First record of the insect pathogenic alga Helicosporidium sp. (Chlorophyta: Trebouxiophyceae) infection in larvae and pupae of Rhizophagusgrandis Gyll. (Coleoptera, Rhizophaginae) from Turkey

The predator beetle Rhizophagus grandis Gyll. (Coleoptera, Rhizophaginae) is one of the most important biological control agents, mass-bred and used to suppress populations of an important pest: the great spruce bark beetle, Dendroctonus micans. The achlorophyllous alga Helicosporidium sp. was first discovered in the pest. Later it was also found in the predator, but only in the adults. In this study, the pathogenic alga Helicosporidium sp. was discovered in larvae and early pupae of R. grandis for the first time. The morphological characteristics of the pathogenic alga were revealed by light and electron microscopy. Infection rates of Helicosporidium sp. in the larvae and pupae of R. grandis were 23.5% and 6.25%, respectively.     

The first study on the bacterial flora of the european spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

The European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera, Scolytidae), is one of the most serious pests of oriental spruce (Picea orientalis L.) in Turkey. In this study, we investigated bacterial flora of D. micans collected from different populations of the forests of Eastern Black Sea Region of Turkey from 2002 to 2004. Seven different bacteria were isolated from healthy, diseased and dead specimens based on the color of colony and morphology. According to morphological, physiological and biochemical properties, metobolic enyzme profile by BIOLOG microtiter plate system, and total cellular fatty acid profile by Microbial Identification System (MIS), isolates were identified as Micrococcus luteus, Bacillus thuringiensis subsp. morrisoni, Serratia grimesii, Enterobacter cloaceae, Enterobacter intermedius, Streptococcus sp. and Pseudomonas putida. This is the first study on the bacterial flora of D. micans.     

北部湾海洋原甲藻的形态特征及其系统进化分析

为探讨广西北部湾海洋原甲藻(Prorocentrum micans)的形态特征及其系统进化意义,利用光学显微镜、分子生物学方法,对海洋原甲藻BBW-01 藻株的形态特征进行了描述,并分析了其分子系统进化关系.结果表明,各地理株系的海洋原甲藻的形态特征相似,仅在细胞大小上存在差异.海洋原甲藻BBW-01 与采自于广东大亚湾的海洋原甲藻形态特征最为接近,其壳板后端的7 个呈“V”字形对称排列的大孔可作为海洋原甲藻鉴定的重要指标.18S rDNA 序列同源检索和系统进化分析表明,海洋原甲藻BBW-01 与源自中国南海的海洋原甲藻的亲缘关系最近,并与其他2 株海洋原甲藻聚成一支,属于浮游、兼性浮游类原甲藻.因此,对赤潮原因种的准确识别有助于预防和减轻赤潮对海洋环境和人类带来的危害.     

Pyrrolizidine alkaloid profiles in Crotalaria species from Brazil: Chemotaxonomic significance

Gas chromatography–mass spectrometry analysis of seeds from 28 species of Crotalaria from Brazil (sections Calycinae, Crotalaria, Chrysocalycinae and Hedriocarpae) showed that pyrrolizidine alkaloids (PAs) are important as chemotaxonomic markers at the infrageneric level. The sections Calycinae and Crotalaria were characterized by 11-membered macrocyclic monocrotaline-type PAs. In the section Chrysocalycinae, a single species in the subsection Incanae, C. incana, showed integerrimine, a 12-membered macrocyclic senecionine-type, as main PA. The group close to the subsection Stipulosae (C. micans and C. maypurensis) showed distinctive PA patterns: C. micans presented mainly the 12-membered macrocyclic integerrimine, and C. maypurensis the unusual 7-hydroxy-1-methylene-8-pyrrolizidine. In the group close to the subsection Glaucae, the PAs with otonecine as the necine base were the main alkaloids, except in C. rufipila which showed an assamicadine-like PA (monocrotaline-type). The section Hedriocarpae showed main 12-membered macrocyclic senecionine-type PAs.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Ecology and impact of Allorhogas sp. (Hymenoptera: Braconidae) and Apion sp. (Coleoptera: Curculionoidea) on fruits of Miconia calvescens DC (Melastomataceae) in Brazil

