Quantification of Wolbachia copy number in Trichogramma eggs (Hymenoptera: Trichogrammatidae): Lysozyme treatment significantly improves total gene yield from the Gram-negative bacterium

The Wolbachia bacterium is one of the most prevalent endosymbionts of arthropods. The bacterium is known to induce four distinct reproductive manipulations in its various hosts; namely, cytoplasmic incompatibility, feminization, male killing and parthenogenesis induction. The bacterial titer has been thought to have an important role in expression of these phenotypes. Although host eggs of the bacterium are the place where all of the manipulations take place, in most cases, they are not big enough to be subjected to mechanical disruption to extract genetic materials from them. Furthermore, DNA extraction protocols generally involve washing steps in which part of the DNA is lost. With the simple three step digestion protocol we developed, we were able to preserve the whole genome and measure the titer of Wolbachia in individual Trichogramma eggs by real-time quantitative polymerase chain reaction (PCR), which was comparable to the titer by visual observation.     

Biological studies on two neotropical egg parasitoid species: Trichogramma nerudai and Trichogramma sp. (Hymenoptera: Trichogrammatidae)

The main biological attributes of two Neotropical egg parasitoids, the arrhenotokous Trichogramma nerudai and the thelytokous Trichogramma sp., were assessed under controlled laboratory conditions. Developmental time from egg to adult, and parasitoid survival, fecundity and fertility were studied using life tables. Results showed that T. nerudai had a faster developmental time than Trichogramma sp. (13.014±0.4019 and 13.595±0.4931 days, respectively). Both species showed similar life table statistics, r_m was 0.222 and 0.225 for T. nerudai and Trichogramma sp., respectively. Parasitoid survival averaged 95% for both species. The results obtained are discussed in the context of selecting one of these natural enemies as a potential biological control agent for the European pine shoot moth Rhyacionia buoliana (Lepidoptera: Tortricidae) in pine forests and the codling moth Cydia pomonella (Lepidoptera: Tortricidae) in apple orchards in Argentina.     

Comparison Between the Control Efficacy of Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) and Two Trichogramma cacoeciae Marchal Strains Against Grapevine Moth ( Lobesia botrana Den. & Schiff.), Depending on Their Release Density

In the first generation of the grape moth Lobesia botrana, Trichogramma evanescens spread to a greater extent than Trichogramma cacoeciae and parasitized most of the grape moth eggs. Reduction of damage to grapes was improved by doubling the number of T. evanescens release points in the first and second generations. In the second generation of the grape moth, T. cacoeciae was more efficient than T. evanescens . The highest proportion of individuals belonging to the reared and natural T. cacoeciae strains were found in the plot in which they were initially released. The total number of individuals emerging from parasitized eggs eggs was the same for each strain, which would suggest that neither predominated. Whichever T. cacoeciae strain considered, a density of 800 release points ha -1 did not induce greater parasitization than a density of 400 release points ha -1 .     

Effect of host egg viability on reproduction and development of Trichogramma cacoeciae and T. principium (Hymenoptera: Trichogrammatidae)

A combination of a sterile insect technique, resulting in infertile eggs, and of an egg parasitoid, should provide better control of codling moth than either alone. Non-choice laboratory experiments were conducted with infertile and fertile codling moth eggs to evaluate the potential parasitism and reproduction of Trichogramma cacoeciae Marchal and T. principium Sug. et Sor. The tendency of T. cacoeciae females to attack infertile eggs was similar to that for fertile eggs, whereas T. principium showed a greater preference for infertile eggs than fertile eggs. The fertility status of the host did not affect the number of eggs that were parasitized but fewer F₁ progeny emerged from infertile eggs when parasitized than from fertile eggs. When T. cacoeciae and T. principium parasitized infertile host eggs their mean developmental time was prolonged but the viability and quality of parasitoid progeny was not influenced by the fertility status of the host eggs. The study demonstrates the compatibility of use of T. cacoeciae and T. principium in an integrated program employing the sterile insect technique for codling moth management.     

