Morphological and optical features of the apposition compound eye of Monochamus alternatus Hope (Coleoptera: Cerambycidae)

Journal Pre-proof Morphological and optical features of the apposition compound eye of Monochamus alternatus Hope (Coleoptera: Cerambycidae) Chao Wen, Tao Ma, Yangxiao Deng, Chuanhe Liu, Shiping Liang, Junbao Wen, Cai Wang, Xiujun Wen

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S0968-4328(19)30222-7

DOI:

https://doi.org/10.1016/j.micron.2019.102769

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JMIC 102769

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Micron

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18 July 2019

Revised Date:

3 October 2019

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4 October 2019

Please cite this article as: Wen C, Ma T, Deng Y, Liu C, Liang S, Wen J, Wang C, Wen X, Morphological and optical features of the apposition compound eye of Monochamus alternatus Hope (Coleoptera: Cerambycidae), Micron (2019), doi: https://doi.org/10.1016/j.micron.2019.102769

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Morphological and optical features of the apposition compound eye of Monochamus alternatus Hope (Coleoptera: Cerambycidae) Chao Wena, 1, Tao Maa, Yangxiao Denga, Chuanhe Liub, Shiping Lianga, Junbao Wenc, Cai Wanga,∗ , Xiujun Wena, ∗ a

Guangdong Key Laboratory for Innovation Development and Utilization of Forest Plant

Germplasm, College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou, China. b

Instrumental Analysis and Research Center, South China Agricultural university.

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Beijing Key Laboratory for Forest Pest Control, College of Forestry, Beijing Forestry University,

Beijing 100083, China. ∗

Correspondence: Cai Wang, Guangdong Key Laboratory for Innovation Development and

Utilization of Forest Plant Germplasm, College of Forestry and Landscape Architecture, South

China Agricultural University, Guangzhou 510642, China. Tel: +15521034689; email:

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[email protected].

Correspondence: Xiu-Jun Wen, Guangdong Key Laboratory for Innovation Development and

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Utilization of Forest Plant Germplasm, College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou 510642, China. Tel: +86 20 85280256; email:

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[email protected]. Highlights

The compound eyes of M. alternatus are characterized by high absolute sensitivity, but relatively poor spatial resolution.

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Compound eye area and number of ommatidia of M. alternatus are correlated with body size. The longhorn beetle M. alternatus possesses an acone apposition eye with a semi-fused type of rhabdom. Dark/light adaptational changes affect cone length, the position of pigment grains and the crosssectional area of the rhabdoms.

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Abstract

The Japanese pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae) is currently the most destructive forest pest as it transmits the pine wilt nematode Bursaphelenchus xylophilus. Morphological, optical features and dark/light adaptational changes of the compound eyes of M. alternatus adults were examined by light, scanning and transmission electron microscopy. The eye of M. alternatus is apposition type and contains 489-712 ommatidia, depending on the beetle’s body size. Each ommatidium features a large corneal lens, composed of a thick inner lens (ILU) and a thin outer lens unit (OLU); an acone-type of cone of four cone cells, a semi-fused type

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of rhabdom formed by eight retinular cells (two central cells: R7-R8 surrounded by six peripheral cells: R1-R6). Dark/light adaptational changes affect size and shape of the cones as well as the rhabdom’s cross-sectional area and outline, to optimize the amount of light that reaches the photopigment. The compound eyes of M. alternatus have an F-number of 0.94, an interommatidial angle of 5.34°, an eye parameter P of 4.98 μm·rad and a ratio of acceptance to interommatidial angle of 0.45. The eye is characterized by relatively poor spatial resolution, but can be expected to exhibit high absolute sensitivity and contrast in dim light.

Keywords: Monochamus alternatus; photoreceptor; dark/light adaptation; fine structure; scanning

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electron microscopy; transmission electron microscopy.

1. Introduction

How animals generally and insects in particular see the external world ultimately depends on

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how they process the signals they receive from their visual organs (Toh and Okamura, 2007). Compound eyes are the main photoreceptors in insects and are involved in sensing an object’s movement, size, shape and colour, all of which known to play important roles in the insect’s foraging

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activity, prey and predator detection, reproductive and homing behaviour (Chapman, 2007; Jia and Liang, 2015). The compound eye of insects, irrespective of the size of a species (Fischer et al., 2015), consist of a variable number of ommatidia, and each ommatidium consists of a corneal lens, a cone,

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a rhabdom formed by usually eight retinular cells and a certain number of pigment cells. According to their optical design, most compound eyes can be assigned to one of two basic types: apposition and superposition eyes (Exner, 1891). In superposition eyes, the dioptric elements,

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i.e. cornea and cones, are separated from the receptors, i.e. retinular cells and rhabdom, by a wide pigment-free gap, known as the clear-zone (Horridge, 1975). The result of this design and the presence of dioptric structures with radial refractive index gradients (Meyer-Rochow and Horridge,

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1975; McIntyre and Caveney, 1985) is that an ommatidial receptor can receive not just light that has entered through its own cornea above, but from a much wider area across the eye. By contrast, in the apposition eye, each ommatidium is isolated from its neighbours by a sleeve of light-absorbing

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screening pigments and only light that has entered the ommatidium through its own window can excite the receptors. This can lead to the formation of a sharper image, but it comes at the expense of absolute sensitivity. Therefore, the sensitivity of a superposition eye is greater than that of an apposition compound eye, albeit in combination with decreased resolving power (Warrant and McIntyre, 1993; Land, 1997; Meyer-Rochow and Gal, 2004). Unsurprisingly, insects considered nocturnal tend to possess eyes of the superposition type while most diurnally-active insects possess apposition eyes (Exner, 1891; Land, 1981; Nilsson, 1989). The Japanese pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae) is currently the most destructive forest pest in China, Japan and many other regions of the world