Two fruit-feeding insects, a gall wasp, Allorhogas sp. (Hymenoptera: Braconidae), and a beetle, Apion sp. (Coleoptera: Curculionoidea), were evaluated in their native habitat in Brazil as potential biological control agents of Miconia calvescens DC (Melastomataceae). Allorhogas sp. occurred at two out of three field sites with native populations of M. calvescens, and Apion sp. occurred at all three sites. Both species exhibited aggregated distributions among M. calvescens trees sampled at each site. Allorhogas sp. infested 9.0% and 3.8% of fruits at each of two sites. The number of larvae and pupae of Allorhogas sp. and/or an unidentified parasitoid (Hymenopetera: Eulophidae: Tetrastichinae) ranged from one to five per infested fruit. Fruits infested with Allorhogas sp. were 20% larger and had 79% fewer seeds than healthy fruits. Although adults of Apion sp. were found on leaves and inflorescences of M. calvescens at all three sites, larvae and pupae were found in fruits at only one site, where a maximum of 1.4% of fruits were infested. Fruits infested by Apion sp. contained only one larva or pupa, and were 15% smaller and had 62% fewer seeds than healthy fruits. While a variety of apionids have been used for biological control in the past, this is the first time a braconid wasp has been considered for biological control of a weed.     

Resource Partitioning among Parasitoids (Hymenoptera: Braconidae) of Phoracantha semipunctata in Their Native Range

The Eucalyptus longhorned borer, Phoracantha semipunctata (F.), is native to Australia, but it has been introduced without its natural enemies into many parts of the world in which its Eucalyptus spp. host has been planted. The beetle has developed large populations in these novel habitats and has been responsible for the mortality of large numbers of trees. Although there is a considerable catalogue of the parasitoids of the beetle in Australia, limited ecological information on the assemblage of parasitoids attacking P. semipunctata is available. We removed bark from 40 felled trees, recorded gallery width and bark thickness over parasitized larvae, and removed all parasitoids. Adult size, sex, and species were recorded when the parasitoid pupae eclosed. Syngaster lepidus Brullè, Jarra phoracantha Austin, Quicke, and Marsh, J. maculipennis Austin, Quicke, and Marsh, and J. painei Austin and Dangerfield were most commonly collected. The solitary parasitoid S. lepidus preferred smaller larvae than did the gregarious Jarra spp. The two species with shorter ovipositors, J. maculipennis and J. painei, parasitized larvae under thinner bark than did the other two species with longer ovipositors. There was a significant positive correlation between host larval size and number of parasitoid pupae of the gregarious species. Also, there was a significant positive correlation between host larval size and parasitoid adult size. The ecological relationships between this assemblage of parasitoids and their beetle host may be useful in establishing an effective biological control program.     

入侵害虫悬铃木方翅网蝽越冬后期在豫北寄主树干上的分布研究

【目的】入侵害虫悬铃木方翅网蝽近年来严重威胁我国城市行道树二球悬铃木的健康,受害严重时造成树木死亡。在越冬后期对悬铃木树干上的悬铃木方翅网蝽成虫的越冬习性开展研究,能为冬季防治积累生物学研究基础。【方法】早春随机调查河南省新乡市行道树二球悬铃木树干栓皮层外层下方隐藏的悬铃木方翅网蝽成虫,比较阳侧和阴侧虫口密度。同时记载悬铃木树干0.00~0.50 m、0.51~1.00 m、1.01~1.50 m、1.51~2.00 m 4个不同高度区段范围和树干皮层外层皲裂程度在5%以下、5%~25%、25%~50%、50%~75%、75%~95%、95%以上6个等级条件下害虫的虫口密度。【结果】栖息在悬铃木树干树皮下方的悬铃木方翅网蝽在阴面更多,阴面和阳面的虫口密度差异达极其显著水平[(F=6.63)(F0.01(1,19)=1.0132),P=0.00]。对寄主树干4个不同高度区段的虫口密度比较后,发现在树干1.0~1.5 m区段间的虫口密度最大,平均11.5头;且虫口密度在悬铃木不同高度区段间存在极显著差异[(F=26.91)(F0.01(3,56)=4.1519),P=0.00]。悬铃木树皮的不同皲裂程度对悬铃木方翅网蝽虫口密度的影响达极其显著水平[(F=31.02)(F0.01(3,56)=4.1519),P=0.00],在树皮外皮50%~75%皲裂程度条件下的虫口密度最大,平均9.8头。利用双因素方差分析方法分析树干不同高度和树皮不同皲裂等级的互作对虫口密度的分布影响达显著水平[(F=2.46)(F0.05(9,56)=2.0519),P=0.0195]。【结论】华北地区害虫秋冬防治时,在寄主悬铃木树干的最好涂白时机是在害虫全部下树后的12月上中旬进行,涂白高度以树干1.0~2.0 m范围为宜。本研究对悬铃木方翅网蝽在越冬期的预防和治理有较为重要的参考价值。     