wFlu: Characterization and Evaluation of a Native Wolbachia from the Mosquito Aedes fluviatilis as a Potential Vector Control Agent

There is currently considerable interest and practical progress in using the endosymbiotic bacteria Wolbachia as a vector control agent for human vector-borne diseases. Such vector control strategies may require the introduction of multiple, different Wolbachia strains into target vector populations, necessitating the identification and characterization of appropriate endosymbiont variants. Here, we report preliminary characterization of wFlu, a native Wolbachia from the neotropical mosquito Aedes fluviatilis, and evaluate its potential as a vector control agent by confirming its ability to cause cytoplasmic incompatibility, and measuring its effect on three parameters determining host fitness (survival, fecundity and fertility), as well as vector competence (susceptibility) for pathogen infection. Using an aposymbiotic strain of Ae. fluviatilis cured of its native Wolbachia by antibiotic treatment, we show that in its natural host wFlu causes incomplete, but high levels of, unidirectional cytoplasmic incompatibility, has high rates of maternal transmission, and no detectable fitness costs, indicating a high capacity to rapidly spread through host populations. However, wFlu does not inhibit, and even enhances, oocyst infection with the avian malaria parasite Plasmodium gallinaceum. The stage- and sex-specific density of wFlu was relatively low, and with limited tissue distribution, consistent with the lack of virulence and pathogen interference/symbiont-mediated protection observed. Unexpectedly, the density of wFlu was also shown to be specifically-reduced in the ovaries after bloodfeeding Ae. fluviatilis. Overall, our observations indicate that the Wolbachia strain wFlu has the potential to be used as a vector control agent, and suggests that appreciable mutualistic coevolution has occurred between this endosymbiont and its natural host. Future work will be needed to determine whether wFlu has virulent host effects and/or exhibits pathogen interference when artificially-transfected to the novel mosquito hosts that are the vectors of human pathogens.     

Review of Pasteuria penetrans: Biology,Ecology, and Biological Control Potential

Pasteuria penetrans is a mycelial, endospore-forming, bacterial parasite that has shown great potential as a biological control agent of root-knot nematodes. Considerable progress has been made during the last 10 years in understanding its biology and importance as an agent capable of effectively suppressing root-knot nematodes in field soil. The objective of this review is to summarize the current knowledge of the biology, ecology, and biological control potential of P. penetrans and other Pasteuria members. Pasteuria spp. are distributed worldwide and have been reported from 323 nematode species belonging to 116 genera of free-living, predatory, plant-parasitic, and entomopathogenic nematodes. Artificial cultivation of P. penetrans has met with limited success; large-scale production of endospores depends on in vivo cultivation. Temperature affects endospore attachment, germination, pathogenesis, and completion of the life cycle in the nematode pseudocoelom. The biological control potential of Pasteuria spp. have been demonstrated on 20 crops; host nematodes include Belonolaimus longicaudatus, Heterodera spp., Meloidogyne spp., and Xiphinema diversicaudatum. Pasteuria penetrans plays an important role in some suppressive soils. The efficacy of the bacterium as a biological control agent has been examined. Approximately 100,000 endospores/g of soil provided immediate control of the peanut root-knot nematode, whereas 1,000 and 5,000 endospores/g of soil each amplified in the host nematode and became suppressive after 3 years.     

