Loss of Genetic Variability Induced by Agroecosystems: Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae) as a Case Study

Four species of green lacewings occur in Brazil, of which Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae) exhibits the widest geographical distribution. Chrysoperla externa is a predatory insect that is potentially useful as a biological control agent of agricultural pests. Studies on the genetic diversity of lacewing populations are essential to reduce the environmental and economic harm that may be caused by organisms with a low ability to adapt to the adverse and/or different environmental conditions to which they are exposed. We used the cytochrome oxidase I mitochondrial gene as a molecular marker to investigate the genetic diversity of green lacewing species collected from native and agroecosystem environments. Populations derived from native areas showed higher rates of genetic variability compared to populations from agroecosystems. Demographic changes in the form of population expansion were observed in agroecosystems, whereas populations in the native environment appeared stable over time. A statistical analysis showed significant genetic structure between each of the sampled groups, combined with its complete absence within each group, corroborating each group’s identity. We infer that the loss of variability exhibited by populations from the agroecosystems is the result of genetic drift by means of the founder effect, a similar effect that has been observed in other introduced populations. Agroecosystems might therefore function as exotic areas for green lacewings, even when these areas are within the normal range of the species.     

Predation capacity and prey preference of <Emphasis Type="Italic">Chrysoperla carnea</Emphasis> on <Emphasis Type="Italic">Pieris brassicae</Emphasis>

The predation capacity and prey preference of larvae of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) on eggs or larvae of Pieris brassicae (Linnaeus) (Lepidoptera: Pieridae) in the absence and presence of cabbage aphids as an alternative prey were evaluated in laboratory experiments at 25°C. Both instars preyed upon butterfly eggs and larvae as well as on cabbage aphids with the third instar being the most voracious. The lacewings had a strong preference for caterpillars to butterfly eggs. In the presence of the aphids the predation on P. brassicae eggs or larvae was either completely abandoned or reduced by about 70%, respectively, by second instar lacewings and either reduced by about 80% or maintained, respectively, by third instar lacewings. Both instars thus had a clear preference for aphids compared to eggs of P. brassicae. However, second instar lacewings preferred aphids to caterpillars whereas the opposite was the case for third instar lacewings. The results indicate that 3rd instar C. carnea has a potential as biocontrol agent against P. brassicae.     

Metschnikowia noctiluminum sp. nov., Metschnikowia corniflorae sp. nov., and Candida chrysomelidarum sp. nov., isolated from green lacewings and beetles

Fourteen yeast isolates belonging to the Metschnikowia clade were isolated from the digestive tracts of lacewings (Neuroptera: Chrysopidae), soldier beetles and leaf beetles (Coleoptera: Cantharidae and Chrysomelidae), and a caddisfly (Trichoptera: Hydropsychidae). The insect hosts were associated with sugary substances of plants, a typical habitat for yeasts in this clade. Based on DNA sequence comparisons and phenetic characters, the yeasts were identified as Candida picachoensis, Candida pimensis, and four undescribed taxa. Among the undescribed taxa, three yeasts were distinguished from one another and from other described taxa by nucleotide differences in the ribosomal DNA repeat, which were sufficient to consider them as new species. Two of the novel yeast species are described as Metschnikowia noctiluminum (NRRL Y-27753^T) and M. corniflorae spp. nov. (NRRL Y-27750^T) based in part on production of needle-shaped ascospores, which are found in most Metschnikowia species. Sexual reproduction was not observed in the third new yeast, Candida chrysomelidarum sp. nov. (NRRL Y-27749^T). A fourth isolate, NRRL Y-27752, was not significantly distinct from Metschnikowia viticola and Candida kofuensis to be described as a new species. Phylogenetic analysis of the D1/D2 loop sequences placed M. noctiluminum within the M. viticola clade, while C. chrysomelidarum was a sister taxon of Candida rancensis. Metschnikowia corniflorae was phylogenetically distinct from other new species and fell outside of the large-spored Metschnikowia group.     

