Phytoplasma Transmission: Insect Rearing and Infection Protocols.

Phytoplasmas are obligate pathogens and thus they can be studied only in association with their plants or insect hosts. In this chapter, we present protocols for rearing some phytoplasma insect vectors, to obtain infected insects and plants under controlled environmental conditions. We focus on Euscelidius variegatus and Macrosteles quadripunctulatus that can infect Arabidopsis thaliana, and Hyalesthes obsoletus and Scaphoideus titanus, that can infect grapevine.

Indigenous strains of Beauveria and Metharizium as potential biological control agents against the invasive hornet Vespa velutina.

Alien species often miss parasites in their invaded area, and this is the case in Vespa velutina. This invasive hornet predator of bees was accidentally introduced in Europe from East China in 2004. The control of this species is still problematic. Indeed to destroy nests, applicators currently use large spectrum insecticides, which is too costly or dangerous to applicators and also to the environment, affecting non-targeted arthropods (one period). Studying the potential interest of biological control methods may help to propose alternatives in V. velutina control. We present here the bioassays in which we assessed the potential control efficiency of different indigenous French isolates of entomopathogenic fungi. We inoculated adults V. velutina by different ways: being directly, by walking on a contaminated surface, in the food, or by inter-individual transfers. We tested differences between the isolates and the application methods using two parameters mortality and LT50. The direct inoculation method was the most efficient modality, then the contact, transfer and food. Considering all contamination methods, there was no difference on susceptibility or mortality among different isolates. Still the LT50 was quite short in all isolates (average 5.8±0.44d), and their virulence was quite high: we conclude that there is high potential in using such entomopathogens as a biological control agent against V. velutina.

Evaluation of competition between a native and an invasive hornet species: do seasonal phenologies overlap?

One common dogma in ecology is based on the competitive exclusion principle. Hence, competition is often considered to be one of the primary determinants of the structure and functioning of ecosystems. In this paper, we investigate how the native Vespa crabro and the recently introduced Vespa velutina show some degree of niche differentiation that potentially minimizes their interspecific competition, the two dimensions investigated here being seasonal activity patterns and preferences for food. These two species share common characteristics: they are closely related, live in the same areas, belong to the same guild (predators), exploit the same kind of food sources, and exhibit a similar annual life cycle. Considering all these similarities, interspecific competition may occur if the two species exhibit identical seasonal phenologies. Our data show that their seasonal phenologies overlap to some extent probably due to biological constraints common to Vespinae. The shifts in time observed here allow the hornet species to not directly compete for food sources at the same time. It does not however exclude indirect competition, especially in a 'first-come, first-served' fashion.

Phenological synchrony between Scaphoideus titanus (Hemiptera: Cicadellidae) hatchings and grapevine bud break: could this explain the insect's expansion?

Scaphoideus titanus is the invasive vector of the phytoplasma causing the Flavescence dorée in European vineyards. This epidemic is a serious threat to viticulture that has been increasing for more than 60 years in Europe. We studied the effect of synchrony with the plant phenology and the effect of plant-sap quality on the individual fitness. Thus, we conducted laboratory experiments to determine if insect hatchings were synchronized with grapevine bud break. We used two natural populations: one from a cold winter vineyard and one from a mild winter vineyard. In both cases, egg hatching was synchronized with bud break and leaf appearance. The phloem quality of the young and old leaves as a food source was analysed by high-performance liquid chromatography, and the effects on S. titanus growth were evaluated. Phloem composition varied with the grapevine cutting's age but also varied between leaves of different ages from the same plant. The older leaves were less nutritious because they had the highest carbon-to-nitrogen ratio and the lowest content of essential amino acids. Despite diverse phloem qualities, no fitness difference was observed. We found that the synchronization of egg hatchings with bud break is well regulated. However, the nymphs are not affected by the phloem-sap quality, suggesting that S. titanus may accept different food qualities and that egg hatching synchrony could contribute to population expansion in vineyards.

Larval intraspecific competition for food in the European grapevine moth Lobesia botrana.

