Use of tetracycline in larval diet to study the effect of Wolbachia on host fecundity and clarify taxonomic status of Trichogramma species in cured bisexual lines

Endosymbionts of the genus Wolbachia were efficiently cured from Trichogramma species by incorporating 0.02% tetracycline into the artificial diet used to rear larvae. Use of this technique yielded stable cured lines (bisexual and arrhenotokous lines) in which no Wolbachia organisms were detected by PCR for up to 14 generations after curing. Four cured strains of Trichogramma pretiosum showed a significantly lower total fecundity compared to their Wolbachia-infected counterpart. However, the fecundity of a single cured strain of Trichogramma evanescens was similar to its Wolbachia-infected counterpart. These differences in the effect on fecundity may be due to differences between the Wolbachia strains infecting T. pretiosum or T. evanescens, providing additional evidence for the hypothesis that a specific interaction exists between some Trichogramma species and their Wolbachia symbionts. Tetracycline in larval diet was also used to generate bisexual strains of Trichogramma oleae and Trichogramma cordubensis so that these species could be crossed with the closely related species, respectively, T. pretiosum and T. evanescens, to test their compatibility. These crosses showed a lack of compatibility, validating maintenance of these as distinct species.     

Effects of repeated thermic and antibiotic treatments on a Trichogramma (Hym., Trichogrammatidae) symbiont

Each individual of all populations of the thelytokous species Trichogramma cordubensis is infected by Wolbachia . Populations are composed of females, a few rare males and some intersexual or gynandromorphic individuals often capable of normal reproduction as females. Antibiotic (minocycline) and high temperature (30°C) treatments inactivate Wolbachia and induce male production. After these treatments, the sex ratio depending on the remaining frequency of thelytoky shows a genetic variability. Effect of cycles with and without treatments (minocycline or 30°C) on the thelytokous reproduction of 'individuals' (one 'individual' being one Trichogramma + n Wolbachia ) was studied. The resistance of thelytokous reproduction to 30°C but not to minocycline, probably due to Wolbachia genes, increased. It is concluded that potentialities of adaptation to high temperature exist in Wolbachia and can explain a part of the spreading of these symbionts among host populations.     

安荔赤眼蜂在中国野外首次发现及其体内Wolbachia的检测

【目的】为丰富赤眼蜂Trichogramma的种类资源,明确野外新采集的一种赤眼蜂的种类,探明该赤眼蜂所感染Wolbachia的类型。【方法】采用挂米蛾Corcyra cephalonica卵卡法在华南农业大学树木园诱集到两批赤眼蜂,通过形态鉴定和PCR扩增ITS2序列并测序分析的分子鉴定手段对野外采集的赤眼蜂材料进行种类鉴定;通过PCR扩增Wolbachia的外膜蛋白基因(wsp)序列,检测赤眼蜂体内Wolbachia的感染情况;通过PCR扩增wsp序列和多位点序列分型(multilocus sequence typing, MLST)对检测到的赤眼蜂体内的Wolbachia进行同源性分析。【结果】所诱集到的两批赤眼蜂均被鉴定为安荔赤眼蜂Trichogramma oleae Voegelé & Pointel,体内Wolbachia的感染率达100%。该Wolbachia株系与安荔赤眼蜂(前南斯拉夫品系)、短管赤眼蜂T. pretiosum(乌拉圭品系)以及T. deion(荷兰品系)体内Wolbachia亲缘关系较近,属于B超组Sib亚组,对应MLST序列型为ST486。【结论】安荔赤眼蜂T. oleae为中国野外首次发现,是完全感染Wolbachia的产雌孤雌生殖品系。本研究为害虫生物防治提供了一种新的天敌种类资源,并为进一步探明Wolbachia与赤眼蜂的互作提供了研究材料。     

Biological Control Potential of Wolbachia -infected Versus Uninfected Wasps: Laboratory and Greenhouse Evaluation of Trichogramma cordubensis and T. deion Strains

