Integument Tissues Research Articles

Molecular Characterization of UDP-N-Acetylglucosamine Pyrophosphorylase and Its Role in the Growth and Development of the White-Backed Planthopper Sogatella furcifera (Hemiptera: Delphacidae).

UDP-N-acetylglucosamine pyrophosphorylase (UAP) is a key enzyme in the chitin biosynthesis pathway of insects. Here, we described the gene SfUAP in the white-backed planthopper Sogatella furcifera (Horváth) with an open reading frame of 1470 bp. Quantitative real-time polymerase chain reaction (qPCR) suggested that SfUAP exhibits a different developmental expression pattern and a higher expression after molting. The highest expression of SfUAP was observed in the integument tissues of adults, whereas head tissues showed negligible expression. RNAi-based gene silencing decreased the mRNA transcript levels in S. furcifera nymphs injected with double-stranded RNA of SfUAP. Finally, SfUAP silencing led to 84% mortality and malformed phenotypes in nymphs. Thus, our results can help better understand the role of SfUAP in S. furcifera.

RNAi-Mediated Silencing of the Chitinase 5 Gene for Fall Webworm (Hyphantria cunea) Can Inhibit Larval Molting Depending on the Timing of dsRNA Injection.

Simple SummaryThe fall webworm, Hyphantria cunea, is a worldwide invasive pest causing serious ecological and economic damage. The use of RNAi is a feasible strategy for controlling this pest. In this study, we evaluated the importance of the chitinase 5 gene (HcCht5) in the development of H. cunea larvae. We found that the RNAi-mediated silencing of HcCht5 arrested molting and caused larval mortality depending on the dsRNA injection time. The silencing of HcCht5 down-regulated genes that were related to chitin metabolism, molting hormone signaling, and detoxification metabolism. Our findings indicate that HcCht5 is an important gene in regulating larval development and a promising target for RNAi-mediated pest management of the pest H. cunea.Chitinases, which are crucial enzymes required for chitin degradation and reconstruction, are often selectively considered to be effective molecular targets for pest control due to their critical roles in insect development. Although the Hyphantria cunea chitinase gene has been reported previously, its sequence characteristics, gene function, and feasibility as a potential target for pest management were absent. In the present study, we characterized the H. cunea chitinase gene and designated it HcCht5. Phylogenic and domain structure analysis suggested that HcCht5 contained the typical chitinase features and was clustered into chitinase group I. Tissue-specific and developmental expression pattern analysis with Real-Time Quantitative PCR (RT-qPCR) showed that HcCht5 was mainly expressed in the integument tissues and that the transcript levels peaked during molting. RNA interference (RNAi)-mediated silencing of HcCht5 caused 33.3% (2 ug) and 66.7% (4 ug) mortality rates after double-stranded RNA (dsRNA) injection. Importantly, the interference efficiency of HcCht5 depended on the injection time of double-stranded RNA (dsRNA), as the pre-molting treatment achieved molt arrest more effectively. In addition, transcriptome sequencing (RNA-seq) analysis of RNAi samples demonstrated silencing of the down-regulated HcCht5 genes related to chitin metabolism and molting hormone signaling, as well as genes related to detoxification metabolism. Our results indicate the essential role of HcCht5 in H. cunea development and detail the involvement of its gene function in the larval molting process.

Polyphenol Profiling of Chestnut Pericarp, Integument and Curing Water Extracts to Qualify These Food By-Products as a Source of Antioxidants.

