Spatial distribution of opsin-encoding mRNAs in the tiered larval retinas of the sunburst diving beetle Thermonectus marmoratus (Coleoptera: Dytiscidae)

Abstract

Larvae of the sunburst diving beetle, Thermonectus marmoratus, have a cluster of six stemmata (E1-6) and one eye patch on each side of the head. Each eye has two retinas: a distal retina that is closer to the lens, and a proximal retina that lies directly underneath. The distal retinas of E1 and E2 are made of a dorsal and a ventral stack of at least twelve photoreceptor layers. Could this arrangement be used to compensate for lens chromatic aberration, with shorter wavelengths detected by the distal layers and longer wavelengths by the proximal layers? To answer this question we molecularly identified opsins and their expression patterns in these eyes. We found three opsin-encoding genes. The distal retinas of all six eyes express long-wavelength opsin (TmLW) mRNA, whereas the proximal retinas express ultraviolet opsin (TmUV I) mRNA. In the proximal retinas of E1 and E2, the TmUV I mRNA is expressed only in the dorsal stack. A second ultraviolet opsin mRNA (TmUV II), is expressed in the proximal retinas of E1 and E2 (both stacks). The finding that longer-wavelength opsins are expressed distally to shorter-wavelength opsins makes it unlikely that this retinal arrangement is used to compensate for lens chromatic aberration. In addition, we also described opsin expression patterns in the medial retina of E1 and in the non-tiered retina of the lensless eye patch. To our knowledge, this is also the first report of multiple UV opsins being expressed in the same stemma.

Fig. 1.

First instar T. marmoratus larval eyes. (A) Frontodorsal view of a larva showing the principal eyes, E1 and E2, and the eye patch (EP). (B) Left lateral view of the head showing the secondary eyes, E3-6, as well as the two principal eyes. (C) An ethyl gallate-stained sagittal section showing the lenses (L), crystalline cone-like structures (CC), retinular cells of the distal (DRC) and proximal (PRC) retinas with their respective rhabdoms (distal, DRh; proximal, PRh). Different retinas (retinular cells and rhabdoms) are labeled on the principal eyes (E1 and E2) and on E5. Other secondary eyes (E3, E4, and E6) have an organization similar to that of E5, but are situated outside the plane of this section. In general, in all eyes (E1-6), distal retinular cells (DRC) stain darker with ethyl gallate than the proximal retinular cells (PRC). Rhabdoms also appear darker. (D) 3D reconstruction of E1 showing the lens (L), crystalline cone-like structure (CC), medial retina rhabdom (MRh), distal retina rhabdom (DRh), and the proximal retina rhabdom (PRh). Insets a and b are schematic drawings of respective sections through the distal retina (as indicated in the figure by sectional planes a and b), illustrating the orthogonal orientation of the dorsal and ventral retinular cells of the distal retina. The insets illustrate the rhabdoms (red) as well as cell bodies (green) which are not shown in the 3-D reconstruction. Scale bars, A and B, 200 μm; C, 100 μm.

Fig. 2.

Schematic illustration of the three cloned opsin cDNAs in T. marmoratus first instar larvae. (A) TmLW cDNA. (B) TmUV I cDNA. (C) TmUV II cDNA. Coding regions (cds) are shown in green in A, and violet in B and C. Gray regions are positions of the predicted transmembrane segments. Numbers at both ends of each cds indicate the positions of start and stop codons. Nucleotide sequences are of degenerate primers that were used to amplify the internal opsin fragments. Primer positions along with lengths of amplified fragments are also indicated, as well as the size and position of the riboprobes that were used in in situ hybridizations.

Fig. 3.

Amino-acid alignment in ClustalW of the three opsins cloned as cDNA from the first instar larval heads of T. marmoratus. Brackets indicate transmembrane regions predicted from hydropathicity analysis and the alignment with other opsins. Identical residues are in red (*), strongly similar ones are in green (:), weakly similar ones are in blue (.). The arrow indicates the site of the chromophore Schiff-base linkage. Conserved amino-acid residues are in gray (for more details see text). The sequences have been deposited in GenBank under accession numbers EU921225 (TmLW), EU921226 (TmUV I), EU921227 (TmUV II).

Fig. 4.

Phylogenetic reconstruction of T. marmoratus larval opsins and 46 other known arthropod opsins. For accession numbers and spectral absorbencies, see Table 2. The numbers appearing above nodes represent bootstrap percentages of 1000 replications. Spectral clades are delineated. Arrows indicate positions of the T. marmoratus larval opsins.

Fig. 5.