Yeast Modulation of Human Dendritic Cell Cytokine Secretion: An In Vitro Study

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. The concept of individual microorganisms influencing the makeup of T cell subsets via interactions with intestinal dendritic cells (DCs) appears to constitute the foundation for immunoregulatory effects of probiotics, and several studies have reported probiotic strains resulting in reduction of intestinal inflammation through modulation of DC function. Consequent to a focus on Saccharomyces boulardii as the fundamental probiotic yeast, very little is known about hundreds of non-Saccharomyces yeasts in terms of their interaction with the human gastrointestinal immune system. The aim of the present study was to evaluate 170 yeast strains representing 75 diverse species for modulation of inflammatory cytokine secretion by human DCs in vitro, as compared to cytokine responses induced by a S. boulardii reference strain with probiotic properties documented in clinical trials. Furthermore, we investigated whether cytokine inducing interactions between yeasts and human DCs are dependent upon yeast viability or rather a product of membrane interactions regardless of yeast metabolic function. We demonstrate high diversity in yeast induced cytokine profiles and employ multivariate data analysis to reveal distinct clustering of yeasts inducing similar cytokine profiles in DCs, highlighting clear species distinction within specific yeast genera. The observed differences in induced DC cytokine profiles add to the currently very limited knowledge of the cross-talk between yeasts and human immune cells and provide a foundation for selecting yeast strains for further characterization and development toward potentially novel yeast probiotics. Additionally, we present data to support a hypothesis that the interaction between yeasts and human DCs does not solely depend on yeast viability, a concept which may suggest a need for further classifications beyond the current definition of a probiotic.     

Yeasts found on an ephemeral reproductive caste of the leaf-cutting ant Atta sexdens rubropilosa

Winged males of leaf-cutting ants are considered an ephemeral reproductive caste only produced before the mating flight season. Although much is known about the yeast diversity found in fungus gardens of attine ants, no study has focused on the yeasts associated with males of leaf-cutting ants. Here, we surveyed the yeasts on the integuments of males of Atta sexdens rubropilosa and assessed their potential role in the attine ant-microbe symbiosis. Using culture-dependent techniques, we found yeasts to be abundant on the integuments of males (54.5 %, n = 200 alates). A total of 242 yeast strains were obtained representing six orders, ten genera and 25 species. Strains of Aureobasidium, Cryptococcus, Hannaella and Rhodotorula were prevalent on the integuments and likely originated from the fungus garden of the parental nest or from the soil. The majority of strains (87.1 %) produced at least one of the evaluated enzymes: pectinase, polygalacturonase, cellulase, xylanase, ligninases and lipase. Aureobasidium pullulans accounted for the highest number of strains that produced all enzymes. In addition, yeasts showed the ability to assimilate the resulting oligosaccharides, supporting observations of other studies that yeasts may be involved in the plant biomass metabolism in the fungus gardens. Because winged males harbor several yeasts with putative functional roles, these fungi may take part and be beneficial in the microbial consortia of the new incipient nest.     

Rearing <Emphasis Type="Italic">Chrysoperla externa</Emphasis> Larvae on Artificial Diets

We tested three artificial diets for rearing larvae of Chrysoperla externa (Hagen) (Neuroptera: Chrysopidae), aiming at reducing the production costs of this predator. Two of the diets come from studies with other species of lacewings, and the third is a modification described in this paper. All diets were based on animal protein and were supplied to 2nd and 3rd instar larvae, whereas 1st instar larvae received eggs of Anagasta kuehniella (Zeller) (Lepidoptera: Pyralidae). We evaluated the preimaginal duration and survival, adult size, longevity and fecundity, egg hatchability, and predatory capacity of larvae produced. The performance of the diets was followed for seven generations. The diet we describe showed to be the best among the artificial diets tested. Our results show that C. externa can be successfully reared on artificial diets during second and third instars, reducing in 90% the dependency on eggs of A. kuehniella.     

