistence, at variable proportions, of different compounds, such as chitin and matrix proteins, involved in ensuring versatility in mechanical properties and resilience to functional efforts.[7,9,77] In both Ae. albopictus and Cx. pipiens, our detection of orange-reddish AF signals indicates the contribution of chitinous material, which has been reported as an established component of mouthparts in larval and adult insects,[81] in agreement with the primarily dependence of the two species on mouth brushes for feeding. [56] Also, it is to recall that both Ae. albopictus and Cx. pipiens are defined as container mosquitoes and their larvae combine browsing along hard surfaces through propulsion by movements of the mouth parts and filtering particles from the water for feeding.[82]

In addition, differences in larval behavior between these two mosquito species have been reported in the literature. Indeed, Cx. pipiens larvae tend to stay at the surface of the water column, while Ae. albopictus individuals move in the middle/bottom,[83] similar to Ae. aegypti, which feed usually in deeper zones of the water column. [56]Moreover, mosquito larvae are known to exhibit differences in exploration, stimulus preference and chemosensory navigation. [47]While both in Culex and Aedes the lateral palatal brushes are reported to extend anterolaterally, in Culex species their orientation is more oblique than in Ae. aegypti.[56] Furthermore, the lateral palatal brush filaments, which create the water and suspended particles’ flow towards the mouth, are shorter and more numerous in Ae. aegypti than in Culex, and move faster. This is reflected in a faster flow generated by the lateral palatal brushes in Ae. aegypti than in Culex.[56] Shannon[84] and Christophers[59] also noticed that Ae. aegypti larvae moved considerably faster than the larvae of most other species of mosquitoes. Mosquito larval locomotion is historically known as an extremely complex process.[59] Filter-feeders such as larvae belonging to the Culicidae family inhabit still waters and thus cannot simply rely on the water in their immediate vicinity to obtain food, as it can be quickly become depleted of the food particles in suspension. Conversely, they need to actively generate a constant particle-bearing current from the area in front of the head, so that the current is directed towards their mouth parts, to be then expelled backwards.[85] In Cx. pipiens, mouth brushes have been suggested to function like a paddle to exploit the feeding current they generate to slowly glide through the water.[85] This enable the larvae to move towards water areas with more food resources, and possibly to optimize foraging by avoiding inefficient filtration due to water recirculation.[85] No specific information about the shape and movements of the lateral palatal brushes are currently available for Ae. albopictus. As it appears from the above recalled literature, different feeding and locomotion modalities are described among Culicidae and can account for the structural differences indicated by the presence of resilin and chitin in the mouth appendages of Ae. albopictus and Cx. pipiens, as suggested by our AF imaging and spectral data. These data stimulate future research aimed at correlating these features with mosquito larval behavior. As to the antennae, the blue AF ascribable to resilin detected at their bases showed differences between the two species both in terms of AF signal distribution and spectral profile. Regarding AF distribution, in Ae. albopictus, the blue emission appears to be delimited to the antacoria, an unmelanized membrane connecting the antenna with the antennal prominence, while in Cx. pipiens the blue emission is localized in a larger area of the cranium, ventrally to the basis of the antennae and in the antennal ridge around the outer margin of the antennal socket. This variance in AF distribution between the two species, as well the spectra with a wider emission profile and a slight shift toward longer wavelengths in Cx. pipiens, may be due to a variability in material composition. These properties may in turn reflect different capacities in facing mechanical stress deriving from diverse types and intensities of antennal movements. This suggestion, together with our images showing that the morphological differences in the antennae of the two species, further stimulates additional studies on the movements of mosquito larval antennae as a non-invasive approach to derive information on larval sensory perception. This is particularly interesting since antennal movements in mosquito larvae are currently unexplored, and would require a multidisciplinary approach, including anatomical, behavioral, and physiological investigations to be elucidated.

Autofluorescence spectra of head structures in mosquito larvae. Typical examples of the AF spectra recorded from A) antennal structures comprising antacoria, sensory cone and scape in Ae. albopictus (Aealb), B) cranial antennal basis, sensory cone and scape in Cx. pipiens (Cpip), C) mouth brushes and tessellated membrane of Ae. albopictus and Cx. pipiens. Spectra are normalized to the maximum peak intensity (100%) to better compare emission profiles. Spectra are identified by colors, as from the inlet legend.

