Differentiation of Non-articulated Laticifers in Poinsettia (Euphorbia pulcherrima Willd.)

Differentiation of non-articulated laticifers in poinsettia(Euphorbia pulcherrima Willd.) was studied ultra-structurally.Growing laticifers show: (1) a multinucleate apical region containingabundant ribosomes but few other differentiated organelles and(2) a sub-apical zone where the cytoplasm is dominated by vacuolesof diverse morphology with latex particles. These particlesappear first within narrow tubular vacuoles developed especiallyin the peripheral cytoplasm. During vacuolation of the laticifer,portions of cytoplasm, including some of the nuclei, becomeisolated by the enlarging and fusing vacuoles; eventually thesebecome lysed, except the latex particles which remain in thecentral vacuole. During differentiation of a laticifer branch,the cytoplasm contains the usual organelles, including a fewmicrobodies and coated vesicles. The plastids that lie withinthe peripheral cytoplasm differentiate into amyloplasts witha single elongated starch grain. Towards the end of differentiationthe cytoplasm becomes restricted to a thin parietal layer, withthe remaining organelles reduced or degenerate, surroundinga central vacuole filled with latex particles. Euphorbia pulcherrima Willd, poinsettia, ultrastructure, differentiation, laticifers     

The chemical nature of the defensive larval secretion of the citrus swallowtail, Papilio demodocus

The eversible cervical gland or osmeterium of the larva of the African citrus swallowtail, Papilio demodocus Esper, produces a secretion containing isobutyric acid and 2-methyl-butyric acid as well as small quantities of the methyl and ethyl esters of these acids. The secretion of final instar larvae was found to differ from that of younger larvae.     

Girdling by notodontid caterpillars: distribution and occurrence

Girdling insects produce a circular groove around a stem or petiole typically before ovipositing or feeding on the distal section. We documented that seven of the 11 species of notodontid caterpillars that we studied, including members of five genera in two subfamilies, chewed girdles in seven families of trees including species of birch, hickory, oak, elm, cherry, willow, and maple. The frequency of girdling varied with notodontid species, tree species, month, and year. Free-ranging final instar larvae of Schizura leptinoides on pecan (Carya illinoensis) spent 4?C11?% of their time cutting and reinspecting girdles over 5-h and 12-h observation periods. Feeding occurred proximal as well as distal to girdles, a result not expected by most hypotheses for the function of girdling. Histological examination of S. leptinoides girdles on river birch (Betula nigra) revealed that only the xylem remained intact; however, on pecan, both the xylem and phloem remained mostly uncut by the girdle. S. leptinoides larvae often rubbed their mouthparts over the surface of finished girdles, anointing them with fluid from the labial salivary glands or possibly the ventral eversible gland. After feeding, S. leptinoides and other notodontids sometimes severed the petiole even when the leaf was only partially eaten; these leaf-clipping larvae were similarly observed rubbing their mouthparts over the severed petiole stub. We propose that notodontids cut girdles and clip leaves at least in part to introduce secretions or their enzymatic products into the vascular system to suppress host defenses and/or enhance nutrition.     

CHARACTERIZATION OF STARCH GRAINS IN THE NONARTICULATED LATICIFER OF EUPHORBIA PULCHERRIMA (POINSETTIA)

Starch grain morphology in laticifer amyloplasts of Euphorbia pulcherrima Willd. (poinsettia) was examined for evidence of starch metabolism in vegetative and flowering plants. Laticifer starch grains in vegetative plants were rod shaped with lengths ranging from 3 to 60 μm. Average grain size was significantly larger in stems than leaves, and in older than younger tissues. Starch grain length frequency was unimodal and approximated a normal probability distribution in stems, but was skewed positively toward smaller grains in leaves. Frequency distributions were shifted toward larger grains in older tissues. Under short-day photoperiod (flowering) conditions, round starch grains formed in latex of stems, and the average length of rod-shaped grains decreased in latex of stems and leaves. Round grains did not occur in laticifers of leaves or bracts. Round starch grains often occurred in aggregates of two or more subunits. Changes in size and shape of latex starch grains indicate that amyloplasts in fully differentiated laticifers metabolize starch. Identification of metabolically active amyloplasts in differentiated laticifers suggests that the function of these organelles may involve starch mobilization under certain physiological conditions.     

Distribution and Organization of Non-articulated Laticifers in Mature Tissues of Poinsettia (Euphorbia pulcherrima Willd.)