The effects of thelytoky-inducing Wolbachia ( &#102 -proteobacteria) on Trichogramma cordubensis and T. deion (Hymenoptera, Trichogrammatidae) were studied in laboratory and greenhouse conditions. One infected (thelytokous, all female) line of each wasp species was compared with its conspecific uninfected (arrhenotokous, sexual) counterpart for several fecundity and dispersal traits. Arrhenotokous lines had a higher fecundity than their thelytokous counterparts, which suggests that Wolbachia negatively affect the fecundity of Trichogramma females. The arrhenotokous females dispersed more in the laboratory than their thelytokous counterparts. In the greenhouse, the opposite effect or no difference between lines was found, indicating that the laboratory set-up used to measure dispersal is not useful to predict relative dispersal of the females in the greenhouse. Calculations show that by releasing 100 adult wasps of both lines, thus including arrhenotokous males in the sexual line, more eggs are parasitized by the thelytokous wasps. Therefore, in spite of their lower individual female fecundity, thelytokous lines have a better potential for biological control than their arrhenotokous counterparts.     

Comparison of Hemolymph and Holotissues of Different Species of Insects as Diet Components forin VitroRearing ofTrichogramma galloiZucchi andT. pretiosumRiley

The aim of this work was to compare pupal holotissues (Diatraea saccharalis, Anticarsia gemmatalis,andBombyx mori) and larval hemolymph of several moth species (Helicoverpa zeaandBombyx mori) commonly produced in Brazilian laboratories as components of artificial diets forin vitrorearing ofTrichogramma galloiandTrichogramma pretiosum.Diets based on larval hemolymph ofH. zeaproduced the best development of both natural enemies, although a diet with pupal holotissues ofB. moriallowed full development ofT. pretiosum.Amino acid analysis showed qualitative differences among the insect derivates and eggs of the natural and alternative hosts, but with a quantitative similarity. Electrophoretic analysis resulting in a diverse protein-stained bands evidenced the qualitative differences among these components.     

Naturally occurring Trichogramma species in olive farms in Egypt

A survey of two-year studies (2001-2003) was carried out in two olive groves sited at two representative olive growing areas, namely Paradise Park (arid area) and Burg E1-Arab farm (semi-arid area) to monitor the frequency of endemic Trichogramma species on olive moth (Prays oleae) and jasmine moth (Palpita unionalis). The suspended host bait traps were found to be a more practical and effective tool for collecting Trichogramma wasps than the attached ones. Four naturally occurring Trichogramma species were collected for the first time in Egypt from the olive groves, where releases have never been conducted. T.bourarachae was collected exclusively from Burg El-Arab farm. It seems that this wasp species adapts well to the semi-arid area. Three species, namely T. cordubensis, T. nr.pretiosum and T. cacociae were isolated from Paradise Park farm. All of these wasps were also bred from naturally parasitized host eggs during favorable and even at unfavorable temperature conditions of June-August. However, these endemic species did not occur naturally in sufficient numbers to keep the pest populations from reaching damaging levels.The excessive usage of insecticides and the oophagous predators (e.g., ants and lacewing larvae) are some factors that affect the performance of Trichogramma wasps in olive farms.The presence of warm weather wasp strains suggests the existence of well-adapted wasp species or swains which may be appropriate candidates for the control of target pests in olive groves. Additional study is required to determine the best “habitat-specific” species/strains of Trichogramma for augmentative release of naturally occurring wasps and to incorporate them into integrated pest management programs. Efforts should be made to conserve these endemic species from oophagous predators, hot weather and insecticides.The olive and jasmine moth-larvae and pupae found under tree canopies were bred and emerged parasitoids were listed.     

