DNA barcoding indicates multiple invasions of the freshwater snail Melanoides tuberculata sensu lato in Florida.

Melanoides tuberculata sensu lato (Thiaridae) are polymorphic female-clonal snails of Asian and African origins that have invaded freshwaters worldwide, including those in Florida. Although the snails have been documented in Florida for at least 70 years, no studies have investigated whether the observed distribution is due to a single introduction or multiple independent invasions. Here, cytochrome oxidase I was used to measure genetic diversity within and among sites in Florida and compare genetic diversity between Florida and other regions of the world. We also examined the relationship between shell morphology and haplotype diversity to determine if shell morphs can serve as a proxy for haplotypes. In total, we recovered 8 haplotypes randomly distributed across populations in Florida. Phylogenetic reconstruction supported the hypothesis of multiple invasions by diverse representatives of the M. tuberculata species complex. In contrast, shell morphology was not found to be a useful phylogeographic character, with divergent haplotypes represented by similar shell forms. These results suggest that the observed invasion patterns in Florida are best explained by serial introductions, and that shell morphology cannot be used to predict haplotypes or reconstruct invasion history of Melanoides tuberculata s.l. and that extensive taxonomic revisions are needed to investigate invasion dynamics.

Maize OPR2 and LOX10 Mediate Defense against Fall Armyworm and Western Corn Rootworm by Tissue-Specific Regulation of Jasmonic Acid and Ketol Metabolism.

Foliage-feeding fall armyworm (FAW; Spodoptera frugiperda) and root-feeding western corn rootworm (WCR; Diabrotica virgifera virgifera) are maize (Zea mays L.) pests that cause significant yield losses. Jasmonic acid (JA) plays a pivotal defense role against insects. 12-oxo-phytodienoic acid (12-OPDA) is converted into JA by peroxisome-localized OPDA reductases (OPR). However, little is known about the physiological functions of cytoplasmic OPRs. Here, we show that disruption of ZmOPR2 reduced wound-induced JA production and defense against FAW while accumulating more JA catabolites. Overexpression of ZmOPR2 in Arabidopsis enhanced JA production and defense against beet armyworm (BAW; Spodoptera exigua). In addition, lox10opr2 double mutants were more susceptible than either single mutant, suggesting that ZmOPR2 and ZmLOX10 uniquely and additively contributed to defense. In contrast to the defensive roles of ZmOPR2 and ZmLOX10 in leaves, single mutants did not display any alteration in root herbivory defense against WCR. Feeding on lox10opr2 double mutants resulted in increased WCR mortality associated with greater herbivory-induced production of insecticidal death acids and ketols. Thus, ZmOPR2 and ZmLOX10 cooperatively inhibit the synthesis of these metabolites during herbivory by WCR. We conclude that ZmOPR2 and ZmLOX10 regulate JA-mediated resistance in leaves against FAW while suppressing insecticidal oxylipin synthesis in roots during WCR infestation.

9,10-KODA, an α-ketol produced by the tonoplast-localized 9-lipoxygenase ZmLOX5, plays a signaling role in maize defense against insect herbivory.

13-Lipoxygenases (LOXs) initiate the synthesis of jasmonic acid (JA), the best-understood oxylipin hormone in herbivory defense. However, the roles of 9-LOX-derived oxylipins in insect resistance remain unclear. Here, we report a novel anti-herbivory mechanism mediated by a tonoplast-localized 9-LOX, ZmLOX5, and its linolenic acid-derived product, 9-hydroxy-10-oxo-12(Z),15(Z)-octadecadienoic acid (9,10-KODA). Transposon-insertional disruption of ZmLOX5 resulted in the loss of resistance to insect herbivory. lox5 knockout mutants displayed greatly reduced wound-induced accumulation of multiple oxylipins and defense metabolites, including benzoxazinoids, abscisic acid (ABA), and JA-isoleucine (JA-Ile). However, exogenous JA-Ile failed to rescue insect defense in lox5 mutants, while applications of 1 µM 9,10-KODA or the JA precursor, 12-oxo-phytodienoic acid (12-OPDA), restored wild-type resistance levels. Metabolite profiling revealed that exogenous 9,10-KODA primed the plants for increased production of ABA and 12-OPDA, but not JA-Ile. While none of the 9-oxylipins were able to rescue JA-Ile induction, the lox5 mutant accumulated lower wound-induced levels of Ca2+, suggesting this as a potential explanation for lower wound-induced JA. Seedlings pretreated with 9,10-KODA exhibited rapid or more robust wound-induced defense gene expression. In addition, an artificial diet supplemented with 9,10-KODA arrested fall armyworm larvae growth. Finally, analysis of single and double lox5 and lox10 mutants showed that ZmLOX5 also contributed to insect defense by modulating ZmLOX10-mediated green leaf volatile signaling. Collectively, our study uncovered a previously unknown anti-herbivore defense and hormone-like signaling activity for a major 9-oxylipin α-ketol.

