Electroantennogram (EAG) responses of Microplitis croceipes and Cotesia marginiventris and their lepidopteran hosts to a wide array of odor stimuli: Correlation between EAG response and degree of host specificity?

In order to test whether the electroantennogram (EAG) response spectrum of an insect correlates to its degree of host specificity, we recorded EAG responses of two parasitoid species with different degrees of host specificity, Microplitis croceipes (specialist) and Cotesia marginiventris (generalist), to a wide array of odor stimuli including compounds representing green leaf volatiles (GLVs), herbivore-induced plant volatiles (HIPV), ecologically irrelevant (not used by the parasitoid species and their hosts for host location) plant volatiles, and host-specific odor stimuli (host sex pheromones, and extracts of host caterpillar body and frass). We also tested the EAG responses of female moths of the caterpillar hosts of the parasitoids, Heliothis virescens and Spodoptera exigua, to some of the odor stimuli. We hypothesized that the specialist parasitoid will have a narrower EAG response spectrum than the generalist, and that the two lepidopteran species, which are similar in their host plant use, will show similar EAG response spectra to plant volatiles. As predicted, the specialist parasitoid showed greater EAG responses than the generalist to host-specific odor and one HIPV (cis-3-hexenyl butyrate), whereas the generalist showed relatively greater EAG responses to the GLVs and unrelated plant volatiles. We detected no differences in the EAG responses of H. virescens and S. exigua to any of the tested odor.     

Discrimination of previously searched,host-free sites byMicroplitis croceipes (Hymenoptera: Braconidae)

The ability of free- ranging parasitoids to discriminate between previously visited and unvisited sites containing host kairomone (caterpillar frass) but not hosts was tested. Females of Microplitis croceipes,a host specialist and plant generalist larval parasitoid of Helicoverpa (Heliothis) zea,were allowed to fly freely in a simulated plant patch in a flight chamber. Wasps spent less time searching frass sites previously searched by themselves or by conspecifics than unsearched frass sites. In addition to chemical marking, spatial memory of visual cues was implicated as a mechanism for discriminating against self-visited, host-free sites.     

Foraging Efficacy of a Larval Parasitoid in a Cotton Patch: Influence of Chemical Cues and Learning

Plant-herbivore chemical signals and behavioral plasticity may enhance parasitoid host-foraging efficacy in the field; however, no studies have quantified the potential benefits from these factors under field-type conditions. The effect of plant-herbivore signals and learning on the foraging efficacy of Microplitis croceipes was quantified by directly observing and recording total and sequential duration of various foraging behaviors relative to 5 randomly placed herbivore-damaged and host-infested cotton plants and 20 undamaged and non-host-infested plants. Microplitis croceipes spent significantly more time searching (flying and antennation) on host infested versus uninfested plants. Antennation time was significantly and negatively correlated with successive host stings. Contrary to expectations of increased duration, flight time remained constant throughout the foraging bout, which may indicate that there was some learning associated with flight. These results suggest that plant-herbivore chemical signals and learning enhances the foraging efficacy of M. croceipes.     

Responses of Microplitis croceipes to host and nonhost plants of Heliothis virescens in a wind tunnel

The relative attractiveness of velvet leaf (Abutilon theophrasti Medicus), cotton (Gossypium hirsutum L.) (host plants) and groundcherry (Physalis angulata L.) (nonhost plant), and cotton plants with or without nectaries and with or without glands to Microplitis croceipes Cresson (Hymenoptera: Braconidae) was determined in a wind tunnel. Female parasitoids flew significantly more to glandless than to glanded cotton; response to nectaried and nectariless cotton was similar. Velvet leaf and cotton were favored significantly over groundcherry; parasitoids being equally responsive to both host plant species. Addition of larval frass alone or in combination with host larvae significantly improved the attractivity of the nonhost plant (groundcherry) to the parasitoids. There was no difference in attractiveness of groundcherry terminals with or without host larvae. Parasitoid search time was significantly increased with addition of larval frass. In the presence of cotton, however, kairomone-treated groundcherry remained unattractive.