Potential for augmentative biological control of black bean aphid in California sugarbeet

In northern California, black bean aphid (Aphis fabae Scopoli complex) can be a major pest of sugarbeet, particularly in spring-planted fields. The major natural enemies of the aphid are predators, including the coccinellidsHippodamia convergens Guerin,Coccinella novemnotata Herbst &C. septempunctata L., and the chrysopidsChrysopa oculata Say &C. nigricornis Burmeister. Augmentative releases of eggs of eitherChrysoperla carnea (Stephens) orC. rufilabris (Burmeister) failed to significantly reduce aphid populations under field conditions. This was attributed in part to incompatibility between black bean aphids and lacewing larvae from commercial sources. Application of food sprays containing yeast hydrolysate, sucrose and molasses resulted in increased densities of eggs and adults ofC. carnea in treated plots. These results suggest that the potential for augmentative biological control of black bean aphid through the application of food sprays is greater than that for release of commercially available lacewings. Regardless of method, an holistic approach to augmentation that takes into account the ecological structure of the target agroecosystem will be required. Some aspects of “pre-emptive” biological control are discussed.     

Yeasts in the flowers of entomophilic plants of the Moscow region

Dynamics of abundance and diversity of epiphytic yeasts in entomophilic flowers of 28 species of meadow, forest, and cultivated plants throughout their blooming period was determined. The number of yeasts in the flowers was shown to increase gradually during the vegetation period, and reached the maximum during summer-autumn. The total abundance and ratio of the yeast species in the flowers depended entirely on the blooming time, rather than on the taxonomic position of the plants. Three stages of development of the entomophilic yeast complexes during the vegetation period may be discerned: predominance of eurybiont nonspecific species (Cryptococcus albidus, Debaryomyces hansenii) in spring, mass development of specific nectar-associated yeasts (Metschnikowia reukaufii) in summer, and their substitution by widespread epiphytic species (Rhodotorula mucilaginosa, Cryptococcus magnus) in autumn.     

Manipulation of oviposition and overwintering site choice of common green lacewings with synthetic lure (Neuroptera: Chrysopidae)

Common green lacewings (Chrysoperla carnea species complex) are important predators of several insect pests, particularly aphids. Due to their potential in biological control, several efforts have been made to find means to retain common green lacewing adults or even to increase their numbers at a given site. In the course of field experiments conducted in Hungary and Norway, we tested the effect of a known ternary bait (phenylacetaldehyde, methyl salicylate, acetic acid) on oviposition and overwintering site choice of common green lacewings. In an orchard in Hungary, overwintering chambers were set up with and without baits. For the experiment on oviposition in Norway, a number of lacewing eggs were compared on trees with and without baits in selected sectors of the canopy. With the ternary bait, more common green lacewings could be attracted to overwintering chambers, and also significantly more eggs were found in the vicinity of baits than in other selected parts of the canopy, suggesting that this bait has the potential to manipulate both overwintering site choice and oviposition site choice of these important beneficial insects.     

Massive isolation and identification of <Emphasis Type="Italic">Saccharomyces paradoxus</Emphasis> yeasts from plant phyllosphere

Year-round studies of epiphytic yeast communities revealed that the number of ascosporogenous yeasts of the genus Saccharomyces inhabiting living and decaying leaves of some plants increased considerably in certain short periods (at the beginning of summer and in winter). Massive isolation of saccharomycetes was performed from 11 plant species; earlier, these yeasts had been revealed mainly in sugar-rich substrates. The isolates were identified as Saccharomyces paradoxus based on their physiological properties and RELP analysis of 5.8S-ITS. Possible reasons for short-term increases in the number of saccharomycetes in plant phyllosphere are discussed.     

Instar-specific defense ofColeomegilla maculata lengi (Col.: Coccinellidae): Influence on attack success of the intraguild predatorChrysoperla rufilabris (Neur.: Chrysopidae)

We conducted a laboratory study of the behavioural interactions between larval stages of the lady beetleColeomegilla maculata lengi Timberlake and an intraguild predator, the lacewingChrysoperla rufilabris Burmeister. Ladybird susceptibility to predation by third instarC. rufilabris is age-specific and influenced by larval defensive strategies. First and second instars ofC. maculata were captured more consistently by chrysopid larvae than third and fourth instars. Young coccinellids generally exhibit escape reactions (dropping, fleeing, retreating) but, do not survive after being caught by lacewings. In addition to escape reactions, older larvae may actively defend themselves by wriggling or biting, even after being pierced. The proportion of physical interactions that resulted in coccinellid death was significantly lower for older instars (<2%) than for first (35%) and second (19%) instars. Capture efficiency byC. rufilabris was related to the coccinellid-chrysopid size ratio. Dropping off the leaves, the most common defensive behaviour displayed by coccinellid larvae, contributed to reduce overall mortality of first instars to a level similar to the one observed for older stages. However, lacewings were able to prevent coccinellid dropping by impaling and holding their prey in the air, or by falling with the ladybirds to the ground where they continued their attack.     