Overexpression of maize ZmLOX6 in Arabidopsis thaliana enhances damage-induced pentyl leaf volatile emissions that affect plant growth and interaction with aphids.

Pentyl leafy volatiles (PLV) are C5 volatiles produced from polyunsaturated fatty acids by plant 13-lipoxygenases (13-LOX) in concert with other lipid metabolizing enzymes. Unlike related C6 volatiles (GLV, green leafy volatiles), little is known about the biosynthesis and physiological function of PLV in plants. Zea mays LOX6 (ZmLOX6) is an unusual plant LOX that lacks lipid oxygenation activity but acts as a hydroperoxide lyase hypothesized to be specifically involved in PLV synthesis. We overexpressed ZmLOX6 in Arabidopsis thaliana and established that it indeed produces PLVs. Overexpression of ZmLOX6 caused a mild chlorotic phenotype, and induced a similar phenotype in untransformed Col-0 plants grown in close proximity, suggesting that airborne signals, such as PLVs, are responsible for the phenotype. PLV production, dependency on the substrate from endogenous 13-LOX(s), and likely competition with endogenous 13-oxylipin pathway were consistent with the model that ZmLOX6 functions as a hydroperoxide lyase. The abundance of individual PLVs was differentially affected by ZmLOX6 overexpression, and the new profile indicated that ZmLOX6 had reaction products distinct from endogenous PLV-producing activities in the Arabidopsis host plants. ZmLOX6 overexpression also induced a new hormonal status, which is likely responsible for increased attraction and propagation of aphids, nonetheless improving host plant tolerance to aphid infestation.

The Synthesis of Pentyl Leaf Volatiles and Their Role in Resistance to Anthracnose Leaf Blight.

Volatiles are important airborne chemical messengers that facilitate plant adaptation to a variety of environmental challenges. Lipoxygenases (LOXs) produce a bouquet of non-volatile and volatile oxylipins, including C6 green leaf volatiles (GLVs), which are involved in a litany of plant physiological processes. GLVs are emitted by a diverse array of plant species, and are the best-known group of LOX-derived volatiles. Five-carbon pentyl leaf volatiles (PLVs) represent another widely emitted group of LOX-derived volatiles that share structural similarity to GLVs, however, relatively little is known about their biosynthesis or biological activity. In this study, we utilized PLV-deficient mutants of maize and Arabidopsis and exogenous PLV applications to elucidate the biosynthetic order of individual PLVs. We further measured PLVs and GLVs after tissue disruption of leaves by two popular methods of volatile elicitation, wounding and freeze-thawing. Freeze-thawing distorted the volatile metabolism of both GLVs and PLVs relative to wounding, though this distortion differed between the two groups of volatiles. These results suggest that despite the structural similarity of these two volatile groups, they are differentially metabolized. Collectively, these results have allowed us to produce the most robust PLV pathway to date. To better elucidate the biological activity of PLVs, we show that PLVs induce maize resistance to the anthracnose pathogen, Colletotrichum graminicola, the effect opposite to that conferred by GLVs. Further analysis of PLV-treated and infected maize leaves revealed that PLV-mediated resistance is associated with early increases of oxylipin α- and γ-ketols, and later increases of oxylipin ketotrienes, hydroxytrienes, and trihydroxydienes. Ultimately, this study has produced the most up-to-date pathway for PLV synthesis, and reveals that PLVs can facilitate pathogen resistance through induction of select oxylipins.

Effects of Parasite Infection and Host Body Size on Habitat Associations of Invasive Aquatic Snails: Implications for Environmental Monitoring.