Host-foraging success of three species of Trichogramma (Hymenoptera: Trichogrammatidae) in a simulated retail environment

Three species of trichogrammatid egg parasitoids (Trichogramma deion Pinto & Oatman, Trichogramma ostriniae Pang & Chen, and Trichogramma pretiosum Riley) (Hymenoptera: Trichogrammatidae) were evaluated under laboratory conditions as potential biological control agents for the Indianmeal moth, Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), on retail shelves. A single shelving unit was used in each trial and a grid of sentinel egg disks was used to evaluate foraging success. The shelving consisted of pallet units with five shelves that were either bare or stocked with empty cereal boxes. In each replicate, approximately 500 female Trichogramma were released at the center of the shelving unit and allowed to forage for 48 h. Percentage of egg parasitism and percentage of host egg mortality were recorded after 7 d. Foraging success as well as the spatial pattern of parasitism differed significantly among the three Trichogramma species. Percentage of egg parasitism was approximately 4 times greater for T. deion than for T. ostriniae or T. pretiosum. The vertical distribution of parasitism by T. deion was also more uniform than for the other two species. In addition, the presence of packaging affected the foraging efficiency of T. ostriniae and T. pretiosum but not T. deion. Based on these findings, Trichogramma deion may be the best-suited candidate for augmentative biological control of P. interpunctella in retail stores, and a central release point of T. deion will likely provide adequate coverage of products on pallet-type shelving.     

米蛾体内Wolbachia的wsp基因序列测定与系统发育分析

Wolbachia是广泛分布于节肢动物体内的一类共生菌, 它们参与多种调控寄主的生殖活动机制。通过对wsp基因的特异性扩增和测序,发现了Wolbachia在米蛾Corcyra cephalonica (Stainton)体内的感染。利用所测序列和其他已发表的序列建立系统树,结果表明米蛾体内Wolbachia属于B大组的Pip类群,与其寄生物茧蜂及赤眼蜂中的Wolbachia各株系遗传距离相差较远。据此推测米蛾体内感染的Wolbachia不是由寄生物（茧蜂、赤眼蜂）水平传播所致。     

Do sympatric Trichogramma species parasitize the pest insect Helicoverpa armigera and the beneficial insect Chrysoperla carnea in different proportions?

Parasitism of two host species by five Trichogramma species (Hymenoptera, Trichogrammatidae) was studied in the laboratory. The host species were: i) the bollworm Helicoverpa armigera Hübner (Lepidoptera, Noctuidae), an important pest of many crops in the tropics and subtropics, and ii) one of its natural enemies, the lacewing Chrysoperla carnea Stephens (Neuroptera, Chrysopidae), a predator often used as a biological control agent. The proportion of H. armigera eggs parasitized from the total number of parasitized hosts differed between Trichogramma species. The average number of parasitized eggs per female in 24 h by Trichogramma pintoi and T. bourarachae was 10 of H. armigera and about 0.5 of C. carnea. For the other three Trichogramma species (T. cordubensis, T. evanescens and T. turkestanica) these averages varied from 6 to 11 H. armigera eggs and from 3 to 4 C. carnea eggs. Total adult offspring production, contacts with hosts, secondary clutch size and sex-ratio of each Trichogramma species were determined as well. The results show that sympatric Trichogramma may parasitize target and non-target species in different proportions. If this difference corresponds to the field situation, simple laboratory tests could be performed to select not only efficient biogical control agents, but also species which are the least detrimental to non-target hosts.     

Influence of Diaphania hyalinata (L.) (Lepidoptera: Crambidae) egg density on the parasitization capacity of Trichogramma exiguum Pinto & Platner and Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae)

The effects of the egg density of Diaphania hyalinata (L.) on several biological parameters of Trichogramma exiguum Pinto & Platner and T. pretiosum Riley were investigated. For that, 24h-old egg masses were isolated in glass tubes (15 replicates; 1 egg mass = 1 replicate), and offered to parasitization by a newly-emerged female of T. pretiosum or T. exiguum 24h at the proportion of one female to 5, 10, 15, 20, 25, 30, 35 and 40 eggs of D. hyalinata. The following parameters were evaluated: number of parasitized eggs, number of individuals per egg, viability and sex ratio. Trichogramma exiguum parasitized more eggs than T. pretiosum when more than 25 eggs were available per female. The percentage of emergence was satisfactory to T. pretiosum in densities up to 15 eggs/female and up to 20 eggs/female for T. exiguum. The number of individuals per egg was not statistically different in both species except in the density of 25 eggs/female. It can be concluded that T. exiguum performed better than T. pretiosum at larger clutch sizes, as T. exiguum parasitization capacity increased as a result of the size of the host clutch size.     

