Selective Toxins and Analogs Produced by Helminthosporium sacchari: Production, Characterization, and Biological Activity

Helminthosporium sacchari toxin and several lower molecular weight, nontoxic analogs were isolated from culture filtrates. Three isomers of the toxin (A, B, and C), each with four galactose units, were separated by high performance liquid chromatography. Isomer C had the highest and isomer A had the lowest toxicity to H. sacchari-susceptible sugarcane; resistant clones were not affected. Each toxin isomer was partially digested with a β-galactofuranosidase and the resulting analogs (seven from each toxin isomer) were separated by reverse phase high performance liquid chromatography and identified. Each isomer of the analogs with 3 galactose units per mole also was partially digested and the arrangement of galactose units was determined. The compound with one galactose attached to position 2 of the bicyclic sesquiterpene and with 2 galactose units attached to position 13 (analog A_1,2) was highly toxic to some but not to all clones of H. sacchari-susceptible sugarcane. Toxin analogs protected sensitive tissue against active toxin; protective effects of the analogs differed, but at least a 10-fold excess of analog was required. Analog C_2,1 was more effective at preventing toxin C-induced electrolyte losses than was any other analog. Each of the 3-galactose analog isomers protected better than did any of the 2-galactose compounds. The 1,1 analogs did not protect as well as did the 2,0 or 0,2 analogs. Thus, the sesquiterpene isomer, the number of galactose units, and the galactose arrangement pattern determine the effectiveness of the compound in induction of electrolyte loss and in prevention of toxininduced loss from sugarcane tissues.     

Pathological response of the parasitoid, Glyptapanteles militaris, to nuclear polyhedrosis virus-infected armyworm hosts

The development of the embryonic and larval stages of the internal gregarious parsitoid, Glyptapanteles (=Apanteles) militaris, is adversely affected by the hypertrophy strain of a nuclear polyhedrosis virus in the armyworm, Pseudaletia unipuncta. The initial effects are cessation of parasitoid growth and general tissue disruption, followed by the melanization of parasitoid tissues. Melanization spreads from the parasitoids' caudal vesicle throughout the body, culminating in eventual encapsulation in virus-infected hosts. Parasitoids in armyworm hosts infected with the typical strain of nuclear polyhedrosis virus exhibited no abnormal development.     

Immature development of Eretmocerus mundus (Hymenoptera: Aphelinidae)

The development from egg to pupation is followed for the wasp Eretmocerus mundus, parasitizing the whitefly Bemisia tabaci. We elucidate and describe structural details, histological developments and changes that the different parasitoid and host tissues have undergone during parasitism. These include the presence and apparent function of very large salivary glands, which probably produce substances that help to regulate the host's decomposition and parasitoid nutrition. Moreover, the gut of all instars is devoid of both peritrophic membrane and microvilli and, in the early instars, it has squamous rather than columnar epithelial cells. Differing from many other parasitoids, the E. mundus larva usually does not come into contact with the host tissues and does not devour the entire host during its development.The possible reasons for the developmental mechanisms, as well as the functions of the host capsule that envelopes the parasitoid, are discussed.     

The Effect ofBacillus thuringiensisM-exotoxin on Trematode Cercariae

The effect ofBacillus thuringiensis israelensiseluate containing water-soluble exotoxin (M-exotoxin) was observed by its use on cercariae of seven trematode species. To the most sensitive species to the toxic effect of the mentioned toxin belonged schistosome furcocercariae (human speciesSchistosoma mansoniand avian parasiteTrichobilharzia szidati). Under the influence of the toxin, surface syncytial structure (tegument) was separated from underlying tissues, with subsequent disintegration of internal organs connected with disruption of acetabular glands and release of their proteolytic content.     