Effective pest management with lower amounts of pesticides relies on accurate prediction of insect pest growth rates. Knowledge of the factors governing this trait and the resulting fitness of individuals is thus necessary to refine predictions and make suitable decisions in crop protection. The European grapevine moth, Lobesia botrana, the major pest of grapes in Europe, is responsible for huge economic losses. Larvae very rarely leave the grape bunch on which they were oviposited and thus cannot avoid intraspecific competition. In this study, we determined the impact of intraspecific competition during the larval stage on development and adult fitness in this species. This was tested by rearing different numbers of larvae on an artificial diet and measuring developmental and reproductive life history traits. We found that intraspecific competition during larval development has a slight impact on the fitness of L. botrana. The principal finding of this work is that larval density has little effect on the life history traits of survivors. Thus, the timing of eclosion, duration of subsequent oviposition, fecundity appears to be more uniform in L. botrana than in other species. The main effect of larval crowding was a strong increase of larval mortality at high densities whereas the probability of emergence, sex ratio, pupal mass, fecundity and longevity of mated females were not affected by larval crowding. Owing to increased larval mortality at high larval densities, we hypothesized that mortality of larvae at high densities provided better access to food for the survivors with the result that more food was available per capita and there were no effect on fitness of survivors. From our results, larval crowding alters the reproductive capacity of this pest less than expected but this single factor should now be tested in interaction with limited resources in the wild.

Different emergence phenology of European grapevine moth (Lobesia botrana, Lepidoptera: Tortricidae) on six varieties of grapes.

The phenology of insect emergence affects reproductive success and is especially critical in short-lived species. An increasing number of studies have documented the effects of thermal and other climatic variations and of unpredictable habitats on the timing of adult insect emergence within and between populations and years. Numerous interacting factors may affect the phenology of adult emergence. Host-plant quality and availability is a key factor that has been largely neglected in studies of the phenology of phytophagous insects. The purpose of this study was to determine the effect of host plant characteristics on the rate of larval growth and the pattern of emergence in a wild population of Lobesia botrana (European grapevine moth), a significant pest in European vineyards. The phenology of emergence differed significantly among the six tested varieties of grapes. The percentage of bunches harboring pupae was similar among the different grape varieties, and the total number of pupae collected was similar to the number of emerging adults per bunch. Among the six varieties of grapes, 0-25 pupae were produced on each bunch. Each of the grape varieties had a single wave of emergence, in which males emerged before females, but their emergence phenology differed significantly in Chardonnay, Chasselas, and Pinot grapes. Both genders had extended durations of emergence in Merlot grapes. Together, the present results show that the characteristics of the grape host plant affect the emergence phenology of L. botrana.

Larval host plant origin modifies the adult oviposition preference of the female European grapevine moth Lobesia botrana.

According to the 'natal habitat preference induction' (NHPI) hypothesis, phytophagous insect females should prefer to lay their eggs on the host species on which they developed as larvae. We tested whether this hypothesis applies to the breeding behaviour of polyphagous European grapevine moth, Lobesia botrana, an important pest in European vineyards. We previously found that different grape cultivars affect several life history traits of the moth. Because the different cultivars of grapes are suspected to provide different plant quality, we tested the NHPI hypothesis by examining oviposition choice of L. botrana among three Vitis vinifera cultivars (Pinot, Chasselas and Chardonnay). In a choice situation, females of L. botrana that had never experienced grapes were able to discriminate between different grape cultivars and preferentially selected Pinot as an oviposition substrate. This 'naive' preference of oviposition could be modified by larval environment: Females raised on grapes as larvae preferred to lay eggs on the cultivar that they had experienced. Furthermore, experience of the host plant during adult emergence could be excluded because when pupae originating from our synthetic diet were exposed to grapes, the emerging adults did not show preference for the cultivar from which they emerged. The NHPI hypothesis that includes the two sub-hypothesis "Hopkins host selection principle" and "chemical legacy" may thus be relevant in this system.

Does natural larval parasitism of Lobesia botrana (Lepidoptera: Tortricidae) vary between years, generation, density of the host and vine cultivar?

Populations of European grapevine moth Lobesia botrana Denis & Schiffermüller and its larval parasitoids were studied for two consecutive years on an experimental insecticide-free vineyard in France planted with adjacent plots of five grape cultivars (Merlot, Cabernet Franc, Sauvignon, Cabernet Sauvignon and Sémillon) using a natural L. botrana population during the first year, and a natural population supplemented with artificially inoculated individuals during the second year. Levels of natural populations of larval parasitoids were measured by their parasitism rate. The ichneumonid Campoplex capitator Aubert was the most common species collected from L. botrana larvae. Its incidence was higher during the spring compared to summer. The overall parasitism rate found on the experimental vineyard varied from 23% in 2000 to 53% in 2001, and was mainly due to C. capitator. Parasitism was not affected by the grape cultivar on which the host developed but was positively correlated with the host density, per bunch or per stock, suggesting that among the five grape cultivars tested, C. capitator females probably do not discriminate between hosts feeding on different grape cultivars, but rather the densities of L. botrana larvae.