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(Kobayashi et al., 1984; Mamiya, 1998). The larva of the beetle drill into stems of Pinus species, weakening the plants and even causing their mortality (Yang et al., 2014). In addition, the adults of this beetle are known to transmit the pine wilt nematode Bursaphelenchus xylophilus, which causes pine wilt disease. This disease is held responsible for the decline of pine trees in large areas and is therefore regarded a serious threat to conifer ecosystems (Abelleira et al., 2011). Compound eyes play important roles in foraging and spawning in longhorn beetles including M. alternatus (Groot and Nott, 2001; Morewood et al., 2002; Yang et al., 2007; Giffard et al., 2017). Field and laboratory observations involving M. alternatus have revealed that visual cues play a crucial role in long-distance host finding and close-range mate selection (Liu et al., 2012). However, little is known about the properties of the visual system in M. alternatus. In order to improve our understanding of this species’ visual behaviour and its mechanism, M. alternatus’ compound eyes’

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external morphology was examined by scanning electron microscopy (SEM). Transmission electron (TEM) and light microscopy (LM) techniques were used to investigate the micro-anatomical structure of the compound eye in M. alternatus adults under dark and light adaptational conditions.

This descriptive work provides the basis for future investigations into the behaviour of M. alternatus.

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2. Materials and methods 2.1 Insects source

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Adult males and females of M. alternatus were collected from Wenquan town (113°37′6′′E, 23°38′59′′N), Guangzhou city, Guang dong province in mid-August, 2018. The collected insects

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were put in a gauze bag (30 × 30 cm) and fed with twigs and leaves of Pinus massoniana. The insects were maintained in the controlled laboratory condition at 26 ± 1°C, a relative humidity of 70-80%, and a photoperiod of 14:10 h (L: D).

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In order to conduct light adaption experiments, the insects were placed under a LED light with high intensity (1000 lux) for at least 20 min in the laboratory, or were kept in complete darkness (dark-adapted) for at least 24 h before fixing. An illuminometer (AC9803A, TaiShi, ChangSha, China) was used for measuring light intensity. The light-adapted insects were decapitated and fixed

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during the daytime, while dark-adapted specimens were decapitated and fixed under dim red light. 2.2 Scanning Electron Microscopy (SEM).

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For scanning electron microscopy (SEM), twenty-five (five males and twenty females) M.

alternatus adults were used. After decapitation, the heads were immediately fixed in 2.5% glutaraldehyde for at least 24 h. Then, the heads were dehydrated in an alcohol series (2 times 20 min each with 70%, 80%, 90%, 95%, 100%). After air-drying for more than 24 h, the specimens were sputter-coated with gold at 30 mA current for 100 s (LEICA EM ACE600, Germany). A scanning electron microscope (EVO MA15, Germany) was used to take micrographs of the eye’s surface. 2.3 Transmission Electron Microscopy (TEM)

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For transmission electron microscopy (TEM) analysis, the samples were decapitated and fixed in 5% glutaraldehyde buffered with 0.1 M cacodylate buffer (pH = 7.4) for 1h (3 times, 20 min each). The heads were postfixed in 1% OsO4 for more than 24 h, and kept in 0.1 M cacodylate buffer for 1 h. The heads were the dehydrated in an alcohol series (2 times 20 min each with 30%, 50%, 70%, 80%, 90%, 100%) and absolute acetone (2 times). The specimens were infiltrated through different acetone/Epon mixtures (3:1, 1:1, 1:3, pure Epon), and hardened at a temperature of 60℃ for 4 d. The embedded samples were cut with a diamond knife on an ultramicrotome (Leica UCT, Germany) and sections of approximately 80 nm for ultrathin sections were obtained. The ultrathin sections were then stained with 2% aqueous uranyl acetate for 15 min and observed in the transmission electron microscope (Talos L120C, America) at 80 kV. 2.4 Light Microscopy (LM)

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For light microscopy (LM), semithin sections at a thickness of about 300 nm were cut on a microtome (Leica UCT, Germany). The sections were stained with 0.5% aqueous solution of

toluidine blue on a hot plate for 100 s. Semi-thin sections of eyes were mounted with rhamsan gum. A fluorescent inverted microscope (Leica, DMI8, Germany) was used to take micrographs.

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2.5 Data analysis

The body sizes of M. alternatus were measured with a digital caliper. Scanning electron microscopy (SEM) was used to determine total facet number per eye, compound eye and facet areas.

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Longitudinal sections for LM were used for measurements of corneal and cone dimensions and to determine the diameter of the distal tip of the rhabdom. Transverse sections were used to measure

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the cross-sectional area of the rhabdoms. TEM micrographs were used to measure the number of corneal laminations, retinular cells and diameters of pigment granules and mitochondria. All histological measurements involved high-resolution photographs and were analyzed by Image J

3. Results

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3.1 Overview

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version 1.52 software.

With bodies measuring 14.50 mm in the smallest and 24.53 mm in the largest specimen (Table 1), a clear size polymorphism exists among M. alternatus adults that were collected in the field.

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Adult M. alternatus possess a pair of crescent-shaped compound eyes on both sides of the head (Fig. 1A, B). The eyes surround the antennal socket, and several facets extend to the anterior side of the head (Fig. 1B). A certain amount of binocular vision can be expected as there is some overlap of the visual fields of the two eyes. Each eye contains an average of 614.20 ± 19.92 facets in males and 630.20 ± 17.05 in females (Table 1). The eye area and number of facets increases with body size (Pearson correlation test, r =0.884, p < 0.001; r = 0.702, p < 0.001; Fig. 2A, B). The facets located in the centre of the eyes are hexagonal in shape, while the facets situated towards the peripheral region of the eyes show pentagonal outlines (Fig. 1C, D). The hexagonal facets’ area covers 2447.33 ± 22.84 μm2, while the pentagonal facets’ area is 2287.25 ± 31.31 μm2 (Table 1).