Effects of conventional and transgenic Bacillus thuringiensis galleriae toxin on Exorista larvarum (Diptera: Tachinidae), a parasitoid of forest defoliating Lepidoptera

The Cry9Aa entomocidal toxin from Bacillus thuringiensis ssp. galleriae (Btg) and an epiphytic Pseudomonas sp. derivative carrying the cloned cry9Aa gene from Btg are active against the pine processionary moth Thaumetopoea pityocampa and the laboratory model species Galleria mellonella. A laboratory study was conducted to investigate the side effects of the Cry9Aa toxin and the engineered bacterium on the post-embryonic development of Exorista larvarum, a larval parasitoid of forest lepidopterous defoliators, cultured in the factitious host G. mellonella. In a first experiment, the purified toxin and the commercial Bt preparation Foray 48B induced a mortality of G. mellonella sixth-instar larvae significantly higher than that of the distilled water control. In parallel, the development of E. larvarum in this host was assessed, but no significant difference was found for any of the parasitoid parameters examined (i.e., eggs oviposited, percentage of puparia and adults and puparial weights). In subsequent experiments, cry9Aa-Pseudomonas suspension significantly increased the mortality of sixth instar G. mellonella larvae compared to untransformed Pseudomonas sp. suspension and distilled water. As to the parasitoid parameters, the cry9Aa-Pseudomonas did not significantly affect the number of oviposited eggs, percentage of puparia and puparial weights. It can be concluded that the post-embryonic development of E. larvarum was not affected by host treatment with either Cry9Aa toxin or cry9Aa-Pseudomonas under the laboratory conditions tested. Although direct effects on parasitoid performance have not been shown, indirect effects could still occur and need to be considered in future studies concerning the effects of genetically modified Bt-derivatives.     

Pea aphid clonal resistance to the endophagous parasitoid Aphidius ervi

The physiological mechanism of resistance to the endophagous braconid Aphidius ervi Haliday (Hymenoptera, Braconidae) by a pink clone (PC) of Acyrthosiphon pisum (Harris) (Homoptera, Aphididae) has been investigated. Comparative data on parasitoid development and associated host biochemical changes in the resistant PC aphids and in a susceptible green clone (GC) of A. pisum are reported. When the PC aphids were attacked as early 4th instars, the developing parasitoid larvae showed a strongly reduced increase in size, compared to those synchronously developing in GC aphids, and were unable to produce a regular mummy. In contrast, parasitism of 2nd instar PC aphids, allowed completion of parasitoid development, but adults had a prolonged developmental time, due to a longer duration of parasitoid’s final (3rd) instar. In all cases, teratocytes, cells deriving from the A. ervi serosal membrane, and the proteins abundantly synthesised by them, were never found in the haemolymph of parasitised PC aphids. Host castration, as demonstrated by total protein incorporation into reproductive tissues, was total in the majority of early (2nd instar) parasitised host aphids, while it was limited when later instars (4th) of PC aphids were parasitised. This is partly due to the absence of the cytolytic activity of teratocytes on host embryos, which, through their persistence, may compete for nutritional resources with the developing parasitoid larvae. In parasitised PC aphids, this competitive effect is further aggravated for the parasitoid by the absence of the regulated amino acid titre increase in the host haemolymph, which is regularly observed in GC aphids. Failure of teratocyte development in the PC clone of the pea aphid is, then, the major functional constraint accounting for the reduction/inhibition of A. ervi larval growth. The reported results allow to assess in vivo the role of teratocytes in the host physiological redirection and nutritional exploitation by the parasitoid, and to integrate and validate the proposed physiological model of host-parasitoid interactions in the system A. pisum-A.ervi.     

Impact of Vairimorpha necatrix and Vairimorpha sp. (Microspora: Microsporida) on Bonnetia comta (Diptera: Tachinidae) within Agrotis ipsilon (Lepidoptera: Noctuidae) hosts