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Résumé L'influence des nectaires et des glandes du coton, et l'attractivité des feuilles veloutées du coton (plante hôte) et de Physalis angulata sur Microplitis croceipes, ont été déterminées dans des expériences avec tunnel à vent. Les résultats ont montré que les parasitoïdes femelles sont significativement plus attirés par les cotons sans glandes que par les cotons glanduleux, tandis que la présence ou l'absence de nectaires ne modifie pas l'attractivité du coton. Elles réagissent de la même façon aux feuilles veloutées. P. angulata traité avec des crottes de chenilles, présenté seul ou en combinaison avec du coton, attire plus de femelles que des pousses de P. angulata non traitées. L'addition de chenilles hôtes seules n'améliore pas significativement l'attractivité de la plante. P. angulata traité avec des kairomones est demeuré inattractif quand il était proposé en même temps que du coton.

     

Transient Expression of β-Glucuronidase in Embryo Axes of Cotton by Agrobacterium and Particle Bombardment Methods

Transient expression of -glucuronidase (GUS) in zygotic embryo axes of two cotton (Gossypium hirsutum L.) cultivars NHH-44 and DCH-32 was induced by Agrobacterium mediated transformation or by particle bombardment. For Agrobacterium transformation, disarmed A. tumefaciens strain GV 2260/p35SGUSINT was used. In cv. NHH-44, the maximum frequency of transient expression (14.28 %) was achieved on spotting Agrobacterium paste on the apical regions of the split embryo axes. The method resulted in a transformed callus line, which showed strong GUS activity. Integration of NPTII gene was confirmed by Southern analysis. Transgene expression by particle bombardment was achieved with p35SGUSINT and pIBGUS plasmids independently. The maximum frequency of GUS expression in 29.16 % explants was observed in cultivar NHH-44 with gold microcarriers (1.1 µm) when bombarded once with rupture disc of 7586 kPa at target cell distance of 6 cm. A transformed callus line was obtained when explants were bombarded with p35SGUSINT and cultured on Murashige and Skoog's medium supplemented with B₅ vitamins, 0.1 mg dm^–3 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea, 0.01 mg dm^–3 -naphthaleneacetic acid, 3 % glucose + 50 mg dm^–3 kanamycin. High GUS activity was observed in callus tissue as well as in somatic embryo like structures achieved in liquid shake cultures.     

Host recognition by the specialist endoparasitoidMicroplitis croceipes (Hymenoptera: Braconidae): Role of host- and plant-related volatiles

The specialist parasitoidMicroplitis croceipes Cresson can parasitize only noctuid larvae in the generaHelicoverpa andHeliothis. To be successful in their search for hosts, the ability to distinguish hosts from nonhosts feeding on the same plant is beneficial. In flight tunnel experiments, we found that prior to landing on the odor sourceM. croceipes were able to distinguish volatiles released from frass of host larvae(Helicoverpa zea Boddie) and nonhost larvae (Spodoptera exigua Hübner andSpodoptera frugiperda J. E. Smith) fed on cotton. However, an initial contact experience with frass of cotton-fed host larvae appeared to be critical for this ability. Wasps that had antennated frass of host larvae fed pinto bean diet were equally attracted to frass of host and nonhost larvae fed on pinto bean diet. In short-range walking experiments, wasps located cotton-fed host larvae faster than diet-fed larvae, regardless of their experience. Wasps that had antennated frass of cotton-fed host larvae were less attracted to cotton-fed nonhost larvae, compared to host larvae, and preferred to sting host larvae. Plant-related volatiles in host frass and larvae appear to play a major role in the successful location of host larvae.     