Interrelationships between yeast fungi and collembolans in soil

The possibility of feeding on green and newly fallen leaves of the small-leaved lime Tilia cordata was studied for the collembolans Protaphorura armata and Vertagopus pseudocinereus. Young leaves grown under sterile conditions and almost free of yeast fungi were found to be toxic to the collembolan V. pseudocinereus: feeding on them led to the death of the animals. Leaves grown under natural conditions were nontoxic: when used by the collembolans as feed, they provided for collembolan growth and fecundity. Feeding preferences of the collembolans in relation to the yeasts attributed to different ecomorphs—epiphytes, litter saprophytes, pedobionts, and saccharobionts—were studied. Of the 24 yeast strains isolated from plant green parts, litter, and soil and assigned to eight species, no strain was revealed that was not used by the collembolans. However, certain yeast strains were preferable for the collembolans. The population of the V. pseudocinereus collembolans feeding on the yeast Rhodotorula glutinis (nss 31–4) exceeded that grown on Cryptococcus terricola (2044) 1.5-fold. Thus, the collembolans have feeding preferences in relation to yeast fungi, as was shown earlier with mycelial micromycetes. The possible mechanisms of the feeding preferences of the collembolans in relation to yeasts are discussed.     

Biotechnology of non-Saccharomyces yeasts—the basidiomycetes

Yeasts are the major producer of biotechnology products worldwide, exceeding production in capacity and economic revenues of other groups of industrial microorganisms. Yeasts have wide-ranging fundamental and industrial importance in scientific, food, medical, and agricultural disciplines (Fig. 1). Saccharomyces is the most important genus of yeast from fundamental and applied perspectives and has been expansively studied. Non-Saccharomyces yeasts (non-conventional yeasts) including members of the Ascomycetes and Basidiomycetes also have substantial current utility and potential applicability in biotechnology. In an earlier mini-review, “Biotechnology of non-Saccharomyces yeasts—the ascomycetes” (Johnson Appl Microb Biotechnol 97: 503–517, 2013), the extensive biotechnological utility and potential of ascomycetous yeasts are described. Ascomycetous yeasts are particularly important in food and ethanol formation, production of single-cell protein, feeds and fodder, heterologous production of proteins and enzymes, and as model and fundamental organisms for the delineation of genes and their function in mammalian and human metabolism and disease processes. In contrast, the roles of basidiomycetous yeasts in biotechnology have mainly been evaluated only in the past few decades and compared to the ascomycetous yeasts and currently have limited industrial utility. From a biotechnology perspective, the basidiomycetous yeasts are known mainly for the production of enzymes used in pharmaceutical and chemical synthesis, for production of certain classes of primary and secondary metabolites such as terpenoids and carotenoids, for aerobic catabolism of complex carbon sources, and for bioremediation of environmental pollutants and xenotoxicants. Notwithstanding, the basidiomycetous yeasts appear to have considerable potential in biotechnology owing to their catabolic utilities, formation of enzymes acting on recalcitrant substrates, and through the production of unique primary and secondary metabolites. This and the earlier mini-review (Johnson Appl Microb Biotechnol 97:503–517, 2013) were motivated during the preparation and publication of the landmark three-volume set of “The yeasts: a taxonomic study, 5th edition” (Kurtzman et al. 2011a, b).     