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3.2 Anatomy Each ommatidium in M. alternatus is composed of two distinct structures: dioptric apparatus and photoreceptive layer. The dioptric apparatus consist of corneal lens and cone, and the photoreceptive layer is made up of retinular cells and their rhabdomeres (Fig. 3A). Screening pigment cells fill the spaces between adjacent rhabdomal units of the compound eyes in M. alternatus and optically isolate ommatidial units from its neighbours (Fig. 4D). The proximal part of the corneal lens is attached to the cone, but the cone is not in direct physical contact with the rhabdom for there is a small region crowded with screening pigment separating the two (Fig. 3A). The absence of a wide clear-zone shows that eyes of M. alternatus conform to the apposition eye type.

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3.2.1 Dioptric apparatus Adult M. alternatus possess corneal lenses measuring 47.71 ± 0.85 μm in thickness. The radius

of curvature of their outer surface is 20.02 ± 0.67 μm (Table 1). Semi-thin sections of the corneal lenses show that each cornea is composed of a thick inner lens unit (ILU) and an outer lens unit (OLU). The ILU is devoid of staining by toluidine blue, while the ILU is stained densely (Fig. 4A,

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B). TEM micrographs show that the cornea has a laminated structure, seemingly composed of many layers with alternatingly differing electron densities (Fig. 4A). TEM micrographs also show a compositional difference between the ILU and the OLU. The laminations of the ILU form a series

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of parabolas, while the OLU appears homogeneous (Fig. 5A). The ILU is shaped as a spindle with a thickness of 35.53 ± 1.38 μm, and the OLU is shaped as a hollow hemisphere measuring 10.19 ± 1.07 μm in thickness (Table 1). Thus, ILU and OLU correspond to two different focal planes. The

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lens has an anterior principal plane H and a posterior principal plane H' (Fig. 6). Insertions seen in the M. alternatus eye, extend proximally to the vicinity of the cone, forming a cuticular sheath which almost completely surrounds and separates each ommatidial dioptric apparatus from that of its

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neighbour (Fig. 4B). Four cone cells located just beneath the cornea are involved in forming the cone. Each cone cell is wedge-shaped and contributes one quarter to the cone (Fig. 5B). 3.2.2 Retinular cells and rhabdom

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The photoreceptive layer in each ommatiduim consists of eight retinular cells: two central cells (R7-R8) surrounded by six peripheral cells (R1-R6, Fig. 4C). The numbering of the cells follows

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the system introduced by Wachmann (1977). Each retinal cell has a rhabdomere on the inner, axial side that links up with its neighbour to form the peripheral, ring-like part of the rhabdom, thus giving the rhabdom its hexagonal or rectangular outline in transverse sections. The peripheral rhabdomeres and the two central to rhabdomeres are in physical contact via narrow bridges with R1 and R4 (Fig. 4C), but the boundaries between R2 and R3, R5 and R6, R7 and R8 are not clearly determinable. Each rhabdomere consists of numerous microvilli, each about 0.06 μm in diameter (Table 1). The microvilli of the central rhabdomeres R7 and R8 are oriented parallel to each other as well as those of R2, R3, R5 and R6, but perpendicular to those of R1 and R4 (Fig. 4C, 5C), a feature characteristic of polarization-sensitive compound eyes (Eguchi, 1999). A variety of cell organelles are present around the rhabdomeres’ edges like mitochondria, measuring in length from 0.22 to 0.76 μm.

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Endoplasmic reticulum was seen to be abundant in the cytoplasm of all retinal cell some distance away from the edge of the rhabdomere (Fig. 4D). 3.2.3 Pigments Ommatidia of M. alternatus contain two primary pigment cells (PPCs) and an undetermined number of secondary pigment (SPCs) (Fig. 4D, 7A). Two PPCs always envelop each group of the four cone cells (Fig. 7A). Both PPCs and SPCs contain numerous spherical electron-dense and opaque screening pigment granules as well as mitochondria in their cytoplasm. The screening pigment granules of the PPCs and SPCs measure 0.77 ± 0.02 μm and 0.62 ± 0.01 μm in diameter, respectively (Table 1). The smaller pigment granules can be seen right down to the basal lamina, demonstrating that the SPCs are elongated and reach the basal lamina (Fig. 3A).

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3.3 Dark/light adaptational changes of the compound eyes In the light-adapted (LA) state, cones measure 14.67 ± 0.51 μm in length (Table 1) and are of conical shapes with a narrow and pointed proximal end, while those pigment cells with the larger

diameter pigment granules, the PPCs, surround the cone (Fig. 7A). In addition, relatively few

mitochondria are noticeable in the two central retinular cells (Fig. 7D). In the dark-adapted (DA)

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state, cones are much shorter (5.57 ± 0.16 μm, Table 1), and form a disc shaped structure with a

round and blunt proximal end (Fig. 7B). Only pigment cells with smaller diameter pigments are

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found peripheral to the cone, and mitochondria are noticed to fill the retinular cells (Fig. 7C）. Comparisons based on transverse sections at identical levels viewed under the transmission electron microscope, show that the cross-sectional areas and shapes of the rhabdoms differ between

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adaptational states. In the dark-adapted (DA) state, rhabdom outlines are roughly hexagonal (Fig. 3C), and the cross-sectional area measures 328.56 ± 8.17 μm2 (Table 1). In the light-adapted state, however, the rhabdom diminishes in size and changes to a more circular outline (Fig. 4D, 5D) with

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a cross-sectional area of 210.74 ± 8.86 μm2 (Table 1). This represents an approximately 35% decrease of the cross-sectional rhabdom area from that of the DA state. 3.4 Optical aspects of the compound eyes

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To estimate the spatial resolution in M. alternatus, the interommatidial angle (△φ) was calculated from the length of a segment (S) transecting the eye and its height (H). (Schwarz et al.,

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2011).

R

(S/2) 2  H 2 2H

Where R is eye radius, S is the baseline length of a segment and H is height. Interommatidial

angle was calculated according to Land (1997) :

φ  D/R Where D is the facet diameter (53.16 ± 0.34, Table 1) and R is the eye radius, with R = 567.83

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μm measured by the method of Schwarz et al (2011). The interommatidial angle is 5.34° (0.09 in rad). The compound eye parameter P according to Horridge (1977) is used to assess the suitability of the compound eye for a particular photic environment, and a high P-value indicates a compound eye adapted to low light intensity.