The distribution and cytological organization of non-articulatedbranched laticifers in the mature root, stem, and leaf tissuesof poinsettia. Euphorbia pulcherrima Willd., were studied bythe use of optical and electron microscopy. The laticifers occurin all parts of the plant body, being well represented in certainparenchymatous tissues and the phloem. The mature region ofthe laticifer has a living protoplast showing a thin parietalcytoplasm, bounded by plasmalemma and a tonoplast, which enclosesa large continuous central vacuole containing the milky latexfluid. The protoplast is multinucleate and possesses large amyloplasts,each enclosing a single elongated starch grain. Sparseness andpoor differentiation of the other components of the protoplast,mostly mitochondria, ribosomes and endoplasmic reticulum, suggestlow metabolism in the mature region of the laticifer. The latexof the central vacuole is dominated by spherical particles,0.3–1 µm in diameter, each with a dense matrix andan eccentric core of lighter staining material. In some laticifersthe latex particles fuse into coagulated masses. Euphorbia pulcherrima Willd, poinsettia, laticifers, ultrastructure, cytology     

Sucrose importation into laticifers of Hevea brasiliensis, in relation to ethylene stimulation of latex production

Background and Aims

The major economic product of Hevea brasiliensis is a rubber-containing cytoplasm (latex), which flows out of laticifers (latex cells) when the bark is tapped. The latex yield is stimulated by ethylene. Sucrose, the unique precursor of rubber synthesis, must cross the plasma membrane through specific sucrose transporters before being metabolized in the laticifers. The relative importance of sucrose transporters in determining latex yield is unknown. Here, the effects of ethylene (by application of Ethrel^®) on sucrose transporter gene expression in the inner bark tissues and latex cells of H. brasiliensis are described.

Methods

Experiments, including cloning sucrose transporters, real time RT-PCR and in situ hybridization, were carried out on virgin (untapped) trees, treated or untreated with the latex yield stimulant Ethrel.

Key Results

Seven putative full-length cDNAs of sucrose transporters were cloned from a latex-specific cDNA library. These transporters belong to all SUT (sucrose transporter) groups and differ by their basal gene expression in latex and inner soft bark, with a predominance of HbSUT1A and HbSUT1B. Of these sucrose transporters, only HbSUT1A and HbSUT2A were distinctly increased by ethylene. Moreover, this increase was shown to be specific to laticifers and to ethylene application.

Conclusion

The data and all previous information on sucrose transport show that HbSUT1A and HbSUT2A are related to the increase in sucrose import into laticifers, required for the stimulation of latex yield by ethylene in virgin trees.Key words: Hevea brasiliensis, laticifers, latex production, ethylene, sucrose transporters     

Hormonal treatment of the bark of rubber trees (Hevea brasiliensis) increases latex yield through latex dilution in relation with the differential expression of two aquaporin genes

Natural rubber is synthesized in laticifers in the inner liber of the rubber tree (Hevea brasiliensis). Upon bark tapping, the latex is expelled due to liber turgor pressure. The mature laticifers are devoid of plasmodesmata; therefore a corresponding decrease in the total latex solid content is likely to occur due to water influx inside the laticifers. Auxins and ethylene used as efficient yield stimulants in mature untapped rubber trees, but, bark treatments with abscisic acid (ABA) and salicylic acid (SA) could also induce a transient increase latex yield. We recently reported that there are three aquaporin genes, HbPIP2;1, HbTIP1;1 and HbPIP1;1, that are regulated differentially after ethylene bark treatment. HbPIP2;1 was up-regulated in both the laticifers and the inner liber tissues, whereas HbTIP1;1 was up-regulated in the latex cells, but very markedly down-regulated in the inner liber tissues. Conversely, HbPIP1;1 was down-regulated in both tissues. In the present study, HbPIP2;1 and HbTIP1;1 showed a similar expression in response to auxin, ABA and SA, as seen in ethylene stimulation, while HbPIP1;1 was slightly regulated by auxin, but neither by ABA nor SA. The analysis of the HbPIP1;1 promoter region indicated the presence of only ethylene and auxin responsive elements. In addition, the poor efficiency of this HbPIP1;1 in increasing plasmalemma water conductance was confirmed in Xenopus oocytes. Thus, an increase in latex yield in response to all of these hormones was proposed to be the major function of aquaporins, HbPIP2;1 and HbTIP1;1. This study emphasized that the circulation of water between the laticifers and their surrounding tissues that result in latex dilution, as well as the probable maintenance of the liber tissues turgor pressure, favor the prolongation of latex flow.     

