Transmission of Metarhizium anisopliae and Beauveria bassiana to adults of Kuschelorhynchus macadamiae (Coleoptera: Curculionidae) from infected adults and conidiated cadavers.

Kuschelorhynchus macadamiae is a major pest of macadamias in Australia, causing yield losses of up to 15%. Our previous studies have shown the weevil is susceptible to Beauveria bassiana and Metarhizium anisopliae. The aim of this study was to investigate horizontal transmission of both fungal species to healthy weevils from both infected adults and weevil cadavers. In a confined environment the mortality of healthy adults caused by the transmission of conidia from live fungus-infected adults was < 50%. Under similar experimental conditions, the mortality of healthy adults reached 100% when exposed to conidiated cadavers. However, when conidiated cadavers were used in more spacious environments (insect cages), the mortality of adults was < 80%. Using scanning electron microscopy, it was observed that all healthy adults had conidia attached to all external parts of the body. This suggests that although the conidia were readily transferred to the adults, the lower mortality in the larger insect cages could be the result of an unfavourable environmental factor such as low humidity. The presence of conidia attached to all the adults indicated that they did not show any discriminatory behaviour such as avoidance of conidiated cadavers infected by these two fungal species. The results from this study show that there is potential for enhanced control of adult K. macadamiae via transmission from either fungus-infected adults or conidiated cadavers and this could strengthen sustainable pest management in macadamias.

Revealing the respiratory system of the coffee berry borer (Hypothenemus hampei; Coleoptera: Curculionidae: Scolytinae) using micro-computed tomography.

The coffee berry borer (Hypothenemus hampei) is the most economically important insect pest of coffee globally. Micro-computed tomography (micro-CT) was used to reconstruct the respiratory system of this species for the first time; this is the smallest insect (ca. 2 mm long) for which this has been done to date. Anatomical details of the spiracles and tracheal tubes are described, images presented, and new terms introduced. The total volume and the relationship between tracheal lumen diameter, length and volume are also presented. The total length of the tracheal tubes are seventy times the length of the entire animal. Videos and a 3D model for use with mobile devices are included as supplementary information; these could be useful for future research and for teaching insect anatomy to students and the public in general.

Evolution of Insect Iridescence: Origins of Three-Dimensional Photonic Crystals in Weevils (Coleoptera: Curculionoidea).

A variety of photonic mechanisms give rise to iridescence and other structural colors in insects. In weevils (Coleoptera: Curculionoidea), iridescence is created by the most complex of these mechanisms, the three-dimensional photonic crystal. These self-assembling crystals take the form of triply periodic networks with single diamond or single gyroid symmetries and have been the subject of many descriptive studies based on individual species (often on a single specimen). To determine how these extraordinary nanostructures have evolved, we conduct the first comparative study of photonic crystals and setal nanostructure across Curculionoidea. By integrating structural data with newly available phylogenetic information, we demonstrate that-despite their widespread geographical and taxonomic distribution-three-dimensional photonic crystals appear to have evolved only once in weevils, in the common ancestor of a clade comprising the current subfamilies Entiminae and Cyclominae. Flattened, hollow setae with an unordered, spongy network in the lumen appear to be a necessary precursor to three-dimensional photonic crystals; we propose an evolutionary pathway by which this transformation has occurred.

Nanoindentation Properties and Finite Element Analysis of the Rostrum of Cyrtotrachelus buqueti Guer (Coleoptera: Curculionidae).

This work focuses on the application of nanoindentation measurements and the finite element method for analyzing the mechanical properties of the rostrum of the outstanding driller Cyrtotrachelus buqueti Guer. Nanoindentation tests were carried out to measure the Young's modulus and hardness of the rostrum, with the results for the "dry" samples being 13.886 ± 0.75 and 0.368 ± 0.0445 GPa, respectively. The values for the "fresh" samples showed no clear difference from those of the "dry" ones. Moreover, field observation was conducted to determine the motion behaviors of the rostrum on the weevil. Micro-computed tomography technology was employed to obtain structural information about the rostrum, using 9 µm slices. A real three-dimensional model of the rostrum was created using the MIMICS application. Finally, the mechanical properties of the rostrum were determined by finite element analysis. It was concluded that the rostrum provides an ideal biological template for the design of biocomposite materials and lightweight tube-shaped structures. The properties determined in this study can potentially be applied in different fields, such as in the design of automotive hybrid transmission shafts, helicopter tail drive shafts, robotic arms, and other sandwich structures in aerospace engineering.

Cuticle network and orientation preference of photonic crystals in the scales of the weevil Lamprocyphus augustus.