Induction of systemic acquired resistance in Zea mays also enhances the plant’s attractiveness to parasitoids

Plants under attack by caterpillars emit volatile compounds that attract the herbivore’s natural enemies. In maize, the caterpillar-induced production of volatiles involves the phytohormone jasmonic acid (JA). In contrast, pathogen attack usually up-regulates the salicylic acid (SA)-pathway and results in systemic acquired resistance (SAR) against plant diseases. Activation of the SA-pathway has often been found to repress JA-dependent direct defenses, but little is known about the effects of SAR induction on indirect defenses such as volatile emission and parasitoid attraction. We examined if induction of SAR in maize, by chemical elicitation with the SA-mimic benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), attenuates the emission of volatiles induced by Spodoptera littoralis or exogenously applied JA. In addition, we determined how these treatments affected the attractiveness of the plants to the parasitoid Microplitis rufiventris in a six-arm-olfactometer. BTH treatment alone resulted in significant systemic resistance of maize seedlings against the pathogen Setosphaeria turcica, but had no detectable effect on volatile emissions. Induction of SAR significantly reduced the emission rates of two compounds (indole and (E)-β-caryophyllene) in JA-treated plants, whereas no such negative cross-talk was found in caterpillar-damaged plants. Surprisingly, however, BTH treatment prior to caterpillar-feeding made the plants far more attractive to the parasitoid than plants that were only damaged by the herbivore. Control experiments showed that this response was due to plant-mediated effects rather than attractiveness of BTH itself. We conclude that in the studied system, plant protection by SAR activation is compatible with and can even enhance indirect defense against herbivores.     

Comparative flight behavior of parasitoids Campoletis sonorensis and Microplitis croceipes

Flight behavior of parasitoids Microsplitis croceipes Cresson (Braconidae) and Campoletis sonorensis (Cameron) (Ichneumonidae) was examined in a wind tunnel. Both species exhibited odor directed, oriented flights to cotton, although flight frequency was related to variation in wind velocity or differential cotton volatile emission with changes in wind velocity, or both. Flight frequency at constant wind velocity was affected by illumination. Microplitis croceipes was attracted to the wind borne odor of Heliothis virescens (F.) frass and larvae, whereas C. sonorensis was not. Possible interaction of the wind borne odors from the habitat and host is suggested for parasitoids.

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Résumé Le comportement de vol de M. croceipes Cres. (Hym. Braconidae) et de C. sonorensis Cam. (Hym. Icheumonidae) a été étudié en olfactométrie dans un tunnel à vent. Les femelles des deux espèces, et non les mâles, ont un vol orienté par l'odeur des feuilles fraîches de coton (Gossypium hirsutum L.). La fréquence d'envol est liée aux changements de vitesse du vent, aux variations d'émissions, par le cotonnier, des substances volatiles, en fonction de la vitesse du vent, ou aux deux. La fréquence d'envol à vitesse de vent constante dépend de l'éclairage. La fréquence d'envol de C. sonorensis augmente avec des intensités lumineuses relativement fortes et diminue avec des intensités relativement faibles. L'inverse est observé avec M. croceipes. Le vent chargé d'odeurs de larves ou d'extréments d'Heliothis virescens F., attire M. croceipes et non C. sonorensis. Nous suggérons une éventuelle interaction des odeurs portées par le vent et provenant de l'habitat et de l'hôte.

     

Characterization of induced resistance in tomato plants

We have characterized, using several types of bioassays, the resistance induced in young tomato plants by feeding of the corn earworm, Helicoverpa zea. Beet armyworm larvae, Spodoptera exigua, and leafminers, Liriomyza trifolii, were used to assay the induced resistance. In whole-plant experiments, damage localized to a single leaflet of fourleaf tomato plants induced a systemic increase in resistance such that beet armyworm larvae confined to previously damaged (induced) plants grew at a rate about half that of larvae raised on control plants and consumed less leaf tissue from induced plants than from control plants. In experiments using excised leaves, beet armyworm larvae suffered increased mortality when reared on leaves from induced plants. The strength of this induced resistance varied spatially relative to the damaged position; moreover, the spatial distribution of induced resistance changed over a three-week period following damage. Other experiments demonstrated that the mechanisms of induced resistance in tomato foliage involves both a decrease in larval preference for and a decrease in the nutritional value of induced foliage. Induction also retarded the oviposition and/or early development of leafminers. Thus, induced resistance has relatively severe effects on the biology of subsequent herbivores. These data should allow us to begin to elucidate cause-effect relationships between induced resistance and induced chemistry in tomato plants.     