Ultrastructure and cytochemical localization of acid phosphatase of laticifers in Euphorbia kansui Liou

Acid phosphatase (AcPase) activities are involved in the degeneration process of cytoplasm in plants. In this study, acid phosphatase was detected by the method of lead nitrate and cytochemical electron microscopy during the development of nonarticulated laticifers in Euphorbia kansui Liou. The most important feature in the differentiation of the laticifers in E. kansui is that the development of small vacuoles arises from endoplasmic reticulum (ER). The mature laticifers possess a thin layer of electron-dense peripheral cytoplasm in which the organelle cannot be distinguished and a large central vacuole filled with latex particles. AcPase cytochemistry studies show AcPase reaction products congregated into heaps are distributed along the tonoplast of central vacuole and around the mitochondria and plastids. Some small vacuoles which develop at later developmental stages of laticifers contain AcPase reaction products. As a result, the central vacuole is formed by cellular autophagy and fusion of small vacuoles which apparently arises from ER.     

Anatomy of the Latex System in the Dormant Embryo of Euphorbia marginata Purch : Correlative Analysis of the Histological Organization and Topographic Distribution of Laticifers

The anatomy, morphology and topography of laticifers in themature embryo of Euphorbia marginata have been studied by conventionaland fluorescence microscopy. The mature embryo consists of awell-developed embryonic axis and two cotyledons and it containsdifferentiating vascular tissue. In the hypocotyl, six procambialstrands are present which branch and extend into the cotyledons,whereas in the root they are fused in a triarch structure. Inthe embryonic axis, the latex system consists of stelar andcortical components that are first anastomized in a cotyledonaryplexus and then are joined above in a cotyledonary system. Thestelar and cortical systems are linked by horizontal branchesto a nodal latex plexus placed under the epicotylary meristem.All the vascular laticifers, 24 in number, end unbranched inthe apex of the radicle. The cortical laticifers are profuselybranched toward the epidermis in tiny veins as far as the sub-epidermallevel. The cotyledonary laticifers follow the vascular tissueand form a network surrounding the mesophyllary areoles. Thetopography of the latex system leads one to hypothesize thatthe criteria which determine the constitution of the latex systemlareonly the essential peculiarities of idioblasts, but also correlativephenomena between growing laticifers and surrounding tissues.The stelar system appears to be under the control of vascularpolar growth, while the cortical and cotyledonary latex systemsseem to be subjected both to endogenous polar stimuli and toextra-embryonal influences. Euphorbia marginata, anatomy, embryo, laticifers     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

Chew and spit: tree-feeding notodontid caterpillars anoint girdles with saliva

Caterpillars of the notodontid Oedemasia leptinoides (formerly Schizura) use their mandibles to cut shallow girdles that encircle the petioles and stems of tree hosts. When girdles are complete, the larvae bathe the girdle surface with fluid. We test whether the fluid originates from the labial salivary glands or ventral eversible gland by blocking the openings to the glands and observing whether fluid is still released onto the girdles. Only larvae with functional labial salivary glands anointed girdles with fluid. Analysis of girdle rinses for a prominent salivary enzyme, glucose oxidase, confirmed that larvae apply saliva and documented that application occurs primarily at the end of girdling. We propose that girdling by notodontids, together with related furrowing and leaf-clipping behaviors exhibited by diverse caterpillar groups, serve at least in part to introduce salivary components to exposed vascular tissues; these compounds presumably function to suppress plant defensive responses normally elicited by caterpillar feeding.     

Morphology of hindwing veins in the shield bug Graphosoma italicum (Heteroptera: Pentatomidae)

Light, fluorescence, and electron microscopy were applied to cross sections and -breakage and whole-mount preparations of the anterior hindwing vein of the shield bug Graphosoma italicum. These analyses were complemented by investigations of the basal part of the forewing Corium and Clavus. The integration of structural, histological, and fluorescence data revealed a complex arrangement of both rigid and elastic structures in the wall of wing veins and provided insights into the constitution of transition zones between rigid and elastic regions. Beneath the exocuticular layers, which are continuous with the dorsal and ventral cuticle of the wing membrane, the lumen of the veins is encompassed by a mesocuticular layer, an internal circular exocuticular layer, and an internal longitudinal endocuticular layer. Separate parallel lumina within the anterior longitudinal vein of the hindwing, arranged side-by-side rostro-caudally, suggest that several veins have fused in the phylogenetic context of vein reduction in the pentatomid hindwing. Gradual structural transition zones and resilin enrichment between sclerotized layers of the vein wall and along the edges of the claval furrow are interpreted as mechanical adaptations to enhance the reliability and durability of the mechanically stressed wing veins.     