Wolbachia感染对拟澳洲赤眼蜂寿命、生殖力和嗅觉反应的影响

研究了通过共享同一寄主卵,短管赤眼蜂Trichogramma pretiosum体内共生的Wolbachia被水平传递到拟澳洲赤眼蜂T. confusum体内后,Wolbachia对新宿主拟澳洲赤眼蜂的影响。结果表明： Wolbachia的侵染能使拟澳洲赤眼蜂进行不完全的产雌孤雌生殖,增加拟澳洲赤眼蜂子代雌性比例,但却导致了雌蜂寿命缩短和繁殖力降低的生理损失。Wolbachia感染的当代处女蜂及其建立的种群连续5代(F₁～F₅),其子代雌蜂百分率分别为79.17%、76.60%、68.66%、72.58%、68.15%和64.06%,基本上呈现出逐代降低的趋势,并越来越接近对照的63.85%。处女蜂及F₁～F₅代雌蜂的平均寿命分别为4.33、5.50、5.60、6.68、7.32和7.50天,而未感染交配雌蜂寿命为7.59 天;处女蜂及F₁～F₅代雌蜂平均产卵量分别为11.33、70.00、86.41、93.90、102.92和124.38粒/雌,除F₅代外,均显著低于未感染交配雌蜂的产卵量134.81粒/雌。用四臂嗅觉仪测定了Wolbachia新宿主拟澳洲赤眼蜂对寄主小菜蛾的嗅觉反应,结果表明Wolbachia的侵染具轻微干扰拟澳洲赤眼蜂嗅觉反应的负面影响。未感染Wolbachia的拟澳洲赤眼蜂及Wolbachia供体短管赤眼蜂对寄主小菜蛾具较强的嗅觉反应,其雌蜂在小菜蛾腹部鳞片正己烷提取液和小菜蛾卵表正己烷提取液处理区内滞留时间显著或极显著长于对照区。而感染Wolbachia的拟澳洲赤眼蜂F₂代和F₃代雌蜂,尽管在小菜蛾腹部鳞片正己烷提取液处理区内滞留时间比对照区长,但没有达到显著水平;其F₂代雌蜂在小菜蛾卵表正己烷提取液处理区内滞留时间与对照区相比,也没有达到显著水平。随Wolbachia在拟澳洲赤眼蜂种群中垂直感染代数的增加,拟澳洲赤眼蜂对寄主小菜蛾的嗅觉反应恢复正常,在Wolbachia感染后的 F₄～F₆代,雌蜂在两种提取液处理区内的滞留时间均显著或极显著长于对照区。     

Collection of Trichogramma Westwood (Hymenoptera: Trichogrammatidae) from tropical northern Australia: a survey of egg parasitoids for potential pest insect biological control in regions of proposed agricultural expansion

Abstract  Trichogramma limit pest damage to crops by killing the developing embryo of their insect host at the egg stage. Their impact on the potentially insecticide resistant species, Helicoverpa armigera (Hübner), is considered integral to the resistance management plan for transgenic cotton production in the Ord River Irrigation Area in northern Western Australia. The dominant species of egg parasitoid in Ord River Irrigation Area cotton crops is the introduced Trichogramma pretiosum (Riley). Surveys of similar northern Australian regions earmarked for agricultural expansion revealed that T. pretiosum has been introduced, or has adventitiously dispersed, to all developing agricultural regions of northern Australia. Several previously unsequenced species were collected during surveys in more pristine habitats. Trichogramma pretiosum demonstrates an apparent ability to supersede native Trichogramma species and is perhaps favoured by agricultural expansion.     