P  D 2 /R  φ D where D is the facet diameter, R is the eye radius, calculated the P-value equal to 4.98 μm·rad. The focal length of the ommatidia was determined by applying the thick lens formula.

P1  (no  n1) / r1 , P2  (n 2  n1) / r 2 , P3  tP1P2 / n1 PL  P1  P2  P3

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f  n0/PL

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(Schwarz et al., 2011; Stavenga, 2003 ).

Where r1 is the outer lens surface radius; r2 is the inner lens surface radius and t is the distance between the vertices of the inner and outer lens surface. As illustrated in Fig. 6, n0 is refractive

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indices for air; n1 the refractive index of the corneal lens; n2 is the refractive index of the internal medium (Toh and Okamura, 2007). Studies have shown that the radial curvatures of the external lenses of insects ocelli or stemmata resembling ommatidia in M. alternatus are 1.45-1.6 (Dethier,

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1942; Meyer-Rochow 1974; Gilbert, 1994). Refractive index values of the dioptric structures in the apposition eyes of Creophilus erythrocephalus and Sartallus signatus were 1.47 and 1.49, respectively (Meyer-Rochow, 1972), and 1.45 for Drosophila lenses (Stavenga, 2003). Therefore,

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we assumed that the refractive index of the corneal lens in M. alternatus was 1.45 and the refractive index of the internal medium for the cone was 1.33 (Jia and Liang, 2017; Schwarz et al., 2011). On this basis the calculated focal length of the M. alternatus facet lens was determined as 49.90 μm.

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The F value as an indicator of the light gathering capacity of s lens system can be calculated as follows if the focal length is known (Jia and Liang, 2015; Land, 1984):

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F  f/D

where f is the focal length, D is the facet diameter. Consequently, the calculated F value equals

0.94 in M alternatus. The approximate angular acceptance function of rhabdoms was estimated by determining the

lens blur in apposition eyes of structurally similar beetles to M. alternatus (Meyer-Rochow, 1972):

△ρ=d/f Where d is the diameter of the distal rhabdom tip (2.11 ± 0.12 μm, Table 1) and f is the focal length. We thus find the acceptance angle of an ommatidium is 2.42° and the ratio of the acceptance

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angle to the interommatidial angle is 0.45. It has to be remembered, though, that the reported values of F, P and △ρ were not based on experimentally determined values of focal lengths and refractive indices, but on assumptions that some features were common to all apposition compound eyes of insects.

4. Discussion The total area of the compound eye and the number of facets in M. alternatus increase in relation to body size, a feature that is consistent with previous reports on 10 species of leafcutter ants, the ants Solenopsis richteri and Melophorus bagoti (Schwarz et al., 2011; Moser et al., 2011; Baker and Ma, 2006). Larger areas and numbers of facets are indicative of wider visual fields, greater sensitivity and better resolution (acuity) of the compound eyes (Schwarz et al., 2011). The

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cornea’s laminated structure has been described by Bouligand (1972) as a growth phenomenon of chitin micelles. It could mean that this structural arrangement in the cornea, the latter being the most

peripheral element of the visual system of the eye, may enhance the sensitivity of the eye by concentrating light and channeling it towards the underlying rhabdom (Toh and Okamura, 2007).

A cuticular sheath was found to be developed in each ommatidium of the compound eye of M.

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alternatus. Adjacent facets were isolated by the cuticular sheath which appeared to exhibit a kind of specialized cuticular construction similar to that of a lens. This structure was also found in other

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compound eyes of longicorn beetles (Koyama et al., 1975; Gokan and Hosobuchi, 1979) and it would seem to be a structure specific of cerambycid coleopterans. The optical significance of the cuticular sheath, however, is still unclear and a matter of speculation.

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In the compound eye of M. alternatus, the corneal lens is attached to the cone, a feature interpreted by Mishra and Meyer-Rochow (2006a) to represent an adaptation to increase the absorption of photons under dim light conditions in Xanthochroa luteipennis (like the cerambycid

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M. alternatus a cucujiform beetle). The eyes of cerambycid coleopterans studied to date all belong to the apposition type with open or semi-open rhabdoms (Wachmann, 1977; Gokan and Hosobuchi, 1979; Meyer-Rochow and Mishra, 2009). The rhabdom pattern in the apposition eye of M. alternatus could be called "semi-fused" (Koyama et al., 1975; Horridge and Giddings, 1971). This

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type of rhabdom occupies a considerable volume in the retinular cells to absorb photons present at very low rates in dim light, even during demanding nocturnal visual tasks (Koyama et al., 1975;

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Stavenga, 1992). On the basis of extensive comparisons Wachmann (1979) concluded that the rhabdoms in cucjiform cerambycid beetles could be divided into two types of pattern, which he termed “Grundmuster 1 and 2”. Our study shows that the compound eye of M. alternatus would belong to “Grundmuster 2”, in which the two central rhabdomeres (R7 and R8) were structurally attached to the peripheral rhabdomeres via two narrow bridges. In the eye of the cerambycid Phytoecia rufiventris, the central two retinula cells (cells 7 and 8), however, did not form rhabdomeres and did not participate in the rhabdom formation (Meyer-Rochow and Mishra, 2009), highlighting the diversity of rhabdom types amongst cerambycid beetles. In order to control the amount of light reaching the rhabdom, pigment grains migrate within