Sabotaging behaviour and minimal latex of Asclepias curassavica incur no cost for larvae of the southern monarch butterfly Danaus erippus

1. The southern monarch, Danaus erippus, uses mainly Asclepias curassavica as its host in the Neotropics, a plant species bearing articulated anastomosing laticifers. When artificially severed, A. curassavica has been shown to release significantly less latex than other Asclepias species. 2. The present study tested the hypothesis that sabotaging behaviour changes during the ontogeny of D. erippus and recorded latex outflow of A. curassavica during sabotaging and feeding. Larvae displayed vein‐cutting behaviour, which was initially observed in the second instar, became more pronounced in the third and fourth instars, and less frequent in the fifth instar. When present, latex outflow was never more than 1 µl at a time during either vein cutting or feeding, regardless of the instar. 3. Mandibular and midrib morphometrics revealed that larvae selected thicker midrib sites for severing as instars progressed; however, no correlation between mandibular size and midrib size severed was found within instars. 4. Costs of sabotaging behaviour and the effects of A. curassavica latex outflow on D. erippus larvae were also examined. Sabotaging behaviour did not incur growth costs for larvae, and only latex exudation volumes at least 10‐fold greater than those observed due to D. erippus sabotaging or feeding, caused significantly higher larval mortality than controls. 5. Since latex outflow is minimal or non‐existent in A. curassavica, sabotaging behaviour in D. erippus is mostly limited by morphological constraints and is probably driven by chemical stimulants rather than latex defence. In turn, latex does not constitute a major defence of A. curassavica against D. erippus.     

Laticifer distribution in fig inflorescence and its potential role in the fig-fig wasp mutualism

Although in Moraceae the presence of laticifers is considered to be a synapomorphy, little is known about the distribution and morphology of this type of secretory structure in the reproductive organs of its species. Ficus, the largest genus of Moraceae, is characterized by an inflorescence known as syconium and by an obligate mutualistic interaction with pollinating wasps. The objectives of the present study were to evaluate the distribution and morphology of laticifers in syconia of 36 species belonging to different Ficus sections and to survey traits of taxonomic and adaptive value for the group. Syconia containing flowers in a receptive state were collected, fixed and processed for anatomical analysis. All species studied have branched laticifers distributed in the syconium receptacle, in the ostiolar bracts and in the pedicel of staminate flowers. Almost all species show laticifers in the pedicel of shorter-styled flowers. Laticifers also occur in the pedicel of longer-styled flowers in most Ficus sections, except F. curtipes (Conosycea section) and more than 75% of the studied species of the Americanae section. Laticifers are observed in the sepals of 25 of the 36 species studied and occasionally in the pistil. The presence of laticifers in the pedicel of shorter-style flowers and its absence in the pistil suggest that the distribution of this secretory structure in the fig flower was selected by pressures imposed by the fig-fig wasp mutualism. The laticifers in the pedicel of shorter-styled flowers may confer protection to the developing wasp larvae against natural enemies. However, the absence of laticifers in the pistil of most Ficus species studied was probably selected by the mutualistic relationship with the agaonid pollinating wasps since the latex could interfere with oviposition through the style, with the larval development of the pollinating fig wasps, and the emergence of pollinator offspring from their galls.     