Pattern of Egg Management by Trichogramma cacoeciae and T. dendrolimi (Hymenoptera: Trichogrammatidae)

The effect of temporary host deprivation on parasitization rates of Trichogramma cacoeciae Marchal and T. dendrolimi Matsumura was investigated. The study was conducted with females that we allowed to engage in 3 days of oviposition after various periods of host deprivation. It seems that the production and management of eggs by the two species is completely different. During the first day of oviposition, parasitization by T. cacoeciae was almost unaffected after 1 to 5 days of host deprivation. As deprivation time increased, however, the number of parasitized hosts decreased from an average of 28.6 ±2.0 hosts provided at emergence to an average of 12.5 ±2.3 hosts when the waiting time was 10 days. The number of hosts parasitized on the first day of parasitization by T. dendrolimi were not affected whatever the waiting test period. During the second or third days of oviposition, the lack of suitable hosts for T. cacoeciae did not depress egg-laying potentiality, whereas a strong reduction in parasitization rates by T. dendrolimi occurred in the next 2 days of oviposition whatever was the waiting period. This leads to ca. 50% reduction in total activity of 3 days of oviposition. Only in T. cacoeciae was it possible to distinguish between ageing and host deprivation. The data suggest that T. dendrolimi is a typical proovigenic species, while T. cacoeciae is neither definitely proovigenic nor synovigenic. A slight decrease in rate of emergence of offspring of T. cacoeciae females that had waited 8 to 10 days for their hosts was observed. As far as biological control is concerned, the efficiency of T. dendrolimi females may be more sensitive to host deprivation than T. cacoeciae.     

Establishment, Spread and Initial Impact of Aphis chloris Koch (Hemiptera: Aphididae), Introduced into Australia for the Biological Control of St John's Wort

The aphid, Aphis chloris Koch, quickly established following field release at numerous sites throughout south-eastern Australia. Local populations built up to levels at which migratory alates were produced within the first season. Dispersal of these alates over large distances led to widespread colonization of St John's wort infested areas within 2-3 years. Detailed observations at the primary release site at Pierce's Creek, ACT, showed that aphid populations undergo a seasonal cycle of population growth throughout the summer, followed by a migratory phase with alate production and dispersal in autumn and a population decline over winter. This pattern was repeated at other release sites monitored over a 5-year period from 1987 to 1991 inclusive, without populations building up to damaging levels. Natural enemies, competition from existing biological control agents, climatic factors and emigration appear sufficient to limit such outbreaks. While caged plants did show a decline in vigour and seed production under heavy aphid attack, such damage was intermittent in open field situations. The present study suggests that, while A. chloris is now widespread, it will not make an important contribution to the overall control of St John's wort.     

Geographic Variation in Egg Load of Microctonus hyperodae Loan (Hymenoptera: Braconidae) and its Implications for Biological Control Success

Eight South American geographic populations of the parthenogenic, proovigenic, koinobiont parasitoid Microctonus hyperodae Loan (Hymenoptera: Braconidae: Euphorinae) were introduced to New Zealand to assist management of the pasture pest Listronotus bonariensis (Kuschel) (Coleoptera: Curculionidae). Geographic variation in fecundity has been suggested as a reason for the populations' differential successes in establishing in New Zealand. This study investigated whether geographic variation in fecundity was due to corresponding variation in pre-oviposition egg load (other possible sources of fecundity variation include searching efficiency, egg survival and female longevity). Variation in egg load accounted for that in fecundity, but also showed that the variation in fecundity was not as great as it had first appeared. Geographic variation in egg load did not explain the pattern of population establishment observed in New Zealand. Egg load was proportional to parasitoid size and this relationship was stronger in populations originating from west, rather than east, of the Andes. A new method for making parasitoid eggs visible is described.     

Comparative biology and life tables of Trichogramma cacoeciae,T. brassicae and T. evanescens (Hymenoptera: Trichogrammatidae) with Ephestia kuehniella and Cadra cautella (Lepidoptera: Pyralidae) as hosts at three constant temperatures