The effect of Vairimorpha necatrix and Vairimorpha sp. on Bonnetia comta developing within pathogen-treated black cutworm, Agrotis ipsilon, hosts was studied. Parasitism by B. comta did not interfere with the near 100% mortality of A. ipsilon caused by either Vairimorpha species. Both microsporidia decreased the number of B. comta able to pupate from a. ipsilon hosts, the days required for adult parasitoid eclosion, and the weights of the puparia. Only in the V. necatrix treatments, however, were these effects significant (P < 0.05) as determined by Duncan's (1955) multiple range test. The impact of each microsporidium on the parasitoid increased with an increase in the concentration of spores to which A. ipsilon was exposed. Both Vairimorpha species had a more detrimental effect on female B. comta than they did on male B. comta. Histological examination of B. comta maggots within V. necatrix- and Vairimorpha sp.-infected A. ipsilon showed the spores primarily restricted to the gut lumen. In older B. comta, dissected from dead Vairimorpha-infected hosts, the guts containing spores were much distended, some to the point that most other parasitoid tissues were absent. This suggests that the detrimental impact the host microsporidian infections had on the parasitoid were related to a nutritional deficiency caused by the accumulation of nondigestable spores in the parasitoid's gut lumen.     

Diverse Phage-Encoded Toxins in a Protective Insect Endosymbiont

The lysogenic bacteriophage APSE infects “Candidatus Hamiltonella defensa,” a facultative endosymbiont of aphids and other sap-feeding insects. This endosymbiont has established a beneficial association with aphids, increasing survivorship following attack by parasitoid wasps. Although APSE and “Ca. Hamiltonella defensa” are effectively maternally transmitted between aphid generations, they can also be horizontally transferred among insect hosts, which results in genetically distinct “Ca. Hamiltonella defensa” strains infecting the same aphid species and sporadic distributions of both APSE and “Ca. Hamiltonella defensa” among hosts. Aphids infected only with “Ca. Hamiltonella defensa” have significantly less protection than those infected with both “Ca. Hamiltonella defensa” and APSE. This protection has been proposed to be connected to eukaryote-targeted toxins previously discovered in the genomes of two characterized APSE strains. In this study, we have sequenced partial genomes from seven additional APSE strains to address the evolution and extent of toxin variation in this phage. The APSE lysis region has been a hot spot for nonhomologous recombination of novel virulence cassettes. We identified four new toxins from three protein families, Shiga-like toxin, cytolethal distending toxin, and YD-repeat toxins. These recombination events have also resulted in reassortment of the downstream lysozyme and holin genes. Analysis of the conserved APSE genes flanking the variable toxin cassettes reveals a close phylogenetic association with phage sequences from two other facultative endosymbionts of insects. Thus, phage may act as a conduit for ongoing gene exchange among heritable endosymbionts.     

Ultrastructure of host tissues exposed to the calyx fluid of the parasitoid,Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae)

Campoletis sonorensis (Cameron) (Hymenoptera: Ichneumonidae) is a solitary endoparasitoid of Heliothis virescens. The lateral oviducts of the female parasitoid contain a particulate suspension called calyx fluid. The particles in calyx fluid are a polydnavirus (CsV) which, when injected into last-instar H. virescens, stimulates degeneration of the host's prothoracic glands. In order to determine if CsV-induced degeneration is specific to prothoracic glands, last-instar H. virescens larvae were injected with C. sonorensis calyx fluid. After 4 days, a variety of host tissues were dissected from both calyx fluid-injected and uninjected control larvae and fixed for transmission electron microscopy. Prothoracic glands from injected larvae were ultrastructurally degenerated by 4 days post-injection, whereas control glands remained intact. Other tissues from calyx fluid-injected larvae (tracheal epithelia, corpora allata, Malpighian tubules, fat body, skeletal muscle, and the brain) showed no signs of ultrastructural degeneration or gross abnormalities as compared with control tissues. These observations suggested that CsV-induced degeneration is specific to the host's prothoracic glands.     