Wolbachia在玉米螟赤眼蜂内的三重感染

Wolbachia是一类广泛存在于节肢动物体内的共生菌。玉米螟赤眼蜂Trichogramma ostriniae是我国玉米田间的优势赤眼蜂种, 据报道, 赤眼蜂种内有Wolbachia感染。本文利用Wolbachia的16s rDNA和wsp基因引物通过PCR方法对玉米螟赤眼蜂的野生种群进行了调查, 发现以wsp基因为鉴定依据, 检测的所有个体都感染了3种Wolbachia ［wOstGDAa (GenBank accession no. EU157103), wOstGDAb (GenBank accession no. EU157104) 和 wOstGDB (GenBank accession no. EU157105)］。本文首次报道了野生赤眼蜂种群内Wolbachia的三重感染率几乎为100%。根据本研究的结果, 可以推测当不同种赤眼蜂寄生同一寄主时, Wolbachia可能会在不同赤眼蜂种间进行横向传播。     

Addition of wsp Sequences to the Wolbachia Phylogenetic Tree and Stability of the Classification

Wolbachia are symbiotic bacteria altering reproductive characters of numerous arthropods. Their most recent phylogeny and classification are based on sequences of the wsp gene. We sequenced wsp gene from six Wolbachia strains infecting six Trichogramma species that live as egg parasitoids on many insects. This allows us to test the effect of the addition of sequences on the Wolbachia phylogeny and to check the classification of Wolbachia infecting Trichogramma. The six Wolbachia studied are classified in the B supergroup. They confirm the monophyletic structure of the B Wolbachia in Trichogramma but introduce small differences in the Wolbachia classification. Modifications include the definition of a new group, Sem, for Wolbachia of T. semblidis and the merging of the two closely related groups, Sib and Kay. Specific primers were determined and tested for the Sem group.     

Parasitism of the Coffee Berry Borer Hypothenemus hampei by Trichogramma pretiosum in the Laboratory

Parasitism of the coffee berry borer, Hypothenemus hampei (Ferrari) by Trichogramma pretiosum Riley resulted in high mortality of developing parasitoids and a low prevalence of adult emergence. A laboratory colony of T. pretiosum reproducing in H. hampei failed after three generations. Adult female T. pretiosum that developed in H. hampei were smaller and produced fewer eggs than conspecifics that developed in a standard lepidopteran host, Sitotroga cerealella (Olivier). Parasitoids that emerged from H. hampei preferentially parasitized S. cerealella over H. hampei. We conclude that T. pretiosum has little potential for biocontrol of the coffee berry borer.     

Wolbachia在我国不同地理种群的玉米螟赤眼蜂中的分布

Wolbachia是一类可以引起寄主生殖行为改变的共生菌,广泛存在于节肢动物内。玉米螟赤眼蜂Trichogramma ostriniae Pang et Chen是我国亚洲玉米螟Ostrinia furnacalis(Guene)卵寄生蜂的优势种。本文利用Wolbachia的16SrDNA基因特异性引物进行PCR扩增以及克隆测序,对我国14个地区的玉米螟赤眼蜂种群中Wolbachia的感染情况做了详细的调查。结果发现,被测的1412头野生玉米螟赤眼蜂全部感染有Wolbachia。系统发育分析表明,北京柳沟、山东菏泽、河南郑州、河南驻马店、湖南长沙5个种群存在超感染现象。这是Wolbachia在玉米螟赤眼蜂种群内分布情况的首次报道。     

两种赤眼蜂在半自然条件下对灰白蚕蛾卵的搜索寄生能力

灰白蚕蛾Ocinara varians Walker是近几年来我国南方发生比较严重的园林害虫之一,赤眼蜂是其卵期的一种重要天敌,为寻找灰白蚕蛾有效的生物防治措施,本实验研究了半自然条件下松毛虫赤眼蜂Trichogramma dendrolimi Matsumura和短管赤眼蜂Trichogramma pretiosum Riley对灰白蚕蛾卵的搜索寄生能力。实验结果表明,在挂卵量相同的情况下,3种不同经历的松毛虫赤眼蜂和短管赤眼蜂对灰白蚕蛾卵的搜索能力都随蜂卵比的减小而降低。在各蜂卵比下,3种不同处理的松毛虫赤眼蜂对灰白蚕蛾卵的寄生率明显高于相应地3种不同处理的短管赤眼蜂。在蜂卵比为1∶1时,具有羽化后学习经历的松毛虫赤眼蜂对灰白蚕蛾卵的搜索能力最强,其寄生率达50.66%,显著高于幼期学习蜂和无经验蜂,而幼期学习经历对其搜索效果无明显地影响。短管赤眼蜂无论是羽化后学习经历还是幼期学习经历对其搜索行为均无明显影响。     