Egg parasitoids of the genus Trichogramma (Hym. Trichogrammatidae) have been successfully utilized for biocontrol of several Lepidopteran pests worldwide. Because of their low host specificity Trichogramma can be mass reared more easily in large numbers and on different natural and factitious hosts. Life table parameters were assessed for Trichogramma cacoeciae Marchall, T. evanescens Westwood and T. brassicae Bezdenko on Ephestia kuehniella Zeller and Cadra cautella Walker at three temperatures (20, 25 and 30°C) as the potential factitious hosts for mass rearing. Female longevity ranged between 9.77 and 2.56 days in our experiments. Fecundity ranged from 73 to 91 parasitized eggs in C. cautella and 76–109 parasitized eggs in E. kuehniella. T. brassicae exhibit similar values of the intrinsic rate of increase ratio (r _m) 0.5407 and 0.5478; the finite rates of increase (λ) were 1.717 and 1.7295 and the doubling times 1.28 and 1.26 at 25 and 30°C, respectively, in C. cautella. The mean duration of one generation (T) varied between 8.25 and 13.37 days for T. cacoeciae, 8.14 and 13.47 days for T. brassicae and 8.23 and 13.18 days for T. evanescens in C. cautella. Generation times for T. cacoeciae varied between 8.23 and 14.68 days, 8.28 and 14.37 days for T. brassicae, and 7.74 and 14.58 days for T. evanescens in E. kuehniella eggs. Both species (T. cacoeciae, T. brassicae) had the same intrinsic rate of increase (r _m; 0.5700 and 0.5704), finite rate of increase (λ; 1.7682, 1.7691) and doubling time (Dt, 1.21) at 30°C in E. kuehniella, respectively.     

Biology and Host Range of the Hawk Moth Nephele densoi Keferstein (Lep.: Sphingidae), a Potential Agent for the Biological Control of Rubbervine Cryptostegia grandiflora R. Br. (Asclepiadaceae)

The larvae of the hawk moth, Nephele densoi Keferstein, feed on the leaves of Cryptostegia grandiflora R. Br. in Madagascar. Host-specificity tests showed that feeding by N. densoi is restricted to plants in the families Asclepiadaceae (subfamily Periplocoideae), Apocynaceae and Moraceae, but only plants in the subfamily Periplocoideae supported larval development. The comparatively wide host range contrasts with the narrow host range demonstrated for the Australian moth N. subvaria (Walker). The possible evolution of host plant specialization in this genus is discussed.     

Laboratory Trials of the Efficacy of the Crown Weevil Mortadelo horridus (Coleoptera: Curculionoidae) for the Biological Control of Slender Thistles Carduus pycnocephalus and C. tenuiflorus in Southwestern Australia

The hypothesis that larval crown weevil (Mortadelo horridus Alonso-Zarazaga and Sanchez-Ruiz) can suppress vegetative growth, seed set and viability in the winter annual slender thistles (Carduus pycnocephalus L. and C. tenuiflorus Curt.) was tested in laboratory gardens. Different densities (eight and 16) of M. horridus larvae and eggs were inoculated onto slender thistle rosettes between one and four times. Controls received no eggs or larvae. Larval feeding destroyed the apical meristems of treated plants, producing multi-crowned rosettes and significant declines in leaf area in both species, irrespective of the number of larvae. Treated thistles bolted at the same time and had a 4-fold increase in stem number compared to controls. The stems of control plants were up to one-third taller and thicker than attacked plants. Relative to controls, the leaves of treated plants had significantly fewer spines, decreased leaf area and decreased leaf perimeter, with less deeply lobed leaf margins. The total number of capitula produced by control and experimental groups was not significantly different, but consideration of fertile capitula only revealed a reduction in fertile capitula production following weevil attack. This was most marked in C. pycnocephalus. Control plants also produced significantly wider capitula. No control plants died, compared to 17 and 26% of the attacked plants of C. pycnocephalus and C. tenuiflorus, respectively. Implications for biocontrol of slender thistles are discussed.     