This paper reports the structural and optical investigations of the structural colour of the weevil Lamprocyphusaugustus The photonic crystal structure within the weevil's scales was investigated using sequential focused ion-beam milling and scanning electron microscopy imaging. We carefully analysed the reconstructed three-dimensional structure to determine the unit cell of the photonic crystal. It was found that the cuticle network of the cubic unit cell perfectly matches the previously reported diamond-based network. However, different results were obtained for the crystal orientations of the small crystal domains that comprise the entire photonic crystal structure in the scales: <111> directions are highly preferred along the surface normal of the scale. This finding explains the fact that the scale is almost uniformly coloured despite the multi-domain structure. It is confirmed experimentally and theoretically that the wavelength range of the reflection band corresponds to the gap of the photonic band.

Photonic crystal micro-pixelation and additive color mixing in weevil scales.

The origin of the brilliant near angle-independent coloration of the weevil Eupholus chevrolati was investigated by a combination of optical and electron microscopy tools, photonic band structure calculations, and color mixing analysis. Optical microscopy and scanning micro-spectroscopy revealed the presence of micrometer-sized red, yellow, green, and blue reflective pixels covering the entire exoskeleton of the weevil. Scanning electron microscopy in combination with focused ion beam milling showed that each micro-pixel consisted of a diamond-based photonic crystal structure and the different reflective colors were the result of different orientations of the photonic crystal. Color mixing analysis was used to study the collective behavior of the reflective micro-pixels. A pointillist, additive color-mixing scheme of the reflective photonic crystal micro-pixels was determined as the origin of the weevil's bright and near angle-independent yellow-green coloration.

Morphology and nanoindentation properties of mouthparts in Cyrtotrachelus longimanus (Coleoptera: curculionidae).

Microstructure and nanoindentation properties of the mouthparts for the skillful driller Cyrtotrachelus longimanus are presented. The composition and morphological examinations are made using light, fluorescent, scanning electron microscopy, and Energy Disperse Spectroscopy, respectively. Nanoindentation was carried out to measure the elastic modulus and the hardness of mouthparts. The hardness and modulus for "dry" samples is 0.202 ± 0.065 and 8.604 ± 0.838 GPa, respectively, and the values of "fresh" ones is 0.126 ± 0.0196 and 6.951 ± 0.065 GPa, respectively. These results are critical for analyze the drilling mechanism of the weevil and provide a biological template to inspire the biomimetic design.

Morphology of the Male Reproductive System and Spermiogenesis of Dendroctonus armandi Tsai and Li (Coleoptera: Curculionidae: Scolytinae).

Studying the reproductive attributes of pests is central to understanding their life cycle history and in crafting management strategies to regulate, if not bring down, their population below threshold levels. In this article, the morphology of the male reproductive tract, topology of the spermatozoa, and salient features of spermiogenesis in the Chinese white pine beetle, Dendroctonus armandi Tsai and Li was studied to provide baseline information for further pest management studies. Results showed that male reproductive tract of this species differs from those documented in other Coleopterans by having 20 testicular tubules in each testis and the presence of two types of accessory glands. The spermatozoon is seen having peculiar characteristics such as an "h"-shaped acrosomal vesicle with a "puff"-like expansion, one centriole, one large spongy body, and two accessory bodies. Despite with some morphological differences of the male reproductive organ, spermatogenesis in this organism is similar to other Coleopterans. Overall, detailed studies regarding the components of the primary male reproductive organ of this beetle species would expand the knowledge on the less-understood biology of Coleopteran pests and would help in designing regulatory measures to conserve endemic and indigenous pine trees in China.

Velcro-Like System Used to Fix a Protective Faecal Shield on Weevil Larvae.

The last instar larva and pupa of Eucoeliodes mirabilis (A. Villa & G. B. Villa, 1835) (Curculionidae: Ceutorhynchini) are described using drawings and SEM images and are compared and keyed with already described larvae of 58 other ceutorhynchinae taxa. The larval body has an effective combination of morphological adaptations that assist a unique biological defensive strategy. All larval stages of E. mirabilis feed ectophytically on leaves of Euonymus europaeus L. (Celastraceae), and the larval body is covered with a thick faecal shield. The fixation of this protective shield on the larval back is performed by a peculiar dorsal microsculpture composed of a dense carpet of microtrichia on the thorax and abdomen, which serves effectively as a velcro system. Because of this strategy, macrosetae on the larval and pupal body of E. mirabilis are completely reduced. Larvae of E. mirabilis also have distinct morphological adaptations for protecting the spiracles against intrusion of faeces and avoiding occlusion of the tracheal system: a) microtrichia around spiracles are slightly shorter, distinctly stronger and are arranged with high-density and in clusters and b) spiracles are protected by an external safety valve. This strategy of E. mirabilis larvae is unique, although somewhat similar to that of Criocerinae and Blepharida-group leave beetles (Galerucinae) (both Coleoptera: Chrysomelidae), but with distinctly different morphological adaptations.