Plant polyphenols have beneficial antioxidant effects on human health; practices aimed at preserving their content in foods and/or reusing food by-products are encouraged. The impact of the traditional practice of the water curing procedure of chestnuts, which prevents insect/mould damage during storage, was studied to assess the release of polyphenols from the fruit. Metabolites extracted from pericarp and integument tissues or released in the medium from the water curing process were analyzed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray-quadrupole-time of flight-mass spectrometry (ESI-qTOF-MS). This identified: (i) condensed and hydrolyzable tannins made of (epi)catechin (procyanidins) and acid ellagic units in pericarp tissues; (ii) polyphenols made of gallocatechin and catechin units condensed with gallate (prodelphinidins) in integument counterparts; (iii) metabolites resembling those reported above in the wastewater from the chestnut curing process. Comparative experiments were also performed on aqueous media recovered from fruits treated with processes involving: (i) tap water; (ii) tap water containing an antifungal Lb. pentosus strain; (iii) wastewater from a previous curing treatment. These analyses indicated that the former treatment determines a 6–7-fold higher release of polyphenols in the curing water with respect to the other ones. This event has a negative impact on the luster of treated fruits but qualifies the corresponding wastes as a source of antioxidants. Such a phenomenon does not occur in wastewater from the other curing processes, where the release of polyphenols was reduced, thus preserving the chestnut’s appearance. Polyphenol profiling measurements demonstrated that bacterial presence in water hampered the release of pericarp metabolites. This study provides a rationale to traditional processing practices on fruit appearance and qualifies the corresponding wastes as a source of bioactive compounds for other nutraceutical applications.

Photophore Distribution and Enzymatic Diversity Within the Photogenic Integument of the Cookie-Cutter Shark Isistius brasiliensis (Chondrichthyes: Dalatiidae)

The cookie-cutter shark Isistius brasiliensis (Squaliformes: Dalatiidae) is a deep-sea species that emits a blue luminescence ventrally, except at the level of a black band located beneath the jaw. This study aims to (i) investigate the distribution and histology of the photophores (i.e., light-emitting organs) along the shark body, (ii) describe the tissue-specific transcriptomes of the black band integument region (i.e., non-photogenic) and the ventral integument region (i.e., photogenic), (iii) describe the repertoire of enzyme-coding transcripts expressed the two integument regions, and (iv) analyze the potential expression of transcripts coding for luciferase-like enzymes (i.e., close homologs of known luciferases involved in the bioluminescence of other organisms). Our analyses confirm the black band’s non-photogenic status and photophore absence within this region. The sub-rostral area is the region where the photophore density is the highest. In parallel, paired-end Illumina sequencing has been used to generate two pilot transcriptomes, from the black band and the ventral integument tissues of one individual. In total, 68,943 predicted unigenes have been obtained (i.e., 64,606 for the black band transcriptome, 43,996 for the ventral integument transcriptome) with 43,473 unigenes showing significant similarities to known sequences from public databases. BLAST search analyses of known luciferases, coupled with comparative predicted gene expression (i.e., photogenic versus non-photogenic), support the hypothesis that the species uses an unknown luciferase system. An enzymatic repertoire was predicted based on the PRIAM database, and Enzyme Commission numbers were assigned for all detected enzyme-coding unigenes. These pilot transcriptomes based on a single specimen, and the predicted enzyme repertoire, constitute a valuable resource for future investigations on the biology of this enigmatic luminous shark.

Carotenoid coloration and health status of urban Eurasian kestrels (Falco tinnunculus).

As the world experiences rapid urban expansion, natural landscapes are being transformed into cities at an alarming rate. Consequently, urbanization is identified as one of the biggest environmental challenges of our time, yet we lack a clear understanding of how urbanization affects free-living organisms. Urbanization leads to habitat fragmentation and increased impervious surfaces affecting for example availability and quality of food. Urbanization is also associated with increased pollution levels that can affect organisms directly, via ecophysiological constraints and indirectly by disrupting trophic interactions in multi-species networks. Birds are highly mobile, while an individual is not necessarily exposed to urban stressors around the clock, but nestlings of altricial birds are. Such a city-dwelling species with a long nestling phase is the Eurasian kestrel (Falco tinnunculus) in Vienna, Austria, which forage on a diverse diet differing in composition from rural habitats. Furthermore, prey items vary in nutritional value and contents of micronutrients like carotenoids, which might impact the nestlings’ health. Carotenoids are pigments that are incorporated into integument tissues but also have antioxidant and immunostimulatory capacity, resulting in a trade-off between these functions. In nestlings these pigments function in parent-offspring communication or sibling competition by advertising an individual’s physical or physiological condition. Anthropogenic disturbance and pollutants could have disruptive effects on the coloration of these traits. In this study, we measured carotenoid based coloration and other indicators of individual health (body condition and susceptibility to the ectoparasite Carnus hemapterus) of 154 nestling kestrels (n = 91 nests) along an urban gradient from 2010 to 2015. We found skin yellowness of nestlings from nest-sites in the city-center to be least pronounced. This result might indicate that inner-city nestlings are strongly affected by urban stressors and depleted their stores of dietary carotenoids for health-related functions rather than coloration. In addition, skin yellowness intensified with age and was stronger pronounced in earlier nests. Since the immune system of nestlings is still developing, younger chicks might need more antioxidants to combat environmental stress. Additionally, parasite infection intensity was highest in nestlings with less intense skin yellowness (paler or less yellow pigmented integuments) and in earlier nests of the season. In combination with results from previous studies, our findings provide further support for the low quality of the inner-city habitat, both in terms of productivity and individual health.