Developing Methods for Testing Host Specificity of Phymastichus coffea LaSalle (Hym.: Tetrastichinae), a Potential Biological Control Agent of Hypothenemus hampei (Ferrari) (Col.: Scolytidae) in Colombia

In line with the United Nations Food and Agriculture Organization Code of Conduct for the import and release of biological control agents, and taking into consideration the recommendations in the draft IUCN (International Union for Conservation of Nature and Natural Resources) Guidelines for the Prevention of Biodiversity Loss Due to Biological Invasion, a procedure was developed to assess the host range of Phymastichus coffea , an endoparasitoid of coffee berry borer, Hypothenemus hampei . Host specificity was tested by exposing a Kenyan biotype of P. coffea to a selection of Scolytidae under quarantine conditions. The results demonstrated that P. coffea can be considered as oligophagous, attacking and developing on other species of the genus of the target borer. In no-choice tests, three species, H. obscurus , H. seriatus and Araptus sp. yielded parasitism. The risk of attack to potential alternative hosts if P. coffea were to be released as a potential biological control agent of coffee berry borer in Colombia is discussed. A quarantine procedure was developed for the importation of P. coffea to Colombia to deal with specific problems of hyperparasitoids and fungal contamination.     

The Biology and Host Range of Falconia intermedia (Hemiptera: Miridae), a Potential Biological Control Agent for Lantana camara (Verbenaceae) in Australia

The life history and host specificity of Falconia intermedia in Australia were investigated. Adults and nymphs feed on the intercellular tissue on the underside of Lantana camara leaves. Eggs are deposited singly or in small clusters alongside veins and, on average, hatch in 12 days. Development to adult takes about 15 days and there are 5 instars. Females live for approximately 30 days and lay an average of 1.5 eggs/day. Oviposition occurred on all five L. camara phenotypes tested but subsequent development was significantly poorer on the pink-flowering phenotype. Forty-six plant species were tested to determine host specificity. The only species upon which adults fed and oviposited were L. camara and another introduced weed, Lippia alba. Both plant species supported populations of F. intermedia over several generations. F. intermedia did not display any predatory behaviour towards eggs, nymphs or larvae of either Aconophora compressa or Ectaga garcia, two other introduced biocontrol agents of L. camara. F. intermedia was approved for release in Australia in 2000.     

Biological Control of Rice Blast byPseudomonas fluorescensStrainPf7–14: Evaluation of a Marker Gene and Formulations

Pseudomonas fluorescensstrainPf7–14 was evaluated for biological control of rice blast in field experiments. StrainPf7–14 was formulated in methylcellulose:talc (1:4) and applied to IR50 rice (Oryza sativa) seeds as a seed treatment and as foliar sprays in seedbed and field experiments. When applied as a seed treatment followed by three foliar applications, the strain provided a 68.5% suppression of rice blast in the seedbed experiment and a 59.6% suppression in the field experiment. The persistence and migration ofPf7–14 on the rice plant was studied with the aid oflacZYgenes inserted into the bacterium. In greenhouse experiments,Pf7–14gal was detected on rice roots at 10⁶to 10⁵cfu/g of root tissue for 110 days, the duration of the rice crop. Migration of the strain from the seeds to the leaves occurred only until the seedlings were 16 days old. WhenPf7–14 was applied to the rice plants by foliar sprays, 10⁴cfu of the bacterium per gram of leaf tissue was detected for the next 40 days. The limited migration of the bacterial biocontrol agent emphasizes the need for multiple foliar applications of the bacterium to sustain the bacterial population for effective suppression of rice blast.     

Biological Control of the Bulb Mite, Rhizoglyphus robini , by the Predatory Mite, Hypoaspis aculeifer , on Lilies: Predator-Prey Dynamics in the Soil, under Greenhouse and Field Conditions

