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The role of the carapace in the uptake and storage of newly accumulated metals was investigated in the green crab exposed to environmentally relevant concentrations of calcium ([Ca] = 389 mg L-1 or 9.7 mmol L-1), zinc ([Zn] = 82 µg L-1 or 1.25 µmol L-1), and nickel ([Ni] = 8.2 µg L-1 or 0.14 µmol L-1) in 12 °C seawater, using radio-tracers (45Ca, 65Zn, 63Ni). After 24-h exposure, carapace exhibited the highest concentration of newly accumulated Ca, whereas carapace and gills exhibited the highest concentrations of both newly accumulated Zn and Ni relative to other tissues. For all three metals, the carapace accounted for >85 % of the total body burden. Acute temperature changes (to 2 °C and 22 °C) revealed the highest overall temperature coefficient Q10 (2.15) for Ca uptake into the carapace, intermediate Q10 for Ni (1.87) and lowest Q10 (1.45) for Zn. New Ca uptake into the carapace continued linearly with time for 24 h, new Zn uptake gradually deviated from linearity, whereas Ni uptake reached a plateau by 6 h. Attachment of a rubber membrane to the dorsal carapace, thereby shielding about 20 % of the total crab surface area from the external water, eliminated both new Zn and Ni incorporation into the shielded carapace, whereas 36 % of new Ca incorporation persisted. When recently euthanized crabs were exposed, new Zn uptake into the carapace remained unchanged, whereas Ca and Ni uptake were reduced by 89 % and 71 %, respectively. We conclude that the carapace is a very important uptake and storage site for all three metals. All of the uptake of new Zn and new Ni, and most of the uptake of new Ca into this tissue comes directly from the external water. For Zn, the mechanism involves only physicochemical processes, whereas for Ca and Ni, life-dependent processes make the major contribution.

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Insect cuticular lipids, especially epicuticular hydrocarbons (CHC), have a significant role in insect ecology and interactions with other organisms, including fungi. The CHC composition of a specific insect species may influence the outcome of the interaction with a specific fungal strain. Some insects, such as Piezodorus guildinii, have low susceptibility towards fungal infections seemingly due to their CHC composition. The entomopathogenic fungus Beauveria bassiana can assimilate CHC and incorporate them as building blocks via cytochrome P450 monooxygenases (CYPs). However, little is known about other enzymes that promote the degradation/assimilation of these cuticular components. In this study, we performed a transcriptomic analysis to evaluate the in vitro response of two virulence-contrasting B. bassiana strains when grown on three different P. guildinii CHC sources. We found a different expression profile of virulence-related genes, as well as different GO and KEGG parameters enriched at 4 days post-inoculation, which could help account for the intrinsic virulence and for an alkane-priming virulence enhancement effect. The hypovirulent strain predominantly showed higher expression of cuticle penetration genes, including chitinases, proteases, and CYPs, with GO term categories of "heme binding," "monooxygenase activity," and "peroxisome" pathways enriched. The hypervirulent strain showed higher expression of cell wall remodeling and cell cycle genes, and cuticle adhesion and a distinct set of CYPs, with GO categories of "DNA-binding transcription factor activity" and KEGG pathways corresponding to "meiosis-yeast" and "cell cycle" enriched. These results suggest a delay and alternate routes in pathogenicity-related metabolism in the hypovirulent strain in comparison with the hypervirulent strain. KEY POINTS: •Transcriptomics of two B. bassiana strains grown in P. guildinii cuticular components •Virulence-related genes correlated with virulence enhancement towards P. guildinii •Differentially expressed genes, GOs and KEGGs showed different metabolic timelines associated with virulence.