Ontogenia de la semilla de Yucca periculosa (Agavaceae)

Las semillas de algunas especies de Yucca han sido estudiadas por Arnott y Horner. Ellos se refieren principalmente al estado maduro y afirman que el tejido de reserva extraembrionario es un perispermo. En el presente trabajo se muestran evidencias ontogénicas de que se trata de un endospermo con desarrollo de tipo nuclear. La forma de la semilla se aproxima a un prisma triangular de poco menos de 1 cm de longitud, es de color negro y el surco de la rafe se observa fácilmente. La cubierta seminal deriva únicamente del tegumento externo. La pared periclinal externa de la exotesta aparece heterogéneamente engrosada. La mesotesta y enclotesta proliferan desigualmente hacia el interior de la semilla formando un ruminado en el endospermo. Hacia la madurez, desaparecen los derivados del tegumento interno ocasionando la fusión de la cutícula intertegumentaria con la tegmen-nucelar; éstas aportan información ontogénica valiosa, debido a su persistencia aún cuando el tejido que las originó hubiere desaparecido. El embrión acelera su desarrollo a las 10 semanas después de antesis, es cilíndrico, plegado en forma de 'S' y sus dos tercios calaza les corresponden al cotiledón.

카드뮴 노출이 붕어(Carassius auratus) 조직에 미치는 영향

본 연구는 Cd에 노출시킨 붕어를 통하여 각 조직에 축적된 Cd 양과 항산화효소 활성을 통해 생리적인 변화 및 아가미와 신장 조직에서의 미세구조 변화를 조사하였다. Cd을 처리한 실험군 붕어 조직 내 Cd의 함량은 아가미 조직에서 가장 높았고 근육 조직에서 가장 낮았다. 또한 모든 조직에서 Cd의 함량은 노출기간이 길어질수록 증가하여 노출 20일에 가장 높았다. 항산화효소의 활성은 아가미와 근육 조직에서 높았으며, 신장과 간 조직에서 낮았다. 모든 조직에서 SOD의 활성이 가장 높았으며, SOD와 CAT의 활성은 10일에 높은 활성을 보이고 감소하는 유사한 결과를 보였다. Cd에 20일간 노출시킨 붕어의 아가미 조직은 대조군 아가미 조직에 비하여 노출기간이 길어질수록 새변 사이에 점액세포의 수가 증가하였으며, 이차새변에서는 부종, 상피세포의 박리와 새변과 새변이 융합한 형태도 관찰되었다. 또한 아가미 조직의 미토콘드리아와 핵에서는 막의 손상이 확인되었다. 신장 조직은 대조군에서 보우만 주머니 안에 사구체가 꽉 찬 정상구조였으나 노출기간이 길어짐에 따라 사구체가 수축하여 보우만 주머니 안의 공간이 넓은 형태로 관찰되었다. The present study aimed to investigate the level of accumulated heavy metal in various tissues of Carassius auratus after exposure to Cadmium (Cd), histologically and physiologically. After treating C. auratus with Cd, the accumulated Cd in gill tissues was detected to be of the highest content, and showed the lowest content in integument tissues. Also, Cd content increased in a time dependent manner and showed the highest accumulation in the tissues exposed for 20 days. Antioxidant enzyme activities showedhigher activity in the gill and integument than in the kidney and liver tissues. In the case of SOD, antioxidation activity of SOD in all Cd exposed tissues was higher than in unexposed tissues. The activities of SOD and CAT also became higher after Cd exposure. Gill tissues exposed to Cd showed an increased number of mucous cells between lamella in a time dependent manner. In addition, the gills showed morphological changes such as edema, exfoliation of epithelial cells, and fusion of the secondary lamellae. Also, exposure to Cd for 20 days had an effect on gill tissues, causing membrane damage in the mitochondria and nucleus. In kidney tissues, atrophied glomerulus was observed, and the empty space in Bowman's capsule was wider.