Extended in vitro culture of Microplitis croceipes teratocytes and secretion of TSP14 protein

Teratocytes, cells which originate from the serosal membrane of some Braconidae and Scelionidae, can be found in the hemocoel of permissive hosts during part or all of the developmental time of the parasitoid larva. Teratocytes from Microplitis croceipes are known to secrete biologically active proteins, which contribute to developmental arrest and failure to pupate of Heliothis virescens larvae. One such protein, which has a molecular weight of approximately 14 kDa is called TSP14. The presence of parasitoid larvae is essential to maintain teratocytes under in vitro conditions with protein-free EX-CELL 400. The teratocyte viability was maintained in vitro for at least 12 days in the presence of larvae when medium was exchanged every three days. Western blots show that TSP14 was secreted during the entire period of exchanges. In the absence of parasitoid larvae, teratocyte viability was only 30% by day 6 and no TSP14 had been secreted. In the absence of parasitoid larvae, teratocytes maintained in vitro in EX-CELL 400 medium supplemented with 10% FBS remained viable for at least nine days and secreted TSP14 for at least six days. This suggests that parasitoid larval secretions are sufficient but not uniquely essential to maintain teratocyte viability. Parasitoid larvae maintained in the absence of teratocytes did not secrete TSP14 and their secretory products did not inhibit pupation of H. virescens larvae.     

Plant regeneration via somatic embryogenesis in cotton

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and stem explants of abnormal seedling as an explant have been developed in Gossypium hirsutum L. Embryogenic callus and somatic embryos have been obtained directly from the explants of cotton abnormal seedlings. Plant growth regulators influenced the induction of cotton somatic embryogenesis. The optimal medium for direct somatic embryogenesis was modified MS medium supplemented with 0.1 mg l^-1 ZT and 2 g l^-1 activated carbon. On this medium, an average of 28.0 and 28.1 matured somatic embryos formed from per leaf and stem explants respectively. The highest frequency of somatic embryogenesis was 100%. The somatic embryos were converted into normal plantlets when cultured on modified MS medium supplemented with 0.1 mg l^-1 ZT. Upon transfer to soil, plants grew well and appeared normal. Plants could be regenerated within 60–80 days. The system of cotton somatic embryogenesis and plant regeneration described here will facilitate the application of plant tissue culture and genetic engineering on cotton genetic improvement. This revised version was published online in June 2006 with corrections to the Cover Date.     

Coronatine alleviates salinity stress in cotton by improving the antioxidative defense system and radical-scavenging activity

Coronatine (COR) is a chlorosis-inducing phytotoxin that mimics some biological activities of methyl jasmonate. This study investigated whether COR confers salinity tolerance to cotton and whether such tolerance is correlated with changes in the activity of antioxidant enzymes. COR at 0.01muM was applied hydroponically to cotton seedlings at the two-leaf stage for 24h. A salinity stress of 150mM NaCl was imposed after completion of COR treatment for 15d. Salinity stress reduced biomass of seedlings and increased leaf superoxide radicals, hydrogen peroxide, lipid peroxidation, and electrolyte leakage. Activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione reductase (GR), and of the stable free radical, 1,1-diphenyl-2-picrylhydrazyl (DPPH), scavenging activity were altered by salinity to varying degrees. Pretreatment with COR increased the activities of CAT, POD, GR, and DPPH scavenging activity in leaf tissues of salinity-stressed seedlings. Thus, COR might reduce the production of reactive oxygen species by activating antioxidant enzymes and DPPH-radical scavenging, thereby preventing membrane peroxidation and denaturation of bio-molecules.     

Salinity Induced Changes in α-Amylase Activity During Germination and Early Cotton Seedling Growth

Salinity induced changes in -amylase activity in three cotton cultivars (NIAB-Karishma, NIAB-86 and K-115) was studied during germination and early seedling growth under controlled conditions. The increase in NaCl concentration resulted in the decrease in -amylase activity and break down of starch into reducing and non-reducing sugars in all cultivars, however, it was more pronounced in NIAB-86. K-115 showed highest germination followed by NIAB-Karishma and NIAB-86.     