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Infective conidia from entomopathogenic fungi are widely used to control insect pests. Many entomopathogenic fungi also produce yeast-like cells called blastospores under specific liquid culture conditions that can directly infect insects. However, little is known about the biological and genetic factors that allow blastospores to infect insects and make them potentially effective for biological control in the field. Here, we show that while the generalist Metarhizium anisopliae produces a higher number of and smaller blastospores, the Lepidoptera specialist M. rileyi produces fewer propagules with a higher cell volume under high-osmolarity conditions. We compared the virulence of blastospores and conidia of these two Metarhizium species towards the economically important caterpillar pest Spodoptera frugiperda. Conidia and blastospores from M. anisopliae were equally infectious, but acted slower, and killed fewer insects than M. rileyi conidia and blastospores did, where M. rielyi conidia had the highest virulence. Using comparative transcriptomics during propagule penetration of insect cuticles, we show that M. rileyi blastospores express more virulence-related genes towards S. frugiperda than do M. anisopliae blastospores. In contrast, conidia of both fungi express more virulence-related oxidative stress factors than blastospores. Our results highlight that blastospores use a different virulence mechanism than conidia use, which may be explored in new biological control strategies.

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The industrial uses of peptidases have already been consolidated; however, their range of applications is increasing. Thus, the biochemical characterization of new peptidases could increase the range of their biotechnological applications. In silico analysis identified a gene encoding a putative serine peptidase from Purpureocillium lilacinum (Pl_SerPep), annotated as a cuticle-degrading enzyme. The Pl_SerPep gene product was expressed as a recombinant in a Komagataella phaffii (previously Pichia pastoris) expression system. The enzyme (rPl_SerPep) showed optimal pH and temperature of 8.0 and 60 °C, respectively. Moreover, rPl_SerPep has a higher thermal stability than the cuticle-degrading enzymes described elsewhere. The structural analysis indicated a conformational change in the rPl_SerPep secondary structure, which would allow an increase in catalytic activity at 60 °C. Komagataella phaffii secretes rPl_SerPep with the pro peptide in its inactive form. Low-resolution small-angle X-ray scattering (SAXS) analysis showed little mobility of the pro peptide portion, which indicates the apparent stability of the inactive form of the enzyme. The presence of 20 mM guanidine in the reaction resulted in the maintenance of activity, which was apparently a consequence of pro peptide structure flexibilization.

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Rhyzopertha dominica is one of the most important stored grain pests that seriously damage rice and wheat. At present, the method of controlling stored grain pests mainly relies on insecticide fumigation. However, the excessive use of pesticides not only leaves pesticide residues, with harmful effects on human health and the environment, but also induces insect resistance. Ozone is a strong oxidant with the characteristics of easy decomposition and without residue. Although ozone has been widely used in the food industry in recent years, research on the control of stored grain pests is limited. In this research, we used ozone treatment to control R. dominica adults and explore the molecular mechanisms that affect them. Here, we found that ozone treatment on R. dominica adults could decrease life span and increase malondialdehyde (MDA) content, as well as reduce activity of total superoxide dismutase (SOD) and catalase (CAT). Using RNA-seq technology, we identified 641 genes that were differentially expressed between ozone-treated and control R. dominica adults [fold-change of ≥ 2 (q-value < 5%)]. When comparing ozone treatment with control R. dominica adults, 330 genes were significantly upregulated and 311 were downregulated. RT-qPCR confirmed that 11 genes were differentially expressed in ozone-treated and control R. dominica adults. These genes were involved in insect cuticle protein and antioxidant system. This research showed that ozone treatment could reduce the lifespan of R. dominica through antioxidant system. It is an environmentally benign method for the control of stored grain pests and has great development potential.