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the PPCs to widen or narrow the ommatidial aperture; a mechanism that has been likened to the pupil mechanism (Mishra and Meyer-Rochow, 2006a). Compared to the light-adapted state, the pigment particles (small diameter) of M. alternatus mostly migrate to the vicinity of the cone, i.e., out of the way of the light in the dark-adapted state, while the cone becomes shorter and wider, so that less light gets absorbed on the way to the photoreceptive membranes by the protective and dark shielding screening pigments. A very similar adaptation mechanism operates in the staphylinid beetle Creophilus erythrocephalus (Meyer-Rochow, 1972). In the eye of M. alternatus there was no crystalline material in the crystal cone cells, suggesting that the cone’s light-gathering capacity is weak (Wachmann, 1977). However, the shape of the cone changed easily under different light intensities driven by forces exerted by the large number of microtubular organelles present (MeyerRochow, 1999; Mishra and Meyer-Rochow, 2006b), but not through muscle fibres that had been held responsible for adaptational changes in Calliphora (Patterson, 1963). The cross-sectional area

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and shape of the rhabdoms change during dark/light adaptation to control the flux of the light and

to assure that a maximum amount interacts with the photopigment molecules. Rhabdom volume

changes are also a common feature in many other insects with apposition eyes that are in need to

adapt to changing light intensities (Brammer et al, 1978; Meyer-Rochow and Waldvogel, 1979;

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Meyer-Rochow, 1999).

The F-number is used as a simple metric for comparing the light-gathering capacities of different compound eyes. A low F-number indicates high absolute sensitivity in dim light (Warrant

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and Mcintyre, 1993). The F-number in nocturnally-active insects with apposition eyes ranges from 0.5 to1.2 (Warrant and Mcintyre, 1996; Warrant, 2004) and for the eye of M. alternatus we have calculated an F-number of 0.94. This which would indicate an extremely high absolute sensitivity

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in relation to light intended for the single target rhabdom (but not neighbouring rhabdoms) under the facet through which the light has entered. However, the F-value calculated for M. alternatus was calculated on the basis of assumptions not experimentally verified and therefore needs to be

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accepted with caution. A high parameter P likewise indicates a compound eye which is suitable for environments with poor light intensities (Snyder, 1977). That P values in nocturnal insects with apposition compound eyes can range from 2 to 4 μm·rad has been reported (Horridge, 1977). Our calculated P-value for M. alternatus was 4.98 μm·rad, but it may be too high, owing to assumptions

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made on the eyes’ optics (focal length, refractive indices, etc.) without experimental confirmation. However, it would undoubtedly suggest that the eyes of M. alternatus are well-adapted to function

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in the dim photic environment of the night that they can be seen to be active in. The interommatidial angle can indicate a compound eye’s resolution, with larger values being

related to lower spatial resolution (Land, 1997). In scarab beetles interommatidial angles rage from 2-7° (Warrant and McIntyre, 1990; Meyer-Rochow and Gal, 2004), but their eyes belong to the superposition type of eye, in which light entering through may facets is focused onto one receptor. The interommatidial angle of M. alternatus’ apposition eye is 5.34°, such relatively wide interommatidial angles in apposition eyes of herbivorous species that are not terribly fast can nevertheless be deemed to provide these insects with sufficient resolving power. The ratio of △ρ /△φ in M. alternatus is 0.45 and this suggests that these eyes are severely under-sampling image

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clarity but furnished with high contrast vision (Land, 1997). That feature combined with a high P value (in C. morosus, for example, 3.8 μm·rad) appears to be of greater use in finding suitable host trees than detecting fine details.

5. Conclusions This study has shown that the nocturnal longhorn beetle M. alternatus possesses an acone apposition eye with a semi-fused type of rhabdom. The dynamic changes in ommatidial structure during the dark/light adaptation indicate that the eye optimizes its vision in response to different ambient illuminations. The eye of M. alternatus exhibits anatomical and optical features indicative of an adaptation to a low light intensity environment. To achieve high absolute sensitivity at night, M. alternatus had to sacrifice some spatial resolution, so that its eye is thought to form blurred but high contrast images of its surrounding environment. The perpendicular orientation of the microvilli

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in the rhabdom of M. alternatus suggests that the beetle may be sensitive to polarized light.

Conflict of interest

We send you the manuscript titled“Morphological and optical features of superposition compound

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eye in Monochamus alternatus Hope (Coleoptera: Cerambycidae). The authors of this work are

Chao Wen,Tao Ma, Yang-Xiao Deng, Chuan-He Liu, Shi-Ping Liang, Junbao Wen, Cai Wang and

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Xiu-Jun Wen. The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in

Acknowledgments

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connection with the work submitted.

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We sincerely thank Jilei Huang and Xiaoxian Wu (Instrumental analysis & research center, South China Agricultural University) for valuable help with the assistance of the SEM and TEM preparation. This work was funded by the Special Fund for Forestry Science and Technology

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Innovation of Guangdong, China (Grant numbers: 2019KJCX016）.

References

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Abelleira, A., Picoaga, A., Mansilla, J.P., Aguin, O., 2011. Detection of Bursaphelenchus Xylophilus, causal agent of pine wilt disease on Pinus pinaster in northwestern Spain. Plant Dis. 95, 776–776. https://doi.org/10.1094/PDIS-12-10-0902.

Bouligand, Y., 1972. Twisted fibrous arrangements in biological materials and cholesteric mesophases. Tissue Cell, 4, 189–217. https://doi.org/10.1016/S0040-8166(72)80042-9. Baker, G.T., Ma, P.W.K., 2006. Morphology and number of ommatidia in the compound eyes of Solenopsis invicta, Solenopsis richteri, and their hybrid (Hymenoptera: Formicidae). Zool. Anz. 245, 121–12. https://doi.org/10.1016/j.jcz.2006.06.001.

11

Brammer, J.D., Stein, P.J., Anderson, R.A., 1978. Effect of light and dark adaptation upon the rhabdom in the compound eye of the mosquito. J. Exp. Zool. 206, 151–156. https://doi.org/10.1002/jez.1402060204. Chapman, R.F., 2007. The insects: structure and function. Academic Press, New York. pp. 132–141. Eguchi, E., 1999. Polarized light vision and rhabdom. In: Ed. by E Eguchi, Y. Tominaga, Atlas of arthropod sensory receptors. Springer, Tokyo, Berlin, New York, pp. 33–46. Exner, S., 1891. Die Physiologie Der facettirten Augen von Krebsen und Insecten: Eine Studie. Franz Deuticke, Leipzig Wien. pp. 1–38. Fischer, S., Meyer-Rochow, V.B., Mueller, C.H.G., 2015. Compound eye miniaturization in Lepidoptera:

a

comparative

morphological

analysis.