Mycoflora of stored rice in relation to the biological control of Sitophilus oryzae by Anisopteromalus calandrae

In this study, we determined the effect of controlling rice weevils on the mycoflora of stored brown rice by using Anisopteromalus calandrae (Howard), a parasite of the rice weevil. The mycoflora in brown rice with the parasitoid was similar to that without the parasitoid; however, the dominant mold species differed. Without the parasitoid, Aspergillus candidus Link and Penicillium islandicum Sopp were the dominant species, whereas Aspergillus penicilloides Speg. and P. islandicum were dominant when the parasitoid was present. The difference appeared to be due mainly to the difference in the grain moisture content with and without the parasitoid. A. candidus was the dominant species in the communities without the parasitoid and was not observed when the weevils were suppressed. P. islandicum dominated or co-dominated regardless of the presence of the parasitoid which suggested that P. islandicum is tolerant to low grain moisture content or is positively affected by the presence of the rice weevil.     

Effects of Helminthosporium carbonum Toxin on Nitrate Uptake and Reduction by Corn Tissues

Susceptible corn tissues exposed to the host-specific toxin of Helminthosporium carbonum race 1 reduced more nitrate to nitrite than did control tissues, as measured by an in vivo method. There were no differences in nitrate reductase activities extracted from treated and control tissues and assayed by an in vitro method. Toxin-treated susceptible roots removed nitrate from solution and accumulated it in the tissues twice as fast as did control roots. Uptake by resistant roots was stimulated also, provided approximately 100 times higher concentrations of toxin were used. Toxin-stimulated nitrate uptake occurred in the presence of tungstate, which eliminates nitrate reductase activity. Toxin did not cause leakage of nitrate from roots under these conditions. Thus, toxin-enhanced nitrate accumulation was caused by increased nitrate uptake rather than by decreased nitrate metabolism or decreased nitrate leakage. The data indicate that toxin increases the rate of nitrate reduction in vivo by increasing the availability of substrate, not by stimulation of enzyme synthesis.     

Biosystematics of the Psyttalia concolor Species Complex (Hymenoptera: Braconidae: Opiinae): The Identity of Populations Attacking Ceratitis capitata (Diptera: Tephritidae) in Coffee in Kenya

The identity of a species of Psyttalia, a parasitoid of tephritid fruit flies in Kenya, was investigated. Individuals reared from coffee infested with Ceratitis capitata (Wiedemann) (Medfly) and two other tephritid species in Kenya were compared with individuals of Psyttalia concolor (Szépligeti) from a laboratory culture in Italy used in augmentative biological control of olive fly, Bactrocera oleae (Gmelin). Reciprocal crosses showed full compatibility, with production of viable female offspring. A preliminary morphometric analysis demonstrated that most individuals originating from Italy could be separated from most of those from Kenya based largely on differences in overall size.     

Parasitism capacity and searching efficiency of Diaeretiella rapae parasitizing Brevicoryne brassicae on susceptible and resistant canola cultivars

Different cultivars of aplant species can affect the foraging and efficiency of natural enemies, both directly through physical and biochemical properties or indirectly through the herbivore's diet. In this study, the parasitism capacity and functional response of Diaeretiella rapae McIntosh were determined on the cabbage aphid, Brevicoryne brassicae (L.) reared on susceptible (Opera) and resistant (Okapi) canola cultivars under laboratory conditions at 25?±?1?°C, 60?±?5% RH and a16:8?h L:D photoperiod. The parasitoid exhibited Type II and Type III functional responses on the resistant and susceptible cultivars, respectively. The estimated value of searching efficiency (a) was 0.1637?±?0.1095?h^?1 on the resistant cultivar whereas its value was dependent on host density on the susceptible cultivar. The handling times (T_h) on the susceptible and resistant canola cultivars were 0.108?±?0.040 and 0.320?±?0.048?h, respectively. The net parasitism rate (C₀) of the parasitoid wasp varied from 128.09 hosts per parasitoid lifetime on the susceptible to 71.01hosts on the resistant canola cultivar. The transformation rate from host population to parasitoid offspring (Qp) was equal to 1 on both cultivars (C₀?=?R₀). The finite parasitism rate (ω) on the susceptible cultivar (0.819 hosts per parasitoid per day) was significantly higher than that on the resistant one (0.578 hosts per parasitoid per day). In conclusion, canola cultivars affected the performance of D. rapae in controlled small-scale laboratory experiments and compared with the susceptible cultivar, the resistant one had anadverseeffect on the efficiency of the parasitoid.     