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The development of a hydrophobic cuticle covering the epidermis was a crucial evolutionary novelty ensuring the establishment of land plants. However, there is little information about its structure and chemical composition, as well as its functional implications in avascular lineages such as Anthocerotophyta. The main goal of the present study was to compare the gametophyte and sporophyte cuticles of Phaeoceros laevis. Semithin sections were analyzed through light microscopy (LM), cuticle structure was evaluated by transmission electron microscopy (TEM) and epicuticular wax morphology was analyzed by scanning electron microscopy (SEM). Total waxes were analyzed by CG/MS, and the components were identified based on the mass spectra. A thin lipophilic layer was detected on the sporophyte surface, structured as a stratified cuticular layer, similar to the well-known structure described for vascular plants. On the other hand, the gametophyte cuticle was observed only with TEM as a thin osmiophilic layer. SEM analyses showed a film-type wax on the surface of both life phases. The wax layer was eight-fold thicker on the sporophyte (0.8 µg cm-2) than on gametophyte (0.1 µg cm-2). Possible mechanical and/or drought protection are discussed. Fatty acids, primary alcohols, and steroids were identified in both life phases, while the kauren-16-ene diterpene (3%) was detected only on the sporophyte. Although no alkanes were detected in P. laevis, our findings unveil great similarity of the sporophyte cuticle of this hornwort species with the general data described for vascular plants, reinforcing the conservative condition of this character and supporting the previous idea that the biosynthetic machinery involved in the synthesis of wax compounds is conserved since the ancestor of land plants.

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Exposure to sublethal doses of insecticide may affect biological traits in triatomines. We investigated the effects of toxicological phenotype (pyrethroid resistance status) and exposure to sublethal doses of deltamethrin on two traits of Triatoma infestans Klug (Heteroptera: Reduviidae) using a phenotypic plasticity experimental design. First-instar nymphs from 14 and 10 full-sib families from pyrethroid-susceptible and pyrethroid-resistant populations, respectively, were used. For the susceptible population, we treated first instars topically with acetone (control) or deltamethrin (treatment) once. For the resistant population, instars were treated once, twice and three times as first, third or fifth-instar nymphs, respectively. We measured cuticle thickness, wing size and wing shape of 484 emerging adults, and tested for treatment effects using mixed ANOVA and MANOVA models. Toxicological phenotype, exposure to deltamethrin and full-sib family exerted significant effects on cuticle thickness, wing size and wing shape. Adult triatomines previously treated with deltamethrin developed significantly thicker cuticles than control triatomines only in the resistant population and significantly bigger wings in both populations. Mean cuticle thickness and wing size increased with increasing exposures to deltamethrin. Exposure to sublethal doses of deltamethrin generated morphological modifications that may affect insect survival and flight dispersal, and hence may have evolutionary and epidemiological consequences.

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Fleshy fruits represent a valuable resource of economic and nutritional relevance for humanity. The plant cuticle is the external lipid layer covering the nonwoody aerial organs of land plants, and it is the first contact between fruits and the environment. It has been hypothesized that the cuticle plays a role in the development, ripening, quality, resistance to pathogen attack and postharvest shelf life of fleshy fruits. The cuticle's structure and composition change in response to the fruit's developmental stage, fruit physiology and different postharvest treatments. This review summarizes current information on the physiology and molecular mechanism of cuticle biosynthesis and composition changes during the development, ripening and postharvest stages of fleshy fruits. A discussion and analysis of studies regarding the relationship between cuticle composition, water loss reduction and maintaining fleshy fruits' postharvest quality are presented. An overview of the molecular mechanism of cuticle biosynthesis and efforts to elucidate it in fleshy fruits is included. Enhancing our knowledge about cuticle biosynthesis mechanisms and identifying specific transcripts, proteins and lipids related to quality traits in fleshy fruits could contribute to the design of biotechnological strategies to improve the quality and postharvest shelf life of these important fruit crops.