Zool.

95,

438–464.

ro of

https://doi.org/10.1111/azo.12041.

Acta

Giffard, B., David, G., Joubard, B., Piou, D., Jactel, H., 2017. How do sex and sexual maturation

influence the response of Monochamus galloprovincialis to host odours? J App Entomol. 141, 551–560. https://doi.org/10.1111/jen.12374.

-p

Gokan, N., Hosobuchi K., 1979. Ultrastructure of the compound eye of the Longicorn beetle, Prionus insularis Motschulsky ( Coleoptera:Cerambycidae). Kontyu. 47, 105–116.

(Coleoptera:

re

Groot, P., Nott, R., 2001. Evaluation of traps of six different designs to capture pine sawyer beetles Cerambycidae).

Agric.

Forestry

Entomol.

3,

107–111.

lP

https://doi.org/10.1046/j.1461-9563.2001.00087.x.

Horridge, G.A., 1975. Optical mechanisms of clear-zone eyes. In: The compound eye and vision of insects (GA Horridge, ed.). Clarendon Press, Oxford. pp. 225–298. G.A.,

1977.

Insects

which

na

Horridge,

turn

and

look.

Endeavour.

1,

7–17.

https://doi.org/10.1016/0160-9327(77)90004-7. Horridge, G.A., Giddings, C., 1971. Movement on dark-light adaptation in beetle eyes of the type.

Proc.

R.

Soc.

London,

Ser.

B.

179,

73–85.

ur

neuropteran

https://doi.org/10.1098/rspb.1971.0082.

Jo

Jia, L.P., Liang, A.P., 2015. Fine structure of the compound eyes of Callitettix versicolor (Insecta: Hemiptera). Ann. Entomol. Soc. Am. 108, 316–324. https://doi.org/10.1093/aesa/sav007.

Jia, L.P., Liang, A.P., 2017. An apposition compound eye adapted for nocturnal vision in the moth midge, Clogmia albipunctata, (Williston) (Diptera: Psychodidae). J. Insect Physiol. 98, 188– 198. https://doi.org/10.1016/j.jinsphys.c2017.01.006. Kobayashi, F., Yamane, A., Ikeda, T., 1984. The Japanese pine sawyer beetle as the vector of pine wilt

disease.

Annu.

Rev.

https://doi.org/10.1146/annurev.en.29.010184.000555.

Entomol.

29,

115–135.

12

Koyama, N., Gokan, N., Matsui, M., 1975. The compound eye of the longicorn beetles (Coleoptera: Cerambycidae) with estimation of the activities by its structure. Journal.ser.a.biology. 18, 1– 16． Land, M.F., 1981. Optics and vision in invertebrates. In H. Autrum, (Ed), Hand-book of sensory Physiology. Springer, Berlin. pp. 471–592. Land, M.F., 1984. The resolving power of diurnal superposition eyes measured with an ophthalmoscope. J. Comp. Physiol., A. 154, 515–533. https://doi.org/10.1007/bf00610166. Land, M.F., 1997.

Visual acuity in insects.

Annu. Rev.

Entomol. 42, 147-177.

https://doi.org/10.1146/annurev.ento.42.1.147. Liu, B., Xu, H.C., Meng, JG., Sun, J.H., Fan, J.T., 2012. Visual cues for the host-finding and mating

ro of

locations of Monochamus alternatus (Coleoptera:Cerambycidae). Journal of Zhejiang Agriculture and Forestry University. 29, 617–620 (in Chinese).

Mamiya, Y., 1998. History of pine wilt disease in Japan. Nematology. 20, 219–26. https://doi.org/10.1002/jez.1402440122.

-p

McIntyre, P., Caveney, S., 1985. Graded-index optics are matched to optical geometry in the

superposition eyes of scarab beetles. Philos. Trans. R. Soc. Lond. B. 311, 237–69.

re

https://doi.org/10.1098/rstb.1985.0153.

Meyer-Rochow, V.B., 1972. The eyes of Creophilus erythrocephalus F. and Sartallus signatus sharp (Staphylinidae: Coleoptera). Zeitschrift Für Zellforschung und Mikroskopische

lP

Anatomie. 39, 323–349. https://doi.org/10.1007/BF00307068. Meyer-Rochow, V.B., 1999. The compound eye: circadian rhythmicity, illumination and obscurity. In: Atlas of arthropod sensory receptors, eds. Eguchi E, and Tominaga Y, Springer Verlag,

na

Berlin. Tokyo. New York. pp. 97–125.

Meyer-Rochow, V.B., Gal, J. 2004. Dimensional limits for arthropod eyes with superposition optics.

ur

Vision Res. 44, 2213–2223. https://doi.org/10.1016/s0042-6989(04)00194-4. Meyer-Rochow, V.B., Horridge, G.A., 1975. The eye of Anoplognathus (Coleoptera, Scarabaeidae).

Jo

Proc. R. Soc. B.188, 1–30. https://doi.org/10.1098/rspb.1975.0001. Meyer-Rochow, V.B., Mishra, M., 2009. A six-rhabdomere, open rhabdom arrangement in the eye of the chrysanthemum beetle Phytoecia rufiventris: Some ecophysiological predictions based on eye anatomy. Biocell. 33, 115–120.