Insecticidal action of Bauhinia monandra leaf lectin (BmoLL) against Anagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfasciatus and Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetle larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil (Callosobruchus maculatus) and the Mexican bean weevil (Zabrotes subfasciatus), are pests that damage stored seeds. The Mediterranean flour moth (Anagasta kuehniella) is of major economic importance as a flour and grain feeder; it is often a severe pest in flour mills. Plant lectins have been implicated as antibiosis factors against insects. Bauhinia monandra leaf lectin (BmoLL) was tested for anti-insect activity against C. maculatus, Z. subfasciatus and A. kuehniella larvae. BmoLL produced ca. 50% mortality to Z. subfaciatus and C. maculatus when incorporated into an artificial diet at a level of 0.5% and 0.3% (w/w), respectively. BmooLL up to 1% did not significantly decrease the survival of A. kuehniella larvae, but produced a decrease of 40% in weight. Affinity chromatography showed that BmoLL bound to midgut proteins of the insect C. maculatus. 33 kDa subunit BmoLL was not digested by midgut preparations of these bruchids. BmoLL-fed C. maculatus larvae increased the digestion of potato starch by 25% compared with the control. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

Open field host selection and behavior by tamarisk beetles (Diorhabda spp.) (Coleoptera: Chrysomelidae) in biological control of exotic saltcedars (Tamarix spp.) and risks to non-target athel (T. aphylla) and native Frankenia spp.

Biological control of invasive saltcedars (Tamarix spp.) in the western U.S. by exotic tamarisk leaf beetles, Diorhabda spp., first released in 2001 after 15 years of development, has been successful. In Texas, beetles from Crete, Greece were first released in 2004 and are providing control. However, adults alight, feed and oviposit on athel (Tamarix aphylla), an evergreen tree used for shade and as a windbreak in the southwestern U.S. and México, and occasionally feed on native Frankenia spp. plants. The ability of tamarisk beetles to establish on these potential field hosts was investigated in the field. In no-choice tests in bagged branches, beetle species from Crete and Sfax, Tunisia produced 30–45% as many egg masses and 40–60% as many larvae on athel as on saltcedar. In uncaged choice tests in south Texas, adult, egg mass and larval densities were 10-fold higher on saltcedar than on adjacent athel trees after 2 weeks, and damage by the beetles was 2- to 10-fold greater on saltcedar. At a site near Big Spring, in west-central Texas, adults, egg masses and 1st and 2nd instar larvae were 2- to 8-fold more abundant on saltcedar than on athel planted within a mature saltcedar stand being defoliated by Crete beetles, and beetles were 200-fold or less abundant or not found at all on Frankenia. At a site near Lovelock, Nevada, damage by beetles of a species collected from Fukang, China was 12–78% higher on saltcedar than on athel planted among mature saltcedar trees undergoing defoliation. The results demonstrate that 50–90% reduced oviposition on athel and beetle dispersal patterns within resident saltcedar limit the ability of Diorhabda spp. to establish populations and have impact on athel in the field.     

Interaction dynamics between saprobic lignicolous fungi and Armillaria in controlled environments: Exploring the potential for competitive exclusion of Armillaria on peach