Comparative Analysis of the Reproductive Attributes of Three Commercially-Produced Trichogramma Species (Hymenoptera: Trichogrammatidae)

The reproductive attributes of commercially-produced Trichogramma platneri, T. minutum and T. pretiosum reared from eggs of both Ephestia kuehniella and Sitotroga cerealella were monitored at 25 C. The age-specific fecundity, longevity and progeny sex ratio and adult size were determined for individual females from all six combinations of Trichogramma and rearing host, using E. kuehniella as the experimental host. Rearing host had a significant influence on the lifetime fecundity, 3-day fecundity and longevity of all three Trichogramma species. In general, the performance of T. minutum and T. pretiosum was better when reared from S. cerealella, but that of T. platneri was superior when reared from E. kuehniella. The lifetime fecundity of the Trichogramma species was linearly related to longevity and the ranking between species was T. pretiosum > T. minutum > T. platneri. The age-specific pattern of oviposition for T. platneri was distinctly precocious, with 40% of its lifetime fecundity oviposited on the first day, in contrast to 17-24% for the other two species. Progeny sex ratio over the lifetime of the Trichogramma females was slightly male biased and differed significantly from 0.5 for T. minutum and T. platneri. Daily sex ratio for parasitoids reared from the most productive rearing host was female biased only for the first day of oviposition for T. platneri in contrast to the first 5-6 days of oviposition for the other two species. There was little evidence that any of the reproductive attributes of these Trichogramma species, reared from small host eggs, was dependent on the size of the adult females.     

Behavior of Trichogramma atopovirilia Oatman & Platner and T. pretiosum Riley (Hymenoptera: Trichogrammatidae) on Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) egg masses.

In this study we analyzed the impact of physical barriers of Spodoptera frugiperda (J. E. Smith) egg-masses on the behavior of Trichogramma atopovirilia Oatman & Platner and Trichogramma pretiosum Riley. The duration of drumming, drilling, oviposition, period spent over the egg-mass, and interval between parasitized eggs were timed, and the number of parasitized eggs were recorded. The presence of scales on the egg-masses caused a significant increase in the time spent by both parasitoids on each process and a decrease in the residence time over the egg-mass and in the number of parasitized eggs, with an increase in the number of egg layers. There was a significant decrease in the number of parasitized eggs in relation to egg-masses with one layer and no scales. We observed that the physical barriers in fall armyworm egg-masses changed the behavior of T. atopovirilia and T. pretiosum, affecting their parasitization capacity.     

Estimating parasitoid immature mortality by comparing oviposition and pupal development of Trichogramma galloi Zucchi and T. pretiosum Riley on natural and factitious hosts

Mortality during the immature development of T. galloi and T. pretiosum was estimated on UV-killed and live eggs of a factitious and a natural host, respectively. A staining technique was used to determine the actual parasitization of UV-treated eggs and was compared with the number of parasitoids that emerged per host egg (detectable parasitization). Effects of temperature as a factor of mortality during the immature development of both parasitoids on the factitious host was also assessed. The actual and detectable parasitization of live hosts was measured by recording both the parasitization behavior and the number of eggs where a parasitoid developed successfully. Our data show that mortality during immature development of both parasitoids may occur in live eggs of the natural host. No such mortality was observed when parasitoids developed on UV-killed eggs of the factitious host. Possible causes of parasitoid immature mortality and the effects of using UV-treated eggs of factitious hosts in estimating the parasitism capacity of Trichogramma in field conditions are discussed.