Meyer-Rochow, V.B., Waldvogel, H., 1979. Visual behaviour and the structure of dark and lightadapted larval and adult eyes of the New Zealand glowworm Arachnocampa luminosa (Mycetophilidae: Diptera). J. Insect Physiol. 25, 601–613. https://doi.org/10.1016/00221910(79)90076-3. Mishra, M., Meyer-Rochow V.B., 2006a. Eye ultrastructure in the pollen-feeding beetle,

13

Xanthochroa luteipennis (Coleoptera: Cucujiformia: Oedemeridae). J. Electron Microsc, 55, 289–300. https://doi.org/10.1093/jmicro/dfl035. Mishra, M., Meyer-Rochow, V.B., 2006b. Fine structure of the compound eye of the fungus beetle Neotriplax lewisi (Coleoptera, Cucujiformia, Erotylidae). Invertebr Biol. 125, 265–278. https://doi.org/10.2307/4124907. Morewood, W.D., Hein, K.E., Katinic, P.J., Borden, J.H., 2002. An improved trap for large woodboring insects, with special reference to Monochamus scutellatus (Coleoptera: Cerambycidae). Can J of Forest Res. 32, 519–525. https://doi.org/10.1139/X01-224. Moser, J.C., Reeve, J.D., Bento, S., Lucia, T., Cameron, R.S.; Heck, N.M., 2004. Eye size and behavior of day- and night-flying leafcutting ant alates. Proc. Zool. Soc. London. 264, 69–75.

ro of

https://doi.org/10.1017/S0952836904005527. Nilsson, D.E., 1989. Optics and evolution of the compound eye. In: Facets of vision. Stavenga, D.G., and Hardie, R.C. (eds.). Springer Verlag; Berlin, Heidelberg, New York. pp. 30–73.

Schwarz, S., Narendra, A., Zeil, J., 2011. The properties of the visual system in the Australian desert Melophorus

bagoti.

Arthropod

https://doi.org/10.1016/j.asd.2010.10.003.

Struct.

Dev.

40,

128–134.

-p

ant

Snyder, A.W., 1977. Acuity of compound eyes: physical limitations and design. J. Comp. Physiol.,

re

A. 116, 161–182. https://doi.org/10.1007/bf00605401.

Stavenga, D.G., 1992. Eye regionalization and spectral tuning of retinal pigments in insects. Trends

lP

in Neurosciences. 15, 213–218. https://doi.org/10.1016/0166-2236(92)90038-A. Stavenga, D.G., 2003. Angular and spectral sensitivity of fly photoreceptors. i. integrated facet lens 003-0390-6.

na

and rhabdomere optics. J. Comp. Physiol., A. 189, 189–202. https://doi.org/10.1007/s00359-

Toh, Y., Okamura, J.Y., 2007. Morphological and optical properties of the corneal lens and retinal structure in the posterior large stemma of the tiger beetle larva. Vision Res. 47, 1756–1768.

ur

https://doi.org/10.1016/j.visres.2007.02.023. Wachmann, E., 1977. Vergleichende Analyse der feinstrukturellen Organisation offenr Rhabdome

Jo

in den Augen der Cucujiformia (Insecta, Coleoptera), unter besonderer Berücksichtigung der Chrysomelidae. Zoomorphology. 88, 95–131. https://doi.org/10.1007/BF01880649.

Wachmann, E., 1979. Untersuchungen zur Feinstruktur der Augen von Bockkäfern (Coleoptera, Cerambycidae). Zoomorphology. 92, 19–48. https://doi.org/10.1007/BF00999833.

Warrant, E.J., 2004. Vision in the dimmest habitats on earth. J. Comp. Physiol., A. 190, 765–89. https://doi.org/10.1007/s00359-004-0546-z. Warrant, E.J., McIntyre, P.D., 1990. Limitations to resolution in superposition eyes. J. Comp. Physiol., A. 167, 785–803. https://doi.org/10.1007/bf00189768.

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Warrant, E.J., McIntyre, P.D., 1993. Arthropod eye design and the physical limits to spatial resolving power. Prog. Neurobiol. 40, 413–461. https://doi.org/10.1016/0301-0082(93)90017 Warrant, E.J., McIntyre, P.D., 1996. The visual ecology of pupillary action in superposition eyes. J. Comp. Physiol., A. 178, 75–90. https://doi.org/10.1007/bf00189592. Yang, H., Wang, B.X., Yang, W., Yang, C.P., Chen, Z.M., 2007. The role of vision in hostorientation and mate-finding behaviors of the white-striped longhorned beetle, Batocera lineolata. Entomol News. 126, 358–371. https://doi.org/10.3157/021.126.0504. Yang, Z.Q., Cao, L.M., Zhang, Y.L., Wang, X. Y., Zhan, M. K., 2014. A new egg parasitoid species (Hymenoptera: Pteromalidae) of Monochamus alternatus (Coleoptera: Cerambycidae), with

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notes on its biology. Ann. Entomol. Soc. Am. 107, 407–412. https://doi.org/10.1603/AN13150.

Fig. 1. External morphology of the compound eyes of M. alternatus female. (A) Female adult of M. alternatus: position of compound eye indicated by arrow. (B) SEM photograph of crescent shaped

eye. (C) SEM photograph of hexagonal facets in the centre of the eye. (D) SEM photograph of

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pentagonal and irregular located near the edge of the eye. AS, antennal socket; H, hexagonal facet;

Jo

ur

na

lP

re

P, pentagon facet.

Fig. 2. Relationship between body size and area of compound eye (A) and number of ommatidia (B).

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Fig. 3. Light micrographs at different levels of the compound eye of M. alternatus. (A) Longitudinal sections of the eye showing corneal lenses, cones, rhabdom layer and basement membrane. (B) Transverse sections through the eyes. (C) Transverse sections through the distal rhabdoms in the

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dark-adapted state and pigment particles sparsely around the rhabdoms. (D) Transverse sections through the distal rhabdoms in the light-adapted state, with fewer pigment grains around the rhabdoms. CO, corneal; C, cone; PPC, primary pigment cells; RH, rhabdom; BM, basement

Jo

ur

na

lP

re

membrane; CS, cuticular sheath; CN, cone cell. P, pigment particle.