The Comal River, a spring-fed system in central Texas, was invaded in the 1960s by two Asian aquatic snails (Thiaridae: red-rimmed melania Melanoides tuberculata and quilted melania Tarebia granifera) and subsequently by three of their trematode parasites (the avian eye-fluke Philophthalmus gralli in the 1960s; the gill trematode Centrocestus formosanus in the 1990s; and the intestinal fluke Haplorchis pumilio in the 2000s). Previous snail collections (2001-2002) established that habitat conditions significantly affect the distribution of both snail species. However, the effects of snail size (known to influence infection prevalence) and habitat conditions (known to influence snail size) on trematode infection patterns in this system were not evaluated. In a re-evaluation of this data set, logistic regression analyses with individual snails showed that for both M. tuberculata and T. granifera populations, large snails were more likely to be infected than small snails, and habitat conditions were significantly related to infection in T. granifera. However, only snail size was significant in explaining the probability of infection in M. tuberculata. This result was confirmed by linear regression models, which showed that both infected and noninfected M. tuberculata used similar habitats, as large individuals in both infection categories were found in patches dominated by fine substrates and high levels of aquatic vegetation and detritus. For the large size-class of T. granifera, noninfected individuals were found primarily in habitats with silt/sand substrates and high vegetation and detritus cover, while infected individuals occurred among all available habitats. Using these results, we suggest that targeted sampling of large individuals of M. tuberculata in habitats with high detritus and vegetation and large individuals of T. granifera in any habitat can be used to efficiently ascertain parasite "hot spots" and to evaluate changes in parasite prevalence or detect the invasion of new parasites in these thiarid snails.

Purification and characterization of Arabidopsis thaliana oligosaccharyltransferase complexes from the native host: a protein super-expression system for structural studies.

The oligosaccharyltransferase (OT) complex catalyzes N-glycosylation of nascent secretory polypeptides in the lumen of the endoplasmic reticulum. Despite their importance, little is known about the structure and function of plant OT complexes, mainly due to lack of efficient recombinant protein production systems suitable for studies on large plant protein complexes. Here, we purified Arabidopsis OT complexes using the tandem affinity-tagged OT subunit STAUROSPORINE AND TEMPERATURE SENSITIVE3a (STT3a) expressed by an Arabidopsis protein super-expression platform. Mass-spectrometry analysis of the purified complexes identified three essential OT subunits, OLIGOSACCHARYLTRANSFERASE1 (OST1), HAPLESS6 (HAP6), DEFECTIVE GLYCOSYLATION1 (DGL1), and a number of ribosomal subunits. Transmission-electron microscopy showed that STT3a becomes incorporated into OT-ribosome super-complexes formed in vivo, demonstrating that this expression/purification platform is suitable for analysis of large protein complexes. Pairwise in planta interaction analyses of individual OT subunits demonstrated that all subunits identified in animal OT complexes are conserved in Arabidopsis and physically interact with STT3a. Genetic analysis of newly established OT subunit mutants for OST1 and DEFENDER AGAINST APOTOTIC DEATH (DAD) family genes revealed that OST1 and DAD1/2 subunits are essential for the plant life cycle. However, mutations in these individual isoforms produced much milder growth/underglycosylation phenotypes than previously reported for mutations in DGL1, OST3/6 and STT3a.

Validation of phylogenetic signals in amplified fragment length data: testing the utility and reliability in closely related taxa.

BACKGROUND: Discriminating taxa with the nuclear marker, amplified fragment length polymorphism (AFLP) has been accomplished for various organisms in economic, ecological, and evolutionary studies. The protocol available for AFLP generation does not require prior knowledge of the genome; however, it is often extensively modified to fit the needs of the researcher. Modification of this protocol for new labs is intimidating and time-consuming, particularly for taxa in which AFLP have not been previously developed. Furthermore, determining what constitutes quality output during different stages of fragment generation is not well defined and this may further hinder the use AFLP by new researchers. FINDINGS: We present a step-by-step AFLP protocol, using flourophore-labeled primers for use with automated sequencers, including examples of both successful and unsuccessful results. We sufficiently normalized peak intensity and standardized allele calling across all samples for each primer combination. Repeatability was assessed with a phylogenetic tree in which replicate samples clustered together using the minimum evolution procedure. We found differences greater than 10% in allele position among replicated samples would cause replicates to no longer cluster. To minimize offset allele positions, we suggest that researchers analyze different primer combinations at the same time using multiple dyes with the automated sequencer to minimize mismatched alleles across replicates. CONCLUSION: For researchers wanting to use AFLP, this molecular technique is difficult and time-consuming to develop. Clarifying what constitutes quality output for each step in AFLP generation will help to reduce redundant trials in protocol development and, in turn, advance the discipline of population genetics.