Host Plants Affect the Foraging Success of Two Parasitoids that Attack Light Brown Apple Moth Epiphyas postvittana (Walker) (Lepidoptera: Tortricidae)

The light brown apple moth, Epiphyas postvittana is a key pest of wine grapes in Australia. Two parasitoids, Dolichogenidea tasmanica and Therophilus unimaculatus, attack the larval stage of this pest. D. tasmanica is dominant in vineyards, whereas T. unimaculatus is mainly active in native vegetation. We sought to understand why they differ in their use of habitats. Plants are a major component of habitats of parasitoids, and herbivore-infested plants influence parasitoid foraging efficiency by their architecture and emission of volatile chemicals. We investigated how different plant species infested by E. postvittana could affect the foraging success of the two parasitoid species in both laboratory and field experiments. Four common host-plant species were selected for this study. In paired-choice experiments to determine the innate foraging preferences for plants, both parasitoid species showed differences in innate search preferences among plant species. The plant preference of D. tasmanica was altered by oviposition experience with hosts that were feeding on other plant species. In a behavioral assay, the two parasitoid species allocated their times engaged in various types of behavior differently when foraging on different plant species. For both parasitoids, parasitism on Hardenbergia violacea was the highest of the four plant species. Significantly more larvae dropped from Myoporum insulare when attacked than from the other three host-plant species, which indicates that parasitism is also affected by interactions between plants and host insects. In vineyards, parasitism by D. tasmanica was significantly lower on M. insulare than on the other three host-plant species, but the parasitism rates were similar among the other three plant species. Our results indicate that plants play a role in the habitat preferences of these two parasitoid species by influencing their foraging behavior, and are likely to contribute to their distributions among habitats.     

Ecological Niche Models and Coalescent Analysis of Gene Flow Support Recent Allopatric Isolation of Parasitoid Wasp Populations in the Mediterranean

Background

The integration of multiple complementary approaches is a powerful way to understand the processes of diversification and speciation. The parasitoid wasp Aphidius transcaspicus Telenga (Hymenoptera: Braconidae) is a parasitoid of Hyalopterus aphids across a wide geographic range. This species shows a remarkable degree of genetic structure among western, central, and eastern Mediterranean population clusters. In this paper we attempt to better characterize this genetic structure.

Methodology/Principal Findings

We use a Bayesian coalescent analysis of gene flow under the Isolation with Migration model using mitochondrial and microsatellite markers together with climate-based ecological niche models to better understand the genetic structure of A. transcaspicus in the Mediterranean. The coalescent analysis revealed low levels of migration among western and eastern Mediterranean populations (Nm<1) that were not statistically distinguishable from zero. Niche models showed that localities within population clusters each occupy areas of continuously high environmental suitability, but are separated from each other by large regions of completely unsuitable habitat that could limit dispersal. Overall, environmental characteristics were similar among the population clusters, though significant differences did emerge.

Conclusions/Significance

These results support contemporary allopatric isolation of Mediterranean populations of A. transcaspicus, which together with previous analyses indicating partial behaviorally mediated reproductive isolation, suggest that the early stages of cryptic speciation may be in progress.     

The probable significance of tracheal tufts in the 8th abdominal segment of Heliothis virescens (F.) on the development of its parasitoid, Toxoneuron nigriceps (Viereck)