Evaluating the potential of chestnut ( Castanea sativa Mill.) fruit pericarp and integument as a source of tocopherols, pigments and polyphenols

The chestnut fruit processing generates large amounts of residues as pericarp (outer shell; 8.9–13.5%) and integument (inner shell; 6.3–10.1%). These materials clearly have the potential as sources of valuable co-products. The analyses of the pericarp and integument of four Portuguese chestnut cultivars (Judia, Longal, Martaínha and Lada) revealed significant contents of total phenolics, low molecular weight phenolics (gallic and ellagic acid), condensed tannins and ellagitannins including castalagin, vescalagin, acutissimin A and acutissimin B. The integument tissues had the highest levels of total phenolics and condensed tannins. The most efficient extraction solvent for the total phenolics, total condensed tannins and low molecular weight phenolics (in Longal) was 70:30 acetone:water at 20 °C. The pericarp and integument tissues of the cultivar Longal were richest in gallic acid and castalagin. It is clear that these materials could be used for the extraction of valuable phenolics.

Truncation of a Protein Disulfide Isomerase, PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance inArabidopsis thaliana

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.

A nondestructive method for extracting maternally derived egg yolk carotenoids

Maternally deposited carotenoids are a prominent component of egg yolk and are vital for the development and growth of the embryo. In most studies of avian yolk carotenoids, eggs are destructively sampled and this may limit both the number of clutches studied and the research questions addressed. We describe an empirical field trial for a nondestructive biopsy method to extract small samples (0.05 ml) of egg yolk for high- performance liquid chromatography (HPLC) analysis of yolk carotenoid concentrations. We sampled 180 clutches (N = 44 biopsies) of two species of introduced thrushes (genus Turdus) from agricultural habitats in central North Island, New Zealand. Once the protocol was established, all biopsied eggs from clutches that were not depredated or deserted before candling were found to be developing normally after 3-5 d of incubation (N = 28) and all hatched. Biopsy samples (>0.02 g) produced concentrations of yolk carotenoids (and variances) that were statistically indistinguishable from whole yolk destructive samples. In addition, our samples (>0.02 g) confirmed previously reported differences in yolk carotenoid concentrations between the two thrush species and revealed a significant decline in yolk carotenoid concentration with laying order. Further examination of how variability in yolk carotenoid concentration and identity influences offspring sex, success, and survival or, later in life, reproductive success and ability to efficiently incorporate dietary carotenoids into both integument and immune tissues will require larger sample sizes. Studies to date have been restricted by the number of destructive samples that investigators are willing (or permitted) to obtain from wild species. Thus, we hope that our nondestructive method of sampling yolk will promote further examination of the links between carotenoid uptake from the environment and maternal investment in the avian yolk.

Comparative study on extracts from the tissue covering the stingers of freshwater ( Potamotrygon falkneri) and marine ( Dasyatis guttata) stingrays

Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), tissue extracts have similar bands above 80 kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A 2 and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted with several components of both tissue extracts, noticeably above 22 kDa. Both tissue extracts presented gelatinolytic, caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents.

Occurrence of paralytic shellfish poison (PSP) in the starfish Asterina pectinifera collected from the Kure Bay, Hiroshima Prefecture, Japan

Assays were made for paralytic toxicity of marine invertebrates inhabiting at the coasts of Hiroshima Bay, where the infestation of bivalves such as cultured oysters with paralytic shellfish poison (PSP) has been occurred. The starfish Asterina pectinifera collected at the estuary of Nikoh River, Hiroshima Bay, was found to contain moderate levels of paralytic toxicity. Its highest toxicities as PSP found on July 30, 1999 were 12.5 MU/g for whole body, 11.0 MU/g for integument tissues and 3.9 MU/g for viscera, respectively. The toxicity of integument was changed from 3.6 to 11.0 MU/g in 1 year. Its paralytic toxin principles were identified as PSP toxins, composing mainly from saxitoxin (STX) group toxins such as carbamoyl- N-hydroxy neosaxitoxin (hyneoSTX), and STX, by HPLC and LC–MS, accounting for over 90 mol%. The PSP toxins contained in the starfish A. pectinifera considered to be transferred from bivalves or detritus living in the same area, which were contaminated with PSP. However, the involved pathway may be different from that of Asterias amurensis which was infested directly through food chain from its food bivalves, for its toxin pattern.