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Pigmentation in insects has been linked to mate selection and predator evasion, thus representing an important aspect for natural selection. Insect body color is classically associated to the activity of tyrosine pathway enzymes, and eye color to pigment synthesis through the tryptophan and guanine pathways, and their transport by ATP-binding cassette proteins. Among the hemiptera, the genetic basis for pigmentation in kissing bugs such as Rhodnius prolixus, that transmit Chagas disease to humans, has not been addressed. Here, we report the functional analysis of R. prolixus eye and cuticle pigmentation genes. Consistent with data for most insect clades, we show that knockdown for yellow results in a yellow cuticle, while scarlet and cinnabar knockdowns display red eyes as well as cuticle phenotypes. In addition, tyrosine pathway aaNATpreto knockdown resulted in a striking dark cuticle that displays no color pattern or UV reflectance. In contrast, knockdown of ebony and tan, that encode N-beta-alanyl dopamine hydroxylase branch tyrosine pathway enzymes, did not generate the expected dark and light brown phenotypes, respectively, as reported for other insects. We hypothesize that R. prolixus, which requires tyrosine pathway enzymes for detoxification from the blood diet, evolved an unusual strategy for cuticle pigmentation based on the preferential use of a color erasing function of the aaNATpreto tyrosine pathway branch. We also show that genes classically involved in the generation and transport of eye pigments regulate red body color in R. prolixus. This is the first systematic approach to identify the genes responsible for the generation of color in a blood-feeding hemiptera, providing potential visible markers for future transgenesis.

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RESUMEN El objetivo de este estudio fue evaluar los efectos de diferentes niveles de dureza total del agua en la muda, calcificación del exoesqueleto, crecimiento y supervivencia de Cryphiops caementarius. Los camarones machos fueron colectados del río Pativilca (Lima, Perú) y cultivados en recipientes individuales dentro de acuarios (55 L). Se emplearon cuatro niveles de dureza total del agua (100, 200, 300 y 400 mg CaCO3 L-1), con tres repeticiones, respectivamente. Los camarones cultivados en agua con dureza de 300 mg L-1 tuvieron menor periodo de muda (26,7 días) y mayor frecuencia de mudas (tres mudas). El contenido de calcio del exoesqueleto del camarón incrementó (p < 0,05) de 25 a 31 % en agua con dureza de 100 y 400 mg L-1, respectivamente. El mayor grosor del exoesqueleto (144 -jm en cefalotórax y 131 μm en abdomen) fue obtenido en agua con dureza de 400 mg L-1 y el menor grosor (93 -jm en cefalotórax y abdomen) en 100 mg L-1. El crecimiento en longitud fue mayor (p < 0,05) en agua con durezas de 200 y 300 mg L-1. El crecimiento en peso fue similar (p > 0,05) entre tratamientos. La mayor supervivencia (> 94,4 %) se mantuvo en agua con durezas de hasta 300 mg L-1 y la menor supervivencia (77,8 %) fue con 400 mg L-1. La dureza total del agua de 200 y 300 mg L-1 es conveniente para el cultivo del camarón, pero dureza del agua mayor o menor a este rango afectan la muda, el crecimiento y la supervivencia por deficiencia o exceso de calcio acumulado, respectivamente en el exoesqueleto del camarón.

ABSTRACT The aim of this work was to evaluate the effects of different levels of total hardness on the molting, calcification of exoskeleton, growth and survival of Cryphiops caementarius prawn. Male prawns were collected from Pativilca river (Lima, Peru) and cultivated in individual containers inside aquariums (55 L). Four levels of the total hardness of water (100, 200, 300 and 400 mg CaCO3 L-1) were used, with three repetitions, respectively. Prawns cultured in water with hardness of 300 mg L-1 has a shorter molting period (26.7 days) and a higher frequency of molts (three molts). The calcium content of the prawn exoskeleton increased (p < 0.05) from 25 to 31 % in water with hardness of 100 and 400 mg L-1, respectively. The greatest thickness of the exoskeleton (144 -jm cephalothorax and 131 -jm abdomen) was obtained in water with a hardness of400 mg L-1 and the smallest thickness (93 -jm in the cephalothorax and abdomen) in 100 mg L-1. The growth in length was greater (p < 0.05) in water with hardness of 200 and 300 mg L-1. The weight growth was similar (p > 0.05) between treatments. The highest survival (> 94.4 %) was maintained in water with hardness up to 300 mg L-1 and the lowest survival (77.8 %) was at 400 mg L-1. The total hardness of 200 and 300 mg L-1 is suitable for prawn farming, but hardness high or lower than this range affects the molting, growth and survival due to deficiency or excess of accumulated calcium, respectively, in the prawn exoskeleton.