Fig. 4. TEM micrographs through the distal region of the eye in M. alternatus. (A) Cornea (co) with laminations. (B) Transverse section of cuticular sheath (CS), surrounded by secondory pigment cells (PPC). (C) Transverse section of retinula cells, showing photoreceptive layer consisting of eight retinula cells: two central cells (R7-R8) surrounded by six peripheral cells R1-6. Arrows indicate microvillar direction. (D) Transverse section of photoreceptive layer, showing numerous secondary pigment cells and extensive endoplasmic reticulum (ER) in the retinular cells.

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Fig. 5. TEM micrographs of transverse sections at different levels of the compound eye. (A)

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Transverse section of the corneal lens, showing layers with differed electron densities. (B) Transverse section of quadripartite cone and membrane specializations (arrow). (C) Transverse section of rhabdom showing perpendicular microvillus orientation (arrows). (D) Transverse section

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of light-adapted rhabdom featuring circular outlines, and surrounded by numerous secondary pigment cells (SPC). OLU, outer lens unit; ILU, inner lens unit; CN, cone cell; NCN, nuclei of cone cell; CS, cuticular sheath; RH, rhabdom; SPC, secondary pigment cell; SPCN, nuclei of secondary

Jo

ur

na

lP

pigment cell; MT, mitochondrion.

Fig. 6. Schematic diagram for assumed image formation by the thick biconvex lens of M. alternatus. Yellow lines represent incident light. H, anterior principal point; H', posterior principal point. S,

17

distance to object from H; S', distance to image from H'. n0, n1 and n2, refractive indices of air, corneal lens and internal medium, respectively. A, object size; B, image size.

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Fig. 7. The structure of the cone and the position of pigment during dark/light adaptation in M. alternatus. (A) TEM micrograph of the light-adapted cone showing that the cone is long and possesses a conical shape. Note that the primary pigment cell with darker color located below the

cone. (B) TEM micrograph of the dark-adapted cone showing that the cone is compressed and discshaped. The primary pigment cell with lighter color located below the cone. (C) TEM micrograph

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of the dark-adapted rhabdom showing that there are fewer pigment cells in the central retinula cells

but mitochondrion. (D) TEM micrograph of the light-adapted rhabdom showing that the primary pigment cells full two central cells. CO, cornea; CN, cone cell; NCN: nuclei of cone cell; PPC,

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primary pigment cell; PPCN, nuclei of primary pigment cell; CS, cuticular sheath; SCN: nuclei of

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ur

na

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cone cell; N, nuclei of retinula cell; RH, rhabdom; MT, mitochondrion.

Fig. 8. Semi-schematic drawings of M. alternatus ommatidium. (A) Longitudinal section of ommatidium. (B) Transverse sectlon at cone. (C) Transverse sectlon at rhabdom. CO, cornea; C, cone; PPC, primary pigment cell; cuticular sheath; SPC, secondary pigment cell; RC, retinula cell; RH, rhabdom; RCN, retinula cell nuclei; BM, basement membrane.

18

Table 1. Measured histological and optical parameters of the compound eye of M. alternatus. The number of samples, the range and the average (mean ± SE) are given in each measurement. N

Symbol Unit

Range

Body size in males

5

-

mm

18.12-20.17

19.22 ± 0.37

Body size in females

50

-

mm

14.50-24.53

20.01 ± 0.63

Facet number in males

5

-

-

578-696

614.20 ± 19.92

Facet number in females

20

-

Compound area in males

5

-

Compound area in females

20

Facet diameter

150

-p

-

mm2

94.40-194.20

148.85 ± 7.48

D

μm

43.03-62.49

53.16 ± 0.34

489-712

mm2

134.20-161.17

141.62 ± 4.41

re

-

150

-

μm2

1703.08-3284.29 2447.33 ± 22.84

150

-

μm2

1462.90-3246.63 2287.25± 31.31

na

Pentagonal facet area

630.20 ± 17.05

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Hexagonal facet area

Average

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Parameter

30

-

μm

37.14-58.90

47.71 ± 0.85

Thickness of OLU

30

-

μm

2.05-21.54

10.19 ± 1.07

Thickness of ILU

30

-

μm

17.32-47.83

35.53 ± 1.38

30

-

μm

11.16-29.20

19.72 ± 0.84

Inner lens surface radius

10

r1

μm

9.90-12.86

12.85 ± 0.67

Outer lens surface radius

10

r2

μm

17.96-22.75

20.02 ± 0.67

Diameter of pigment granules prim

100

-

μm

0.28-1.26

0.77 ± 0.02

Diameter of pigment granules sec

100

-

μm

0.28-0.96

0.62 ± 0.01

Cone length in LA

20

-

μm

8.91-20.66

14.67 ± 0.51

ur

Thickness of cornea

Jo

Diameter of cornea

20

-

μm

4.10-6.80

5.57 ± 0.16

Diameter of distal tip rhabdom

30

d

μm

1.15-3.29

2.11 ± 0.12

Diameter of rhabdomere microvilli

30

-

μm

0.03-0.09

0.06 ± 0.003

Rhabdom cross-sectional area in LA

30

-

μm2

115.09-306.19

210.74 ± 8.86

Rhabdom cross-sectional area in DA

30

-

μm2

237.92-424.99

328.56 ± 8.17

Length cone

30

-

μm

11.28-27.26

21.04 ± 0.77

Eye radius (calculated)

10

R

μm

-

567.83

Mitochondrion diameter

30

-

μm

0.22-0.76

0.21 ± 0.02

Number of cone cells

4

-

-

-

Number of retinular cells

8

-

-

-

Focal length (calculated)

-

f

-

-

F-number (calculated)

-

F

-

-

-

△ρ

acceptance

angle

(calculated)

-

Eye parameter (calculated)

-

na ur Jo

-

49.90 0.94 2.42

△φ

degree -

5.34

P

μm·rad -

4.98

lP

Interommatidial angle (calculated)

degree -

re

Ommatidium

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Cone length in DA

-p

19