Armillaria root rot, caused by Armillaria tabescens and Armillaria mellea, is a major cause of premature tree death in peach orchards in the southeastern United States. The root systems of infected trees can become entirely colonized by Armillaria, serving as an inoculum source for adjacent trees and providing massive inoculum levels in replant situations. If dead or dying trees could be colonized by an effective competitor of Armillaria before their removal, the extent of root colonization by the pathogen could be reduced, thus decreasing the threat to adjacent trees and/or subsequent plantings. Interactions between five species of saprobic lignicolous fungi (Ganoderma lucidum, Hypholoma fasciculare, Phanerochaete velutina, Schizophyllum commune, and Xylaria hypoxylon) and the two Armillaria species were examined in controlled conditions to provide proof of concept for competitive exclusion of Armillaria from peach roots. On agar-coated glass slides, all five potential antagonists induced detrimental reactions in >58% of the Armillaria hyphae observed, with the majority resulting in hyphal swelling or granulation. On poplar wood blocks, all antagonists consistently either overgrew Armillaria colonies or—in the case of S. commune—engaged in deadlock reactions; in all cases, the viability of Armillaria colonies was reduced to <30% of that of unchallenged controls. When inoculated simultaneously onto opposite ends of peach root segments, all antagonists consistently reduced growth and viability of Armillaria on and under the bark, whereby reduction of pathogen growth underneath the bark, Armillaria’s primary ecological niche, was most pronounced for G. lucidum, S. commune, and X. hypoxylon. When root segments were allowed to be colonized entirely by Armillaria before being inoculated with the antagonists, the latter were able to overgrow the pathogen on the root surface but unable to pre-empt it from underneath the bark. In summary,G. lucidum, S. commune, and X. hypoxylon caused strong hyphal and mycelial interference reactions and the most pronounced reductions in growth of Armillaria above and below the bark, indicating that they would be the most promising candidates for field-scale evaluations to restrict colonization of dead or dying peach trees by Armillaria in the orchard.     

Elimination of EVE protein by CALI in the short germ band insect Tribolium suggests a conserved pair-rule function for even skipped

The question of the degree of evolutionary conservation of the pair-rule patterning mechanism known from Drosophila is still contentious. We have employed chromophore-assisted laser inactivation (CALI) to inactivate the function of the pair-rule gene even skipped (eve) in the short germ embryo of the flour beetle Tribolium. We show that it is possible to generate pair-rule type phenocopies with defects in alternating segments. Interestingly, we find the defects in odd numbered segments and not in even numbered ones as in Drosophila. However, this apparent discrepancy can be explained if one takes into account that the primary action of eve is at the level of parasegments and that different cuticular markers are used for defining the segment borders in the two species. In this light, we find that eve appears to be required for the formation of the anterior borders of the same odd numbered parasegments in both species. We conclude that the primary function of eve as a pair rule gene is conserved between the two species.     

Virulence of the entomopathogenic nematodes Heterorhabditis bacteriophora, Heterorhabditis zealandica, and Steinernema scarabaei against five white grub species (Coleoptera: Scarabaeidae) of economic importance in turfgrass in North America

We compared the virulence of the entomopathogenic nematodes Steinernema scarabaei, Heterorhabditis zealandica, and Heterorhabditis bacteriophora (GPS11 and TF strains) against third instars of the Japanese beetle, Popillia japonica, the oriental beetle, Anomala (=Exomala) orientalis, the northern masked chafer, Cyclocephala borealis, the European chafer, Rhizotrogus majalis, and the Asiatic garden beetle, Maladera castanea, in laboratory and greenhouse experiments. The virulence of the nematode species relative to each other differed greatly among white grub species. H. bacteriophora and H. zealandica had similar modest virulence to P. japonica, A. orientalis, C. borealis, and M. castanea. But against R. majalis, H. zealandica showed low virulence with a clear concentration response whereas H. bacteriophora caused only erratic and very low mortality. In contrast, S. scarabaei had modest virulence against C. borealis, but was highly virulent against R. majalis, P. japonica, A. orientalis, and M. castanea with R. majalis being the most susceptible and M. castanea the least susceptible.     

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.     

Horizontal transmission of a microsporidium from the convergent lady beetle, Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae), to three coccinellid species of Nova Scotia

Convergent lady beetles, Hippodamia convergens Guérin-Méneville, are a popular choice for aphid control in North America. An unidentified microsporidium was found in H. convergens adults that were purchased from a commercial insectary in 2004. This study examined egg cannibalism and egg predation as a means of horizontal transmission of the unidentified microsporidium among H. convergens larvae and three coccinellid species found in Nova Scotia: Coccinella septempunctata (seven-spotted lady beetle), C. trifasciata perplexa (three-banded lady beetle), and Harmonia axyridis (multicolored Asian lady beetle). The microsporidium was transmitted with 100% efficiency when first instars fed on microsporidia-infected eggs. Mean spore count data from smear preparations of infected beetles suggest that the infection was as heavy in C. trifasciata perplexa (a native coccinellid) (11.2 ± 0.96 spores/100 μm²) as it was in H. convergens (the natural host) (12.8 ± 1.16) but lighter in the introduced species C. septempunctata (7.5 ± 0.65) and H. axyridis (0.8 ± 0.11). For all of the beetle species examined, larval development was significantly longer for microsporidia-infected individuals than for their uninfected cohorts. The microsporidium had no effect on larval mortality. Based on the results of this study, field-collected H. convergens should be examined for microsporidia and uninfected individuals should be used to rear individuals for release in biological control programs. However, this is unlikely to happen because H. convergens are relatively easy and inexpensive to collect from their overwintering sites for redistribution.     