Research of bionic design on tools with rostrum of cyrtotrachelus bugueti guer (coleoptera: curculionidae).

To improve the torsional resistance capability of multilayered composite pipes, a bionic design was presented based on the rostrum of Cyrtotrachelus Bugueti Guer. Morphology and mechanical properties of the weevil's rostrum were studied. Scanning electron microscope (SEM) was employed to observe the macro-/microstructure and inner structure. Nanoindentation was carried out to measure the elastic modulus and the hardness of the biological materials. Based on the structure of rostrum, a mathematical model of multilayered biomimetic pipe was established with the statistical methods. Then, the 3D biomimetic cylinder was built and the torsional resistance capability was analyzed using the finite element analysis. From the SEM and nanoindentation, it was found that the rostrum is a cylindrical hollow multilayered structure composed by three materials. In the transverse section of rostrum, the materials' moduli are 3.07, 3.15, and 8.64 GPa, the hardness are 172.71, 125.32, and 278.99 MPa, respectively. In the longitudinal section, the moduli are 3.01, 4.35, and 7.66 GPa, the hardness are157.46, 149.15, and 253.51 MPa, respectively. The results of simulation showed that the distributional type of lamination has a significant effect on the torsional resistance capability of multilayered structure. The biomimetic structure, which imitates the lamination of rostrum, is superior in the aspect of torsional resistance compared with the nonlaminated or uniform laminated structure. Additionally, the bionic method is feasible and efficient.

From insect scales to sensor design: modelling the mechanochromic properties of bicontinuous cubic structures.

Many of the three-dimensional photonic crystals occurring in the scales of insects have bicontinuous cubic structures. Their optical properties have been studied extensively, however little is known about their mechanical properties and their optical response under deformation. We demonstrated a mechanochromic effect by deforming the scales of a weevil and calculated the elastic, optical and mechanochromic (assuming homogeneous deformation) properties of the three types of bicontinuous cubic structures occurring in nature: P-structure (primitive), G-structure (gyroid) and D-structure (diamond). The results show that all investigated properties of these three structure types strongly depend on their geometry, structural parameters such as volume fractions of the two constituting phases and the directions of the incident light or applied stress, respectively. Interestingly, the mechanochromic simulation results predict that these structures may show blue-shift or even red-shift under compression along certain directions. Our results provide design guidelines for mechanochromic sensing materials operating in the elastic regime, including parameters such as sensitivity and direction of spectral shift.

A multilayer micromechanical model of the cuticle of Curculio longinasus Chittenden, 1927 (Coleoptera: Curculionidae).

Curculio longinasus Chittenden, 1927 (Coleoptera: Curculionidae), is a weevil species common throughout the southwestern United States that uses its rostrum - a very slender, curved, beak-like projection of the head - to excavate tunnels in plant organs (such as acorns) for egg laying (oviposition). Once the apical portion of the rostrum has been inserted into the preferred substrate for oviposition, the female begins rotating around the perimeter of the hole, elevating her head by extending the fore-legs, and rotating the head in place in a drilling motion. This action causes significant elastic deformation of the rostrum, which will bend until it becomes completely straight. To better understand the mechanical behavior of the cuticle as it undergoes deformation during the preparation of oviposition sites, we develop a comprehensive micro/macro model of the micromechanical structure and properties of the cuticle, spanning across all cuticular regions, and reliably mirroring the resultant macroscale properties of the cuticle. Our modeling approach relies on the use of multi-scale, hierarchical biomaterial representation, and employs various micromechanical schemata - e.g., Mori-Tanaka, effective field, and Maxwell - to calculate the homogenized properties of representative volume elements at each level in the hierarchy. We describe the configuration and behavior of this model in detail, and discuss the theoretical implications and limitations of this approach with emphasis on future biomechanical and comparative evolutionary research. Our detailed account of this approach can thereby serve as a methodological template for exploring the biomechanical behavior of new insect structures.

Fluorescent in situ hybridization for the localization of viruses, bacteria and other microorganisms in insect and plant tissues.