棉花GhZIP4基因的克隆及表达分析

根据EST拼接的序列设计引物,利用RT-PCR和PCR方法,从陆地棉‘苏棉18’-cDNA和基因组DNA中分别克隆获得了GhZIP4基因片段.结果表明:(1)GhZIP4基因cDNA序列全长1 487 bp,包含1 269 bp ORF,编码422个氨基酸残基,其氨基酸序列具有典型的ZIP蛋白特征,预测具有8个跨膜结构域,第Ⅲ和第Ⅳ跨膜结构域间存在可变区,在可变区有2个富含His的结构域“HRHSHPHG”和“HSHGHGHD”.(2)氨基酸进化树分析显示,GhZIP4同拟南芥ZIP家族AtZIP4的相似性较高.(3)GhZIP4 DNA序列编码区全长1 778 bp,包含4个外显子和3个内含子,所有外显子/内含子交接点都遵从gt/ag剪接规则.(4)半定量分析显示,GhZIP4基因在茎中表达量最高,表明该基因有可能在某些金属离子地上部和根部的动态平衡分布过程中具有重要作用.     

Three viruses found in the braconid parasitoid Microplitis croceipes and their implications in biological control programs

Productivity and longevity decreased in a laboratory colony of the parasitoid wasp Microplitis croceipes (Cresson) (Hymenoptera: Braconidae). Using light microscopy, it was determined that the colony was free of microsporidia. However, samples of the colony examined for pathogens by electron microscopy revealed three types of viruses: a nonpathogenic polydnavirus which is produced by all female wasps; a nonoccluded baculovirus which is pathogenic to late-stage pupae and adults; and a picorna-like virus which is present in larvae, pupae, and adults. The nonoccluded baculovirus was eliminated from the laboratory colony of M. croceipes by selection of progeny from wasps which had oviposited within 2 to 3 days after emergence from the cocoons and which had lived for at least 14 days post-emergence. Upon death, the wasps were examined by negative stain electron microscopy and only progeny from baculovirus-free wasps were retained. Parasitoid colonies should be systematically examined for pathogenic viruses that may reduce their productivity and efficacy as biological control agents. In addition, exotic parasitoids and predators should be evaluated for viruses and other pathogens while in quarantine.     

Differentially expressed genes in cotton plant genotypes infected with Meloidogyne incognita

Meloidogyne incognita is a nematode responsible for huge losses of economically important crops. The control of this pathogen is heavily centered on chemical nematicides, which are toxic to humans and environment, besides being very expensive. Alternatively, resistant varieties of cotton generated from conventional breeding programs represent an attractive strategy for the control of M. incognita. In this context, the goal of the work reported here was to analyze the gene expression profile of one resistant and one susceptible cotton genotype infected with M. incognita aiming to understand the mechanisms involved in resistance. EST libraries of cotton in both resistant and susceptible to infection by M. incognita were constructed and sequenced, generating 2261 sequences that were assembled into 233 contigs and 1593 singlets. Genes differentially expressed were observed in both resistant and susceptible cotton. Twenty genes were found to be expressed exclusively in the resistant cotton genotype, with functions related to pathogen recognition, signal transduction, defense mechanisms and protein synthesis transport and activation. The coordinated action of these genes suggests the existence of a complex defense pathway towards nematode attack in cotton. Our data indicate some candidate genes for validation and use through transformation in other agronomically important plants.     

Differentiation of cotton fibers from single cells in suspension culture

Summary A cotton cell suspension culture has been developed that provides unique opportunities for plant biologists to investigate early developmental events regulating cotton fiber properties, plant cell elongation, and cell wall biogenesis. The suspension culture was derived from cells of cotton (Gossypium hirsutum L.) ovule callus. These cells undergo the stages of fiber development previously described for in vivo fiber development. Fibers range in length up to 11 mm and have secondary walls. Supported by the U.S. Department of Agriculture, Agricultural Research Service, Southern Regional Research Laboratory, New Orleans, Louisiana, and Cotton Incorporated, Raleigh, North Carolina.