Distribution of opsin mRNAs in the principal eyes as examined by in situ hybridization; all images are of sagittal sections. (A) An overview histological section showing the positions of the in situ images. (B) E1, fluorescent staining of TmLW mRNA illustrating its expression in the distal retina. (C) E1, fluorescent staining of TmUV II mRNA illustrating its expression in the proximal retina. (D) E1, double chromogenic staining of TmLW (purple) TmUV II (brown) mRNAs. (E) E2, double chromogenic staining of TmLW (purple) and TmUV II (brown) mRNAs illustrating primarily the distal retina at larger magnification. (F) E2, double staining for TmLW (purple) and TmUV II (brown) mRNAs illustrating primarily the proximal retina at larger magnification. DRC, retinular cells of the distal retina; PRC, retinular cells of the proximal retina; DRh, distal rhabdom; PRh, proximal rhabdom n, nuclei. Scale bars: A, 100 μm; B-D, 100 μm; E,F, 50 μm.

Fig. 6.

Distribution of TmUV I and TmUV II mRNAs in the proximal retina of the principal eyes as examined by in situ hybridization. A, sagittal section; B-D, cross sections through the proximal retina (see inset in B). (A) E1, fluorescent staining of TmUV I mRNA illustrating its expression in the dorsal stack of the proximal retina. (B) E2, fluorescent staining of TmUV I mRNA in a cross section through the proximal retina as indicated in the inset. Note that the expression is only present in the dorsal stack. (C) E2, fluorescent staining of TmUV I mRNA in a cross section through the dorsal stack of the proximal retina at a higher magnification. (D) E2, fluorescent staining of TmUV II mRNA in a cross section through the proximal retina. Note the stronger hybridization signal of TmUV II mRNA compared with that of TmUV I mRNA in B and C. DRC, retinular cells of the distal retina; PRC, retinular cells of the proximal retina; DRh, distal rhabdom; PRh, proximal rhabdom; n, nuclei. Scale bars: A,B,D, 100 μm; C, 50 μm.

Fig. 7.

Distribution of opsin mRNAs in the medial retina of E1 as examined by in situ hybridization. A-E are cross sections through E1; F is a sagittal section (see inset). (A) An overview histological cross section through E1 showing the position of the medial retina as seen in B-E. (B) Fluorescent staining of TmLW mRNA showing its expression in the dorsal and ventral parts of the medial retina. Note the absence of signal in the central part of the medial retina. (C) Fluorescent staining of TmUV II mRNA showing its expression in the more central parts of the medial retina. (D) Double chromogenic staining of TmLW (purple) and TmUV II (brown) mRNAs. Note how the purple staining (TmLW mRNA) surrounds the brown staining (TmUV II mRNA) from the dorsolateral and ventrolateral sides. (E) Fluorescent staining of TmUV I mRNA. Note that the hybridization signal covers a similar region as the expression pattern of TmUV II mRNA (see C). (F) Double chromogenic staining for TmLW (purple) and TmUV II (brown) mRNAs in a sagittal section, as indicated in the inset. Note the band of UV-expressing tissue (brown) extending along the margin of the distal retina. It appears that the UV-sensitive part of the medial retina is anatomically connected with the UV-sensitive proximal retina. Furthermore, the LW-sensitive part of the medial retina may be an extension of the distal retina (black arrow). CC, crystalline cone like structure. Scale bars: A,F, 100 μm; B-E, 50 μm.

Fig. 8.

Distribution of TmLW and TmUV I mRNAs in the secondary eyes as examined by fluorescence in situ hybridization. Staining of TmLW mRNA is illustrated in the left panels and TmUV I mRNA in the right panels. Each row is a different eye, as indicated on the left side of the figure. The terms ‘sagittal’ and ‘cross’ refer to the sectional plane of the head. All eyes are shown in cross sections, except for E4 in C (sagittal) and E6 in H, (sagittal). E3-5 have comparable arrangements of their retinas with distal retinular cells (DRC) projecting to the periphery on both sides of the rhabdoms, and proximal retinular cells (PRC) extending to the back of the eyes (see Fig. 1, E5). (A, C, E and G) TmLW mRNA is present in the lateral eye region, which is associated with the distal retina. (B, D, F and H) TmUV II mRNA is present in the back of the eyes where the proximal retinular cells are located. In E3 the signal is localized in a relatively small area covering two narrow bands. This is most apparent in the inset in B, which is a section through E3 without the lens. L, lenses. Scale bars: A-H, 100 μm.

Fig. 9.

Distribution of TmLW and TmUV I mRNAs in the eye patch retina as examined by in situ hybridization. (A) Sagittal head section illustrating the fluorescent hybridization signal for TmLW mRNA in the eye patch retina. (B) Sagittal head section illustrating the fluorescent hybridization signal for TmUV I mRNA in the eye patch retina. (C) Frontal (cross) head section illustrating chromogenic double staining for TmLW (purple) and TmUV I (brown) mRNA in the eye patch retina. Rh, rhabdom. Scale bars: A,B, 100 μm; C, 50 μm.

Fig. 10.

Schematic illustration of the spectral sensitivity of the entire T. marmoratus first instar larva visual system based on opsin expression patterns. Rhabdoms are shown as stripped regions. EP, eye patch; MR, medial retina.