Methods for the localization of cellular components such as nucleic acids, proteins, cellular vesicles and more, and the localization of microorganisms including viruses, bacteria and fungi have become an important part of any research program in biological sciences that enable the visualization of these components in fixed and live tissues without the need for complex processing steps. The rapid development of microscopy tools and technologies as well as related fluorescent markers and fluorophores for many cellular components, and the ability to design DNA and RNA sequence-based molecular probes and antibodies which can be visualized fluorescently, have rapidly advanced this field. This review will focus on some of the localizations methods which have been used in plants and insect pests in agriculture, and other microorganisms, which are rapidly advancing the research in agriculture-related fields.

Spinosad Induces Antioxidative Response and Ultrastructure Changes in Males of Red Palm Weevil Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae).

The red palm weevil, Rhynchophorus ferrugineus, is of great concern worldwide, especially in the Middle East, where dates are a strategic crop. Despite their ecological hazard, insecticides remain the most effective means of control. A bioinsecticide of bacterial origin, spinosad is effective against several pests, and its efficacy against male R. ferrugineus was assessed in the present study. The antioxidative responses of key enzymes including catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST) to spinosad were investigated in the midgut and testes, and the effects of this insecticide on the cell ultrastructure of the midgut, Malpighian tubules, and testes were also determined. The lethal concentration 50 of spinosad was measured at 58.8 ppm, and the insecticide inhibited the activities of CAT, SOD, and GST in the midgut. However, no significant changes in the activities of these enzymes were observed in the testes. Spinosad treatment resulted in concentration-dependent changes in the cellular organelles of the midgut, Malpighian tubules, and testes of R. ferrugineus, and some of these effects were similar to those exerted by other xenobiotics. However, specific changes were observed as a result of spinosad treatment, including an increase in the number and size of concretions in Malpighian tubule cells and the occasional absence of the central pair of microtubules in the axonemes of sperm tails. This study introduces spinosad for potential use as an insecticide within an integrated control program against male red palm weevils. Additionally, the study provides biochemical and ultrastructural evidence for use in the development of bioindicators.

Micromorphology of the elytral cuticle of beetles, with an emphasis on weevils (Coleoptera: Curculionoidea).

The elytral cuticle of 40 beetle species, comprising 14 weevils (Curculionoidea) and 26 representatives of other taxa, is examined. All weevils and 18 other species have an endocuticle with prominent macrofibers, which corresponds to a modified pseudo-orthogonal cuticle. Angles between successive layers of macrofibers range between 30° and 90°, but are constantly less than 60° in weevils. In all Curculionoidea, as well as in one buprestid and one erotylid species exo- and endocuticle are densely interlocked. In the weevil Sitophilus granarius, transmission electron microscopy revealed vertical microfibrils extending from the exocuticle between the macrofibers of the underlaying endocuticle. Vertical microfibrils connecting successive macrofiber layers of the endocuticle were observed in S. granarius and Trigonopterus nasutus. Distinct cuticular characters are traced on a beetle phylogeny: the angles between unidirectional endocuticle layers; the presence and the shape of endocuticular macrofibers; and the interlocking of exo- and endocuticle. While character traits seem to be more or less randomly distributed among Coleoptera, the Curculionoidea have a uniform groundplan: The "weevil-specific" combination of characters includes 1) interlocking of exo- and endocuticle, 2) an endocuticle with distinct ovoid macrofibers embedded in a matrix and 3) comparatively small angles between successive endocuticular layers. Thus, phylogenetic constraints appear equally important to functional factors in the construction of the weevil elytron.

Comparative thorax morphology of death-feigning flightless cryptorhynchine weevils (Coleoptera: Curculionidae) based on 3D reconstructions.

The thorax morphology, especially the muscles and the tracheal system of three flightless species of Cryptorhynchinae is examined by digital 3D reconstructions based on synchrotron X-ray microtomography and compared to other Curculionidae. Wings, metanepisternites, and muscles functional in flight are fully reduced in the species examined: Kyklioacalles roboris (Curtis), Trigonopterus scharfi Riedel and Trigonopterus vandekampi Riedel. All three share the same set of thoracic muscles, but differences exist in the shape and size of muscles. Both Trigonopterus species examined have a conspicuous fan-shaped branch of Musculus mesosterni primus contracting pro- and mesothorax, interpreted as an adaption to their thanatosis defense strategy. Trigonopterus vandekampi furthermore shows a marked increase in the size of two metacoxal muscles, which may be functional in this species' thanatosis blocking mechanisms. The metathoracic spiracle of all Trigonopterus species is located at the side of the metaventrite externally and not in the subelytral space as in other beetles. It is hypothesized that this translocation was triggered by the need to improve oxygen supply during thanatosis, when both the mesothoracic spiracle and the subelytral cavity are tightly sealed from the outside.