Detection of sex and sex ratio in Calanus finmarchicus early stage fifth copepodites

Ability to determine sex ratios from developing copepodites collected in the field has, in the past, been limited to observations of overall gonad shape and size. This approach has been useful in distinguishing developing male and female fifth copepodites (C5s), but is limited to individuals in the final stages of gonad development. We have refined the approach by identifying and describing five distinct gonad types found in C. finmarchicus C5s and categorizing the early stages of sexual differentiation in the gonad on the basis of gonad shape and cell size. In Calanus ,t he first meiotic division of cells in the gonad occurs well before it has begun to take on the characteristic shape of an ovary or testis. Cell size can be seen easily after staining the gonads and clearing the integument and somatic tissues. Developing testes and ovaries can be recognized at an early stage by observing relative cell size anterior and posterior to the zone of first meiotic division. We used this technique to make preliminary determinations of the sex ratio in developing C. finmarchicus C5s on and around the Georges Bank during the 1998 GLOBEC Broadscale Survey. These data are compared with monthly sex ratios calculated from adult abundance from the same sample set. Results show that the seasonal patterns of C5 and adult sex ratios are similar, with a temporal offset attributed to developmental timing. In all samples, the proportion of developing male C5s is higher than the proportion of adult males, which we attribute to differential mortality between adult males and females. 2000 International Council for the Exploration of the Sea

Cloning and molecular analyses of a gibberellin 20-oxidase gene expressed specifically in developing seeds of watermelon.

To understand the biosynthesis and functional role of gibberellins (GAs) in developing seeds, we isolated Cv20ox, a cDNA clone from watermelon (Citrullus lanatus) that shows significant amino acid homology with GA 20-oxidases. The complementary DNA clone was expressed in Escherichia coli as a fusion protein, which oxidized GA(12) at C-20 to the C(19) compound GA(9), a precursor of bioactive GAs. RNA-blot analysis showed that the Cv20ox gene was expressed specifically in developing seeds. The gene was strongly expressed in the integument tissues, and it was also expressed weakly in inner seed tissues. In parthenocarpic fruits induced by 1-(2-chloro-4-pyridyl)-3-phenylurea treatment, the expression pattern of Cv20ox did not change, indicating that the GA 20-oxidase gene is expressed primarily in the maternal cells of developing seeds. The promoter of Cv20ox was isolated and fused to the beta-glucuronidase (GUS) gene. In a transient expression system, beta-glucuronidase staining was detectable only in the integument tissues of developing watermelon seeds.

LOCALIZATION OF PHYTOCHROME DURING PEANUT (ARACHIS HYPOGAEA) GYNOPHORE AND OVULE DEVELOPMENT

Previous studies indirectly indicated that phytochrome plays a role in peanut (Arachis hypogaea L. cv. Virginia) gynophore elongation and in ovule and embryo development. Recent advances in the use of monoclonal antibody procedures used in this study have allowed precise localization of phytochrome in the developing peanut gynophore and ovular tissues. Peanut phytochrome from etiolated tissues was found to have a molecular weight of 124 kD as determined by immunoblotting procedures using a monoclonal antibody to pea (Pisum sativum L. cv. Alaska) phytochrome. Immunoblotting procedures revealed that no detectable phytochrome was present in the gynophore tissues or immature ovules during the elongation of the peanut gynophores. After the gynophores penetrated the soil for 8–12 d, phytochrome was detected in increasing amounts in the ovular tissues but not the gynophore tissues. Immunohistological analysis revealed that phytochrome was localized in the developing embryo and adjacent integument tissues. These findings contradict earlier reports that suggested phytochrome was initially present in the gynophore tissues after fertilization where it was believed to inhibit ovular development and stimulate gynophore elongation.

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