Grape variety affects larval performance and also female reproductive performance of the European grapevine moth Lobesia botrana (Lepidoptera: Tortricidae).

For insect herbivores, the quality of the larval host plant is a key determinant of fitness. Therefore, insect populations are supposed to be positively correlated with the nutritional quality of their host plant. This study aimed to determine if and how different varieties of grapes (including the wild grape Lambrusque) affect both larval and adult performance of the polyphagous European grapevine moth Lobesia botrana (Denis & Schiffermüller). Significant differences were found in larval development time, but not in pupal mass, adult emergence rate, or sex ratio. Although the fecundity of females is not different among varieties, females fed on some varieties produced eggs of different sizes which are correlated to their fertility. Thus, females adapt resource allocation to eggs depending on their diet as larvae. Using a fitness index, the average reproductive output was found to be highest for females reared on cv. Chardonnay. Females reared on wild grape produced a fitness index identical to the cultivated grapes. However, Lambrusque and Gewurztraminer separate themselves from the cultivated varieties according to our discriminant analyses. It is emphasized, through this study, that cultivars fed on by larvae should be considered in the population dynamics of L. botrana and that egg number is insufficient to determine host plant quality.

A bioassay to evaluate the activity of chemical stimuli from grape berries on the oviposition of Lobesia botrana (Lepidoptera: Tortricidae).

A two-choice bioassay was developed to evaluate the role of host-plant berry compounds on the oviposition site acceptance of the generalist moth Lobesia botrana (Denis & Shiffermüller). A key feature was the lining of the bioassay arena with felt which focused oviposition on the test substrates. Initial experiments comparing substrates with different physical features indicated that smooth textures and spherical shapes with interstices favour oviposition. Artificial oviposition substrates were thus constructed with glass spheres in order to test the behavioural activity of grapevine berry extracts. Only polar extracts obtained by soaking berries in methanol or water stimulated oviposition (more eggs were laid on the extract-treated substrate than on the control substrate), whereas more apolar ones obtained with chloroform or hexane had no significant effect. The prior removal of epicuticular waxes from grape berries before extraction did not enhance the stimulatory activity of the methanol extract. The oviposition response to this extract was dose-dependent. It is concluded that polar compounds present on grape berries act as oviposition stimulants for L. botrana.

Coincident resection-recession (tenectomy-recession) of the medial rectus muscle, bilaterally, for the treatment of esotropia: results in 18 cases.

PURPOSE: To study the effect of preceding tenectomy of the medial rectus tendon on the results of medial rectus muscle recession. SUBJECTS AND METHODS: Eighteen consecutive cases of incomitant esotropia were retrospectively reviewed. The average preoperative esotropia was 35 PD with an incomitant deviation between 10 PD and 30 PD. All patients underwent 7 mm bilateral medial rectus muscle recession after 4 mm tenectomy of the anterior medial rectus muscle. RESULTS: Fourteen patients (78%) had "satisfactory" results (within 10 PD static esodeviation and 12 PD dynamic deviation [incomitance]). Four (22%) were undercorrected. One showed a postoperative consecutive exotropia of 4 PD at distance only. CONCLUSIONS: Tenectomy of the anterior muscle tendon preceding large recessions of the medial rectus was effective in reducing the frequency of overcorrection (consecutive exotropia). Undercorrection did not appear to be more common.

Oviposition response ofLobesia botrana females to long-chain free fatty acids and esters from its eggs.

Avoidance of occupied ovisposition sites supposes that females perceive information related to their own progency. Fatty acids identified from egg extracts have been reevaluated using a different extraction method, and we have investigated the dose-dependent oviposition response of European grape vine moths (Lobesia botrana) to myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, methyl palmitate, methyl oleate, and ethyl palmitate; all except ethyl palmitate have been identified from eggs ofL. botrana. A methylene dichloride extract of eggs fromL. botrana revealed the presence of saturated free fatty acids (myristic, palmitic, and stearic) and unsaturated acids (palmitoleic, oleic, linoleic, and linolenic) in amounts ranging from 3.9 ng/egg equivalent for myristic acid to 30 ng/egg equivalent for palmitic and oleic acids. The extract also contained traces of methyl palmitate and methyl stearate. The greatest avoidance indexes were observed in response to palmitic, palmitoleic, and oleic acids. All the other compounds tested caused weaker responses. A reduction in the number of eggs laid was observed when moths were exposed to each of the esters applied at 0.3 µg per application spot. Reduction in eggs laid was also observed at a 10-fold higher dose of oleic acid. The present results confirm that general and simple molecules can be involved in the regulation of oviposition site selection and that they may participate in chemical marking of the eggs.