The 8th abdominal segment of Heliothis virescens (Fabricius) larvae contains aerating trachea and tracheole tufts that end in the hemocoel of the 8th segment, unlike the tracheae that invade tissues in other segments. These tracheal tufts from the 8th abdominal segment extend to the tokus region, which along with the telson cavity is known to act as a “lung” for hemocytes in Calpodes ethlius and a few other lepidopteran larvae. The goal of this research was to study the effects of these tracheal tufts in the 8th abdominal segment on parasitoid development inside the host larvae, H. virescens. The first objective was to determine if the eggs of the parasitoid, Toxoneuron nigriceps, are predominantly located among the tracheal tufts of the 8th abdominal segment compared to other body cavity regions irrespective of their oviposition site or the position of the host larvae. The results showed that several hours after oviposition most of the eggs are found in the 8th abdominal segment irrespective of the oviposition site or the position of the host larvae. The second objective was to study the effect of varying oxygen concentrations in vitro on various developmental stages of the egg. The results showed that decreasing oxygen concentrations adversely affects the parasitoid egg development in vitro. A third objective was to determine the oxygen concentration in 8th abdominal segment of the host larvae and compare it to other regions of the body using an oxygen sensor placed in vivo. The results suggested relatively high concentration of oxygen in the 8th abdominal segment compared to other regions of the host, thus supporting our hypothesis that the increased oxygen level in the 8th abdominal segment is important to the development of the parasitoid eggs.     

Metabolic and symbiotic interactions in amino acid pools of the pea aphid,Acyrthosiphon pisum,parasitized by the braconid Aphidius ervi

Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera, Aphididae). This parasitoid strongly redirects host reproduction and metabolism to favour nutrition and development of its juvenile stages. Parasite-regulated biosynthesis and mobilization of nitrogen metabolites determine a significant increase of host nutritional suitability. The aim of the present study was mainly to investigate the temporal changes of A. pisum amino acid pools, as affected by A. ervi parasitism, and to assess the role of the aphid bacterial endosymbiont Buchnera in determining the observed changes. In parasitized aphids, we observed a very significant increase in total free amino acids, compared with synchronous non-parasitized controls, starting from day 4 after parasitization (+51%). This trend culminated with more than doubling the control value (+152%) on day 6 after parasitization. However, a significant “parasitism” effect was observed only for 10 of the 28 amino acids detected. Tyrosine accumulation was the most prominent parasitoid-induced alteration, with a fourfold increase over control levels registered on day 6. In parasitized hosts, the amino acid biosynthetic capacity of Buchnera was unaltered, or even enhanced for the phenolic pool, and contributed greatly to the definition and maintainance of host free amino acid pools. The hypertyrosinemic syndrome was not dependent on food supply of the aromatic nucleus but was induced by parasitism, which likely enhanced the aromatic shuttle mediating phenylalanine transfer from bacteria to the host tissues, where tyrosine conversion occurs. This process is likely associated with a selective disruption of the host’s functions requiring tyrosine, leading to the remarkable accumulation of this amino acid. The possible mechanisms determining these parasitism-induced host alterations, and their nutritional significance for the developing parasitoid larva, are discussed.     

Variation in paralytic shellfish toxin composition within the Protogonyaulax tamaronsis/catenella species complex; red tide dinoflagellates

Unialgal isolates of the Protogonyaulax (—Gonyaulax) tamarensis/catenella species complex, a group of dinoflagellates which causes paralytic shellfish poisoning (PSP), were subjected to toxin analysis by HPLC. Protogonyaulax isolates from widely separated geographical locations were compared, including the northeastern Pacific (British Columbia and Washington State), eastern Canada, Portugal, the United Kingdom and New Zealand. Two distantly related gonyaulacoid species were also analyzed, but the presence of PSP toxins was not detected. Although Protogonyaulax isolates varied markedly in total toxin concentration and toxicity, even through the culture cycle, the toxin ratios of individual isolates were distinctive and relatively constant. No toxins were detected in the Plymouth (U.K.) isolate of P. tamarensis, from the species type locality. Two isolates from Vancouver Island (British Columbia), which were previously considered to be non-toxic according to the mouse bioassay, revealed weak toxin spectra by HPLC. Within populations from English Bay (British Columbia) the toxin profiles of tamarensoid isolates tended to be conservative. However, this was not the case for the catenelloid forms from Washington State, which displayed a greater degree of toxin heterogeneity. Significantly, there was no identifiable relationship between toxicity or toxin profiles and the morphological characteristics conventionally used to separate the two dominant morphotypes into species within this species complex.