We tested the capacity of the soil-dwelling predatory mite, Hypoapsis aculeifer , to control mites attacking lily bulbs. Experiments in the greenhouse and in the field showed that in the absence of predatory mites populations of the bulb mite, Rhizoglyphus robini , on lily bulbs increased, whereas the release of predatory mites either slowed down the increase - as observed in the field - or caused the bulb mites populations to decrease - as observed in the greenhouse. In all cases the population of predatory mites increased as long as bulb mite densities were not too low. However, within the first week after predator release there was usually a sharp decline to 10-40% of the original number released. Greenhouse experiments on intact lily bulbs in pots, boxes and 1 m 2 plots with peat soil showed that when released in a ratio of 1 predator to 2 or 5 prey, the predatory mite, Hypoaspis aculeifer , suppressed populations of bulb mites to less than 10 individual per bulb within 6 weeks. Elimination of bulb mites was observed only when the predator-to-prey ratio at release was equal to 3:1. Field experiments in 2 m 2 plots with intact bulbs in rather compact sandy soil showed that when released in ratio of 1 predator to 1 or 2 prey, the predatory mite, H. aculeifer , did not cause the population of bulb mites to decrease, but it did reduce their population growth. The initial predator-to-prey ratios required to achieve suppression (ca 1:2) or elimination (3:1) in the soil environment are much higher than those required for bulb mite elimination when lily bulb scales were embedded in a medium of vermiculite (ca 1:20). Among the possible causes are: (1) the initial losses of predators in the greenhouse and even more so in the field due to mortality and/or emigration from the experimental plots; (2) the lower temperatures in the greenhouse and especially in the field, which slow down the growth and predation processes and thereby delay prey extinction; and (3) the spatial complexity of the soil environment which creates refuges for the bulb mites.     

Evaluation of the Predatory Wasp, Ancistrocerus gazella, for Biological Control of Leafrollers in Otago Fruit Crops: 1. Prey Composition, Nest Structure and Wasp Productivity from Artificial Nests

Groups of artificial nest tubes were deployed in the field for colonization by the accidentallyimported wasp, Ancistrocerus gazella (Panzer), a reported important predator of lepidopterous larvae, especially pest tortricids. There were 3-4 nest sites in each of seven study areas, and four tube lengths (75, 100, 125, 150 mm) were compared. Nests were dissected at various stages after occupancy to determine the prey composition and nest structure, and the numbers and sex of adults produced. The wasps collected larvae of nine lepidopterous families, including many tortricids, but pest leafrollers comprised only 4.1% and 1.5% of all larvae collected respectively in 1995-96 and 1996-97. Prey included beneficial species feeding on weeds and the larvae of some non-pest native insects. Prey species composition was highly site-dependent and was a major determinant of the numbers of larvae per cell. Cells for male wasps were smaller and contained fewer larvae than those for females. Tube length did not affect colonization rates but more cells and adult wasps were produced in longer tubes. The results cast serious doubt on the proposed management of A. gazella for biological control of leafrollers, but suggest that this approach could be useful for other pests, such as Coleophora spp., which were collected in large numbers.     

Potential Non-target Effects of a Biological Control Agent, Prickly Pear Moth, Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), in North America, and Possible Management Actions

Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), the poster child of biological control, has recently invaded the United States. The first US record was at Big Pine Key, Florida, in 1989. Since then it has moved rapidly northward into South Carolina. Fears are high that it will disperse, either on its own, or with human help, into the US desert southwest and Mexico. There are at least 31 species of prickly pear in the US that are likely to be attacked by Cactoblastis and 56 species in Mexico. As well as the threat to wild cacti, there are over 250,000 ha of Opuntia plantations in Mexico that support a thriving agricultural industry, most of which is centered on harvesting fruits or pads. Possible control measures include classical biological control using parasitoids or pathogens from South America, chemical control or F₁ sterility, as has been used successfully for the codling moth. However, most of these options appear to have insurmountable difficulties. Classical biological control raises the fear of further non-target effects of natural enemies on native cactus herbivores. Chemical control has potential non-target effects on other (rare) insects and is expensive. F₁ sterility is also expensive and is not self-sustaining, requiring considerable and continual human input. Nevertheless, research on control options is vital as is further work on the rate of spread and impact of Cactoblastis in the United States Southeast, so that we can be as well prepared as possible to deal with this threat when it arrives in Arizona, California, and Mexico.