Egg dispersion in codling moth: Influence of egg extract and of its fatty acid constituents.

Codling moth females (Cydia pomonella, Lepidoptera: Tortricidae) (CM) usually lay single eggs and have a tendency to disperse. In a first experiment we observed that single females exposed to 20 apples distribute their eggs regularly among apples, suggesting a dispersive oviposition behavior. In a dual-choice situation, isolated females avoided oviposition on areas of cardboard treated with a methylene dichloride egg extract at the dose of ca. 1.0 egg equivalent/cm(2). A strong avoidance was obtained in response to a 20-fold dose, which was accompanied by a significant reduction of total oviposition. Seven major compounds found by GC analyses in the methylene dichloride extract of 2 to 3-day-old eggs were saturated or unsaturated C14-C18 straight-chain fatty acids: myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid. This was confirmed by GC-MS analysis of an extract made by ethyl ether. Smaller amounts of three methyl esters were also identified as methyl myristate, methyl palmitate, and methyl stearate. A blend of the seven fatty acids (FA) mimicked rather well the avoidance provoked by the extract, and this avoidance was confirmed by choice between treated and untreated fruits by single females. The treatment of apples with the 7FA mixture induced an aggregative distribution of the eggs among apples. We also found that the amounts of fatty acids harvested in the extract depend on the egg age. Amounts of fatty acids increased until eggs were 4 days old and then decreased before hatching. In this paper we discuss the possible role of simple molecules, such as fatty acids and their esters, as semiochemicals indicative of juvenile tissues.

Fluid air exchange in vitreo retinal surgery.

This study evaluates the efficiency of fluid-air exchange on the reattachment of the retina and clarifies the possibility that a posterior retinotomy is a cause for intra- and postoperative complications. A consecutive series of 211 eyes with retinal detachments due to P.V.R. (47%), diabetic traction, perforating trauma, macular hole or giant tears is presented. All eyes underwent pars plana vitrectomy, fluid-air exchange, internal drainage of subretinal fluid, laser endophotocoagulation and scleral buckling of the tears; 56% of the eyes were phakic and 55% underwent a posterior retinotomy, 54% underwent tamponade with C3F8 and 46% with silicone oil. Intraoperatively the retina was completely flattened in 91% cases. The causes of incomplete reattachment were residual membranes (6.6%), poor visualization (1.4%) and suprachoroidal hemorrhage (1%). These complications were isolated as being the cause of the bad final results (p < 0.001). Postoperatively, the retina remained attached in 66% of cases after the first procedure and with further surgery in 81% (mean follow up 16 months). Best corrected visual acuity was improved in 73% of eyes, unchanged in 17%, and worse in 10%. Complications were retraction of the retinotomy site in 3 cases and peripheral choroidal hemorrhage in 4 cases. We concluded that fluid air exchange with internal subretinal fluid drainage was an efficient and safe technique even if a posterior retinotomy was necessary.

To penetrate or not to penetrate? A behavioral choice by bean beetle first-instar larvae in response toPhaseolus vulgaris seed surface quality.

Survival ofAcanthoscelides obtectus larvae depends on the ability of the first instar to pierce the seed coat ofPhaseolus vulgaris, which represents a critical sequence because of physical characteristics and toxicity. We have investigated the influence of seed surface quality on larval boring behavior by the usual method of surface washing with different solvents, or by removing the testa, or by coating the testa with a polymer spray. Observations were made on isolated larvae in no-choice and dual-choice bioassays. In the no-choice situation, larval penetration was reduced after seed coats were soaked with chloroform, whereas water, diethyl ether, or methanol had no significant effect. The ratio of boring attempts to successful penetrations was increased on seeds washed with chloroform. In the dual-choice situation, larvae avoided artificially coated seeds and preferred untreated seeds over those washed with chloroform or methanol. These results indicate that boring stimulants exist on the seed coat and that they are removed by chloroform and methanol or made inaccessible by artificial coating. The high mortality of first instars on seeds washed with these two solvents is attributed to a lack of chemicals necessary to initiate and sustain boring behavior, causing larval stress, possibly due to prolonged locomotory activity and starvation. Thus, seed coat quality may influence the population dynamics ofA. obtectus.