Molecular characterization,virulence and horizontal transmission of Beauveria pseudobassiana from Dendroctonus micans (Kug.) (Coleoptera: Curculionidae)

The great spruce bark beetle, Dendroctonus micans (Kugelann) (Coleoptera: Curculionidae), has been a potential threat for Turkey and the entire Eurasian spruce forests for many years. Control strategies which have been applied so far are still insufficient to prevent its damage. A previous study has shown that a Beauveria isolate (ARSEF 9271) proved to be an efficient microbial control agent against the great spruce bark beetle. In this study, this isolate was identified as B. pseudobassiana based on the partial sequence of EF1‐α and ITS sequence. A conidial suspension (1 × 10⁸/ml) of this fungus caused 100% mortality on both larvae and adults of D. micans within 5 and 6 days, respectively. Also, it caused 100% mycosis value on both larvae and adults. Mortality values of horizontal transmission experiments between larvae and adults which were contaminated with 1 × 10⁶/ml spore suspension at 25%, 50%, 75% and 100% rates were determined as 100% after 15 days at 20°C under the laboratory conditions. We also determined the decrease of the damage in spruce wood block (15 × 25 cm) when the contamination rate of the larvae was increased. Our results indicate that B. pseudobassiana ARSEF 9271 seems to be a very promising biocontrol agent against D. micans.     

The interactions of tropical soda apple mosaic tobamovirus and Gratiana boliviana (Coleoptera: Chrysomelidae), an introduced biological control agent of tropical soda apple (Solanum viarum)

Tropical soda apple (Solanum viarum Dunal (Solanaceae) is a South American invasive plant of rangelands, pastures and natural areas in Florida. A chrysomelid beetle from South America, Gratiana boliviana Spaeth, has been released at >300 locations in Florida for biological control of tropical soda apple since 2003. Tropical soda apple is a host of several plant viruses, including the newly described tropical soda apple mosaic virus (TSAMV). We investigated the influence of TSAMV infection of tropical soda apple plants on developmental time, leaf tissue consumption, longevity, fecundity, and feeding preference of G. boliviana, and also tested transmission of the virus by the beetle. Developmental time was approximately 10% slower, and adults consumed only about 50% as much leaf tissue, for beetles fed on infected plants compared to uninfected plants. Longevity did not differ between females reared on infected and uninfected plants, but females fed on uninfected plants produced 71% more eggs than those fed on infected plants. Adult G. boliviana preferentially fed on uninfected plants when given a choice. There was no evidence of TSAMV transmission by G. boliviana. The potential impacts of TSAMV infection on the effectiveness of G. boliviana as a biological control agent are discussed.     

Prior encounters modulate subsequent choices in host acceptance behavior by the bark beetle Ips pini

Laboratory bioassays indicate that the bark beetle Ips pini employs flexible, rather than absolute, responses to phytochemicals in its host acceptance behavior. Each beetle's decision to enter substrate was influenced by the types and concentrations of monoterpenes present. However, previous rejection of a simulated host containing a moderate concentration of monoterpenes increased the likelihood that the same concentration would be accepted upon a second or third encounter. This flexibility more than offsets any loss of vigor due to starvation and age that accompanies a process of trial and error. Starvation decreased beetles' total lipid content, but beetles can recover some energetic losses by a small amount of feeding during each trial. In addition to its adaptive value, a flexible host acceptance strategy may yield population level consequences. That is, bark beetles preferentially enter trees having low concentrations of monoterpenes, but may modify their acceptance thresholds when cues associated with stressed trees are not available. This could partially explain how some tree-killing bark beetles colonize a broader physiological range of trees during outbreaks. The adaptive value of relating individual decisions to population density may arise from two ecological relationships: first, as populations rise, the pool of stressed trees is rapidly depleted; secondly, healthy trees are attainable through pheromone – mediated mass attacks when adequate numbers of beetles are present. Flexible host acceptance behaviors may also reduce the advantage of relying exclusively on pre-landing cues to distinguish between susceptible and non-susceptible trees.