The effect of aminopterin (4-amino folic acid) on growth and cell division of fermentative versus oxidative yeasts

In a related brewing study detailed characteristics of fermentations displaying effective yeastaminopterin interaction were presented.Fermentative yeast types (certain Saccharomyces species and Selenotila intestinalis) proved effective aminopterin reactors whereas oxidative yeasts (certain Candida, Cryptococcus, Pichia, Rhodotorula, Saccharomyces, and Trigonopsis species) proved ineffective reactors. In general effective reactors were polyploids characterized by the lack of film or pellicle formation and ineffective reactors the opposite. In stationary fermentations the Fleischmann 139 strain of S. cerevisiae proved a fair reactor. When aerated it proved an ineffective reactor and aminopterin or products there-of stimulated growth. Conversely aeration enhanced aminopterin activity of effective reactor yeasts.The positive effect of biotin on aminopterin activity and the negative effect of yeast extract, L-asparagine, adenine and thymine is shown and compared and contrasted with earlier reported studies.These findings supported by outside data suggest that oxidative yeasts (and bacteria) can readily elicit enzymes capable of inactivating aminopterin whereas fermentative types are lacking in this capability. Finally that past yeast-aminopterin studies were conducted with oxidative yeast types.Advantages of effective aminopterin reactor yeasts to be published elsewhere include improved ultrastructure using KMnO₄–OsO₄ fixation, a yeast bioassay procedure for detecting aminopterin in plasma and urine, and cell synchronization.Non-Standard Abbreviation apt aminopterin     

Nectar Yeasts in the Tall Larkspur Delphinium barbeyi (Ranunculaceae) and Effects on Components of Pollinator Foraging Behavior

Microorganisms frequently colonize the nectar of angiosperm species. Though capable of altering a suite of traits important for pollinator attraction, few studies exist that test the degree to which they mediate pollinator foraging behavior. The objective of our study was to fill this gap by assessing the abundance and diversity of yeasts associated with the perennial larkspur Delphinium barbeyi (Ranunculaceae) and testing whether their presence affected components of pollinator foraging behavior. Yeasts frequently colonized D. barbeyi nectar, populating 54–77% of flowers examined depending on site. Though common, the yeast community was species-poor, represented by a single species, Metschnikowia reukaufii. Female-phase flowers of D. barbeyi were more likely to have higher densities of yeasts in comparison to male-phase flowers. Pollinators were likely vectors of yeasts, as virgin (unvisited) flowers rarely contained yeasts compared to flowers open to pollinator visitation, which were frequently colonized. Finally, pollinators responded positively to the presence of yeasts. Bombus foragers both visited and probed more flowers inoculated with yeasts in comparison to uninoculated controls. Taken together, our results suggest that variation in the occurrence and density of nectar-inhabiting yeasts have the potential to alter components of pollinator foraging behavior linked to pollen transfer and plant fitness.     

Evaluation of herbivore-induced plant volatiles for monitoring green lacewings in Washington apple orchards

We evaluated five herbivore-induced plant volatiles plus a male-produced pheromone as attractants for adult green lacewings in Washington apple orchards in 2008. We found at least five attractants or combinations of attractants were attractive to the three most abundant green lacewing species in our trials. Chrysopa nigricornis and Chrysopa oculata were attracted to the combination of methyl salicylate and iridodial with iridodial alone being the second best attractant. Chrysoperla plorabunda was found in lower numbers than C. nigricornis and C. oculata, but did exhibit a significant attraction to benzaldehyde. In mid-summer, we added the herbivore-induced plant volatile squalene to the study and found it to be exceedingly attractive, but only to male C. nigricornis. Whether alone or in combination, squalene attracted 4–5-fold more C. nigricornis than any other compound tested. Our data have revealed C. nigricornis to be an abundant orchard predator that can be readily monitored with squalene-baited traps. Despite the obvious promise of HIPVs in biological control programs, we urge caution in their deployment as large-scale attractants, at least until further studies have investigated potential disruption of natural enemy population dynamics.     

Scent of a killer: How could killer yeast boost its dispersal?

Vector‐borne parasites often manipulate hosts to attract uninfected vectors. For example, parasites causing malaria alter host odor to attract mosquitoes. Here, we discuss the ecology and evolution of fruit‐colonizing yeast in a tripartite symbiosis—the so‐called “killer yeast” system. “Killer yeast” consists of Saccharomyces cerevisiae yeast hosting two double‐stranded RNA viruses (M satellite dsRNAs, L‐A dsRNA helper virus). When both dsRNA viruses occur in a yeast cell, the yeast converts to lethal toxin‑producing “killer yeast” phenotype that kills uninfected yeasts. Yeasts on ephemeral fruits attract insect vectors to colonize new habitats. As the viruses have no extracellular stage, they depend on the same insect vectors as yeast for their dispersal. Viruses also benefit from yeast dispersal as this promotes yeast to reproduce sexually, which is how viruses can transmit to uninfected yeast strains. We tested whether insect vectors are more attracted to killer yeasts than to non‑killer yeasts. In our field experiment, we found that killer yeasts were more attractive to Drosophila than non‐killer yeasts. This suggests that vectors foraging on yeast are more likely to transmit yeast with a killer phenotype, allowing the viruses to colonize those uninfected yeast strains that engage in sexual reproduction with the killer yeast. Beyond insights into the basic ecology of the killer yeast system, our results suggest that viruses could increase transmission success by manipulating the insect vectors of their host.     