The shape of the antennal sensory cone in insect larvae has been reported to be unique to each species.[61] This structure has been suggested to result from the fusion of different basiconic sensilla, [86] and its morphology and ultrastructure have been described in many species belonging to Coleoptera, Trichoptera and Diptera (see Akent’eva and colleagues[61] for a review), but not yet in Ae. albopictus. In insect larvae, the antennal sensory cone is an extremely complex sensory organ composed of a cone-shaped cuticular portion that may contain pores and poral tubes and is known to be innervated by receptor cells.[61] Also, in the sensory cone of Ae. aegypti, a potential role as osmoreceptor for its peg organ was suggested by Zacharuk and Blue.[66] The absence of visible pores in our SEM images of the antennal sensory cone of both Ae. albopictus and Cx. pipiens lead us to suggest that it may work as a contact chemoreceptor organ, although functional studies are required to confirm such a role.

In Cx. pipiens, the long trichoid sensillum with a thickened base and a rounded tip, located lateral to the larval antennal sensory cone, displays strong blue AF typical of resilin, similar to the long trichoid sensillum at the antennal tip of Ae. albopictus. In a previous study on the AF of Ae. albopictus adult mosquitoes, sensilla trichoidea, the primary olfactory sensilla located along the flagellomers in the female and in the terminal flagellomere in males, were found to be fluorescent too.[17] Taken together, this evidence on AF signals recorded from the larval antennal sensilla may suggest the presence of a functional conservation either across developmental stages and between Cx. pipiens and Ae. albopictus.

Resilin is well assessed to be present in mobile joints and veins walls, where it may be involved in specific folding/unfolding mechanisms and/or be present at positions where particular elasticity is required to avoid structural damage.[7,87-90] In this respect, the presence of blue AF in the sutures (e.g., in the maxillary sutures in Cx. pipiens) relatable to the presence of resilin is not unexpected, given the need to absorb shock derived from mechanical impacts taking place during feeding[7]. Also larval antennae can be potentially under constant mechanical stress as they are sensory organs exposed to the external environment, as it has been proposed for adult antennae.[15] The role of resilin in these structures may clarify the relationships between functional morphology and biomechanics in larval heads, as already proposed following the characterization of the antennae of male and female adults of Ae. albopictus, which shows marked morphological differences.[17]

In conclusion, in this exploratory study we found that AF analysis may allow to visualize body structures and tissues otherwise hardly observable in bright field conditions, revealing both shared and specific traits of fourth instar larvae of the two mosquito species Ae. albopictus and Cx. pipiens. Our AF data show the presence, common to both mosquito species, of a strong blue emission attributable to resilin in the tessellated membrane, the head sutures, as well as in the antennal bases, sensory cone and sensilla. In addition, a reddish AF emission attributable to chitinous material was detected in the mouth brushes. Within these common traits, AF revealed differences in signal distribution and spectral profiles between the larvae of the two considered species. In particular, AF showed a more extended tessellated membrane in Cx. pipiens than in Ae. albopictus, along with morphological differences in the antennal basis area. In addition, the wider emission profile and the slight shift toward longer wavelengths of AF spectra in Cx. pipiens with respect to Ae. albopictus suggested a variability in material composition, in a likely dependence on the functional role of the corresponding body structures mainly involved in larval foraging, navigation and sensory behaviors. AF-based investigations, similar to a label-free histochemical approach, are expected to produce key knowledge in these complex mosquito larval functions, with ecological and evolutionary implications, providing a powerful tool to advance biomechanical, biochemical, behavioral and taxonomical integrated studies. Such a potential is testified by the various reports published over the last decade on other insects species focusing on correlating biomechanical features to predatory and feeding behaviors, as well as flight ability, and on revealing anatomical traits useful in comparative morphology and evolutionary studies.[9,11,12,91-96]

Finally, such expanded knowledge may have applied relevance to develop new strategies based on the use of chemicals reproducing the olfactory cues emitted by conspecific larvae and able to affect adult oviposition,[97,98] and to better inform larvicide application to improve mosquito control in the field.[47]