Acute haematogenous osteomyelitis of the patella revealed by a pathological fracture in multiple myeloma.

A 57-year-old man was followed up for multiple myeloma with no lytic bone lesions. During a septicaema, he presented with a pathological fracture of the patella. Despite a poor haematological status, he received surgical treatment, which allowed diagnosis of haematogenous osteomyelitis of the patella. The outcome was favourable, with a 6-week plaster immobilization after cerclage and 4 months of intravenous antibiotic therapy. This case emphasizes the fact that one well-known cause of bone lesions may lead to an erroneous diagnosis. The successful clinical course suggests that accurate therapeutic management may be discussed in haematological malignancies when intensive-care support is available.

Floral volatiles ofTanacetum vulgare L. attractive toLobesia botrana den. et schiff. females.

The European grapevine moth (EGVM),Lobesia botrana, is a major pest of grapes in Europe. Females are attracted to a nonhost plant: tansy (Tanacetum vulgare L.), which is a common weed in Slovakian vineyards. A steam distillate extract of tansy flowers was analyzed by means of a GC-EAG technique to screen constituents detected by the olfactory receptors of EGVM females. From more than 200 GC peaks, nine peaks corresponding to monoterpenoids released an EAG response in more than 70% of the females (N=15):p-cymene,d-limonene,α-thujene,α-thujone,ß-thujone, thujyl alcohol, terpinene-4-ol, (Z)-verbenol, and piperitone. The steam distillate of tansy as well as a synthetic blend of identified compounds released consistent attraction in a field cage. The use of nonhost plants and host plant odors in integrated pest management is discussed.

Biological evidence of an oviposition-deterring pheromone inLobesia botrana Den. et Schiff. (Lepidoptera, Tortricidae).

Females of the European grapevine moth (Lobesia botrana Den. et Schiff.) usually deposit isolated eggs on flowers and berries of the grapevine (Vitis vinifera L.). We have investigated whether an epideictic pheromone could be present on the egg surface to explain this spacing behavior. About 21,000 eggs ofL. botrana were washed in cold methanol, and the biological activity was tested in a two-choice bioassay offering treated and nontreated areas. Different dilutions of the extract were tested in methanol, which was inactive alone. At the dose of four egg equivalents per microliter of extract, the number of eggs laid by theL. botrana females was reduced by as much as 57% on the treated areas compared to nontreated areas. A longer-term suppression of oviposition (at least 24 hr) following an exposure to the extract occurred for two doses (0.6 and 4.0 eggs/µl) of egg extract. Our results strongly suggest the occurrence of an oviposition-deterring pheromone (ODP) on the eggs ofL. botrana. The ecological value of these results is discussed.

Sunflower aroma detection by the honeybee : Study by coupling gas chromatography and electroantennography.

Combined electrophysiological recordings (EAG) and gas chromatographic separation were performed in order to investigate which volatile chemical components of a sunflower extract could be detected by honeybee workers and thus are likely to trigger the foraging behavior. A direct coupling device allowed for the stimulation of the antennal receptors with individual constituents of a polar fraction of the flower aroma shown to be attractive to bees. More than 100 compounds were separated from the extract. Twenty-four compounds elicited clear EAG responses. These compounds were identified by mass spectrometry (electronic impact and chemical ionisation). Both short- and long-chain aliphatic alcohols, one short-chain aliphatic aldehyde, one acid, two esters, and terpenic compounds were found to stimulate the antennal receptors. Six compounds identified in previous behavioral experiments were found to exhibit EAG activity. The chemicals screened by this method may be used for recognition of the plant odor and the selective behavior of honeybees.

Misleading the Colorado potato beetle with an odor blend.

Walking tracks of Colorado potato beetles,Leptinotarsa decemlineata Say, were recorded on a locomotion-compensator in response to wind, odors of host plantsSolanum tuberosum L. and nonhost plantsLycopersicon hirsutum f.glabratum C.H. Mull, and to mixtures of these plant species. Host-plant odor induced positive anemotactic responses in starved females, whereas odor of the nonhostL. hirsutum was neither repellent nor attractive. The attractiveness of host-plant odor, however, was neutralized in the odor blend of plant species. Masking the attractive host-plant odor will hinder the beetle's searching for host-plant patches, and this principle may be exploited in pest control by mixed cropping.