The vectoring of cactophilic yeasts by Drosophila

Summary At two locations in the Sonoran Desert, yeasts were sampled from species of Drosophila, the flies' cactus hosts, and other neighboring sources of cactophilic yeasts to determine the relation between the yeasts vectored by the fly and the yeasts found in their breeding sites. D. mojavensis, D. nigrospiracula, and D. mettleri vectored yeast assemblages significantly more similar to the yeast species found on the rot from which the flies were collected than to the yeasts found on other rots from the flies host cactus or other rotting cactus at the same site. Rots with Drosophila had fewer yeast species than those without flies, suggesting that flies were associated with younger rots. Rots with flies and the Drosophila also had more yeast species with the capability to produce ethyl acetate than rots without flies. The results support the contention that cactophilic Drosophila feed on a subset of the yeasts available in an area, and may act to maintain differences among the yeast communities found on different species of cactus.     

The costs and benefits of killer toxin production by the yeast Pichia kluyveri

Numerous yeast species in many genera are able to produce and excrete extracellular toxic proteins (mycocins) that can kill other specific sensitive yeasts. Natural distributions of killer yeasts suggest that they may be important in maintaining community composition and provide a benefit to the toxin producing cells. The fact that not all yeasts are killers and that polymorphisms exist within some killer species suggests there may be a cost associated with killer toxin production. This study focuses on the costs and benefits associated with toxin production by the yeast Pichia kluyveri. Strains differing in their ability to kill were obtained by tetrad dissection. One parent strain produced spores that exhibited a trade-off between killing ability and intrinsic growth rate. A killer clone from this strain was able to maintain a higher proportion of cells than a non-killer when grown with the same sensitive yeast under laboratory-simulated natural conditions. On the other hand, when grown with a yeast not sensitive to Pichia kluyveri toxin, the non-killer maintained a higher proportion of the total community than did the killer clone. The data support the hypothesis that there are both costs and benefits to producing killer toxin, and based on this, selection may favor different phenotypes in different conditions.     

Nectar yeasts in Delphinium nuttallianum (Ranunculaceae) and their effects on nectar quality

Microorganisms colonize the nectar of many angiosperms. Variable diversity and spatio-temporal dynamics of nectar-inhabiting microorganisms (e.g., yeasts) may drive variation in nectar sugar composition and subsequent plant–pollinator interactions. We assessed yeast frequency of occurrence and density in the nectar of the perennial herb, Delphinium nuttallianum, across multiple spatio-temporal scales, including flower lifetime and sex-phase transition, flowering season, populations, and years. We tested the hypothesis that pollinators vector yeasts by comparing densities between virgin flowers and those open to visitation. Finally, we identified yeasts using molecular methods and tested for an association between yeast density and nectar composition using ultra-performance liquid chromatography. Yeasts were frequent colonists of Delphinium nectar, occurring in all populations and years sampled. Yeast frequency of occurrence and density varied across most spatio-temporal scales examined. Pollinators were vectors of yeast: virgin flowers remained yeast-free, while those open to visitation became inoculated. Nectar samples were species-poor, with a majority colonized by Metschnikowia reukaufii. Finally, increasing yeast density was correlated with a decrease in sucrose and an increase in monosaccharides. Our results document that yeasts form species-poor communities in populations of this hermaphroditic perennial, in addition to highlighting their spatio-temporal dynamics and effects on nectar quality. Spatio-temporal variation in frequency of occurrence, density, and changes in nectar may have important implications for the nature and strength of interactions between Delphinium and its pollinators.