A Latex Metabolite Benefits Plant Fitness under Root Herbivore Attack

Plants produce large amounts of secondary metabolites in their shoots and roots and store them in specialized secretory structures. Although secondary metabolites and their secretory structures are commonly assumed to have a defensive function, evidence that they benefit plant fitness under herbivore attack is scarce, especially below ground. Here, we tested whether latex secondary metabolites produced by the common dandelion (Taraxacum officinale agg.) decrease the performance of its major native insect root herbivore, the larvae of the common cockchafer (Melolontha melolontha), and benefit plant vegetative and reproductive fitness under M. melolontha attack. Across 17 T. officinale genotypes screened by gas and liquid chromatography, latex concentrations of the sesquiterpene lactone taraxinic acid β-D-glucopyranosyl ester (TA-G) were negatively associated with M. melolontha larval growth. Adding purified TA-G to artificial diet at ecologically relevant concentrations reduced larval feeding. Silencing the germacrene A synthase ToGAS1, an enzyme that was identified to catalyze the first committed step of TA-G biosynthesis, resulted in a 90% reduction of TA-G levels and a pronounced increase in M. melolontha feeding. Transgenic, TA-G-deficient lines were preferred by M. melolontha and suffered three times more root biomass reduction than control lines. In a common garden experiment involving over 2,000 T. officinale individuals belonging to 17 different genotypes, high TA-G concentrations were associated with the maintenance of high vegetative and reproductive fitness under M. melolontha attack. Taken together, our study demonstrates that a latex secondary metabolite benefits plants under herbivore attack, a result that provides a mechanistic framework for root herbivore driven natural selection and evolution of plant defenses below ground.     

Malaria Mosquitoes Host-Locate and Feed upon Caterpillars

Adult female mosquitoes need blood to develop their eggs and both sexes use nectar and honeydew as carbohydrate resources for flight, survival and to enhance reproduction. However, there are also a few reports in the literature of mosquitoes feeding on haemolymph of soft-bodied insects such as caterpillars. The frequency and significance of this entomophagous behavior is not well understood, but is thought to be a vestige of ancestral feeding behavior or an opportunistic behavior that has evolved over time. In our current paper we investigated the extent to which the malaria mosquito, Anopheles stephensi, is attracted to, and can successfully feed on, larvae of two common moth species, Manduca sexta and Heliothis subflexa. Using y-tube olfactometer assays we found that female An. stephensi readily flew upwind to and landed on the caterpillars of both moth species. The nature of the volatile cues used in host location remains unclear but respirometer studies suggest a possible role of CO₂. Laboratory cage assays further showed that the female mosquitoes were able to actively feed on moth larvae and gain sufficient nutritional benefit to influence survival. The extent to which such an opportunistic behavior occurs in the field has yet to be explored but our results suggest that this haemolymph feeding behavior could play a role in malaria mosquito life history and could provide a novel mechanism for horizontal transmission of pathogens and other micro-organisms between hosts.     

Rapid Identification of the Foodborne Pathogen Trichinella spp. by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS) reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da) from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP) representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.     

Localization and Behaviour of Actin Microfilaments in Laticiferous Cells of Hevea brasiliensis in Latex Exploitation

Actin microfilaments of laticiferous cells and bark wounds in Hevea brasiliensis were studied using TRITC-phalloidin fluorescent microscopy. Actin in latex from mature rubber trees was also investigated using SDS-PAGE and western-boltting. TRITC-fluorescent substance plugged the end of laticifers when latex flow stopped. Actin was detected only in C serum among the four latex fractions. Higher actin content was found in the latex collected at the beginning of tapping than in that collected just before latex flow stopped. Lower actin content was detected in the latex from rubber trees with more intensive exploitation. The present study indicated that actin microfilaments might play an important role in regulation of latex flow and plugging of the laticifers wounds.     

Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo

Cytohesin Arf-GEFs are conserved plasma membrane regulators. The sole Drosophila cytohesin, Steppke, restrains Rho1-dependent membrane cytoskeleton activity at the base of plasma membrane furrows of the syncytial embryo. By mass spectrometry, we identified a single major Steppke-interacting protein from syncytial embryos, which we named Stepping stone (Sstn). By sequence, Sstn seems to be a divergent homologue of the mammalian cytohesin adaptor FRMD4A. Our experiments supported this relationship. Specifically, heterophilic coiled-coil interactions linked Sstn and Steppke in vivo and in vitro, whereas a separate C-terminal region was required for Sstn localization to furrows. Sstn mutant and RNAi embryos displayed abnormal, Rho1-dependent membrane cytoskeleton expansion from the base of pseudocleavage and cellularization furrows, closely mimicking Steppke loss-of-function embryos. Elevating Sstn furrow levels had no effect on the steppke phenotype, but elevating Steppke furrow levels reversed the sstn phenotype, suggesting that Steppke acts downstream of Sstn and that additional mechanisms can recruit Steppke to furrows. Finally, the coiled-coil domain of Steppke was required for Sstn binding and in addition homodimerization, and its removal disrupted Steppke furrow localization and activity in vivo. Overall we propose that Sstn acts as a cytohesin adaptor that promotes Steppke activity for localized membrane cytoskeleton restraint in the syncytial Drosophila embryo.