Farming of a defensive fungal mutualist by an attelabid weevil.

The mutualism between fungus-growing animals and fungi is a classic example of a complex interspecies association. A handful of insects, notably the well-recognized fungus-farming ants, termites and beetles, have developed advanced agriculture, which includes seeding new gardens with crop propagules, improving growth conditions and protecting and harvesting the fungal crop. More examples, which could be called 'proto-fungiculture', involve fewer adaptations, as exemplified by marine snails that farm intertidal fungi on marsh grass. Recent work has indicated that the solitary leaf-rolling weevil Euops chinensis (family Attelabidae) has a protofarming symbiosis with the mycangial fungus Penicillium herquei (family Trichocomaceae). In this study, we investigated how the weevils create cradles (leaf-rolls) for their offspring and protect the fungal garden. We describe new specialized structures and behaviors that E. chinensis females use for leaf-rolling and fungus inoculation. The fungus P. herquei produces the antibiotic (+)-scleroderolide in laboratory culture and in leaf-rolls, which can serve to inhibit microbial 'weeds' and pests, thus protecting the fungal garden against potential infection. The fungiculture of E. chinensis differs from other advanced insect fungiculture systems because female weevils do not continuously tend the inoculated microbe and do not depend nutritionally on the fungus. The defensive role of the cultivated fungus makes the attelabid weevils exceptional in 'proto-fungiculture' animals.

Morphology of the male reproductive system, sperm ultrastructure and γ-irradiation of the red palm weevil Rhynchophorus ferrugineus Oliv. (Coleoptera: Dryophthoridae).

In the present study we describe the morphology of the male reproductive apparatus and sperm ultrastructure of the red palm weevil - an invasive pest of several palm tree species - as well as the most important steps of spermatogenesis. The reproductive apparatus consists of a pair of testes (each formed by two lobes) a long tube-like accessory gland, a prostate gland and a small accessory gland. Characteristic features of the sperm are 90-100 µm total length, 10 µm nucleus, two mitochondrial derivatives, two accessory bodies, one well-developed puff-like structure and a typical insect 9+9+2 flagellar axoneme. One of the methods used for the biological control of pests is the Sterile Insect Technique (SIT), for the evaluation of which we make a preliminary comparison of the sperm ultrastructure of non-irradiated and irradiated weevils (at a dose of 80 Gray).

Effect of allyl isothiocyanate on ultra-structure and the activities of four enzymes in adult Sitophilus zeamais.

Rarefaction and vacuolization of the mitochondrial matrix of AITC-treated (allyl isothiocyanate-treated) adult Sitophilus zeamais were evident according to the ultra-structural by TEM. Four important enzymes in adult S. zeamais were further studied after fumigation treatment with allyl isothiocyanate (AITC) extracted from Armoracia rusticana roots and shoots. The enzymes were glutathione S-transferase (GST), catalase (CAT), cytochrome c oxidase, and acetylcholinesterase (AChE). The results indicated that the activities of the four enzymes were strongly time and dose depended. With prolonged exposure time, treatment with 0.74µg/mL AITC inhibited the activities of cytochrome c oxidase, AChE, and CAT, but induced the activity of GST. The activities of cytochrome c oxidase, AChE, and CAT were remarkably induced at a low AITC dosage (0.25µg/mL), but were restrained with increased AITC dosage. The activity of GST was inhibited at a low AITC dosage (0.5µg/mL), but was induced at a high AITC dosage (1.5µg/mL). According to the results of TEM, toxic symptoms and enzymes activities, it suggested that mitochondrial maybe the one site of action of AITC against the adult S. zeamais and it also suggested that cytochrome c oxidase maybe one target protein of AITC against the adult S. zeamais, which need to further confirmed by protein function tested.

In vivo X-ray cine-tomography for tracking morphological dynamics.

Scientific cinematography using ultrafast optical imaging is a common tool to study motion. In opaque organisms or structures, X-ray radiography captures sequences of 2D projections to visualize morphological dynamics, but for many applications full four-dimensional (4D) spatiotemporal information is highly desirable. We introduce in vivo X-ray cine-tomography as a 4D imaging technique developed to study real-time dynamics in small living organisms with micrometer spatial resolution and subsecond time resolution. The method enables insights into the physiology of small animals by tracking the 4D morphological dynamics of minute anatomical features as demonstrated in this work by the analysis of fast-moving screw-and-nut-type weevil hip joints. The presented method can be applied to a broad range of biological specimens and biotechnological processes.