Saprophagous insect larvae,Drosophila melanogaster,profit from increased species richness in beneficial microbes

Female fruit flies, Drosophila melanogaster, lay their eggs on decaying plant material. Foraging fly larvae strongly depend on the availability of dietary microbes, such as yeasts, to reach the adult stage. In contrast, strong interference competition with filamentous fungi can cause high mortality among Drosophila larvae. Given that many insects are known for employing beneficial microbes to combat antagonistic ones, we hypothesized that fly larvae engaged in competition with the noxious mould Aspergillus nidulans benefit from the presence of dietary yeast species, especially when they are associated with increasingly species rich yeast communities (ranging from one to six yeast species per community). On a nutrient‐limited fruit substrate infested with A. nidulans, both larval survival and development time were positively affected by more diverse yeast communities. On a mould‐free fruit substrate, merely larval development but not survival was found to be affected by increasing species richness of dietary yeasts. Not only yeast diversity had an effect on D. melanogaster life‐history traits, but also the identity of the yeast combinations. These findings demonstrate the importance of the structure and diversity of microbial communities in mutualistic animal–microbe interactions.     

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 role of indigenous yeasts in traditional Irish cider fermentations

AIMS: To study the role of the indigenous yeast flora in traditional Irish cider fermentations. METHODS AND RESULTS: Wallerstein laboratory nutrient agar supplemented with biotin, ferric ammonium citrate, calcium carbonate and ethanol was employed together with PCR-restriction fragment length polymorphism analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene in the identification of indigenous yeasts at the species level, from traditional Irish cider fermentations. By combining the molecular approach and the presumptive media it was possible to distinguish between a large number of yeast species, and to track them within cider fermentations. The Irish cider fermentation process can be divided into three sequential phases based on the predominant yeast type present. Kloeckera/Hanseniaspora uvarum type yeasts predominate in the initial 'fruit yeast phase'. Thereafter Saccharomyces cerevisiae type yeast dominate in the 'fermentation phase', where the alcoholic fermentation takes place. Finally the 'maturation phase' which follows, is dominated by Dekkera and Brettanomyces type yeasts. H. uvarum type yeast were found to have originated from the fruit. Brettanomyces type yeast could be traced back to the press house, and also to the fruit. The press house was identified as having high levels of S. cerevisiae type yeast. A strong link was noted between the temperature profile of the cider fermentations, which ranged from 22 to 35 degrees C and the yeast strain population dynamics. CONCLUSIONS: Many different indigenous yeast species were identified. The mycology of Irish cider fermentations appears to be very similar to that which has previously been reported in the wine industry. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has allowed us to gain a better understanding of the role of indigenous yeast species in 'Natural' Irish cider fermentations.     

High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster

Among the most common parasites of Drosophila in nature are parasitoid wasps, which lay their eggs in fly larvae and pupae. D. melanogaster larvae can mount a cellular immune response against wasp eggs, but female wasps inject venom along with their eggs to block this immune response. Genetic variation in flies for immune resistance against wasps and genetic variation in wasps for virulence against flies largely determines the outcome of any fly-wasp interaction. Interestingly, up to 90% of the variation in fly resistance against wasp parasitism has been linked to a very simple mechanism: flies with increased constitutive blood cell (hemocyte) production are more resistant. However, this relationship has not been tested for Drosophila hosts outside of the melanogaster subgroup, nor has it been tested across a diversity of parasitoid wasp species and strains. We compared hemocyte levels in two fly species from different subgroups, D. melanogaster and D. suzukii, and found that D. suzukii constitutively produces up to five times more hemocytes than D. melanogaster. Using a panel of 24 parasitoid wasp strains representing fifteen species, four families, and multiple virulence strategies, we found that D. suzukii was significantly more resistant to wasp parasitism than D. melanogaster. Thus, our data suggest that the relationship between hemocyte production and wasp resistance is general. However, at least one sympatric wasp species was a highly successful infector of D. suzukii, suggesting specialists can overcome the general resistance afforded to hosts by excessive hemocyte production. Given that D. suzukii is an emerging agricultural pest, identification of the few parasitoid wasps that successfully infect D. suzukii may have value for biocontrol.     

Host-marking by female pepper weevils, Anthonomus eugenii

Pepper weevils, Anthonomus eugenii Cano (Coleoptera: Curculionidae), feed and oviposit in flower buds and small fruits of plants in the genus Capsicum, as well as several species of Solanum (Solanaceae). Females chew a small hole into the fruit, deposit a single egg within the cavity, and seal the hole with a clear anal secretion that hardens into an ‘oviposition plug’. Female oviposition behavior was studied in a series of small‐arena bioassays to determine whether previous oviposition in Jalapeño pepper fruit deterred subsequent oviposition and to determine what specific cues from an infested fruit influence female behavior. In choice and no‐choice tests, females preferred clean fruit to fruit that had received four eggs 24 h previously (i.e., infested fruit), whether the fruit was infested with conspecific eggs or their own eggs. Further bioassays demonstrated that the presence of female frass, or oviposition plugs alone, in the absence of eggs or any fruit damage, was sufficient to deter oviposition. In addition, females given the choice between an infested fruit with the oviposition plug removed or an unaltered infested fruit preferred the fruit with no plugs, even when eggs, frass, and feeding damage were still present. To determine whether females would avoid infested peppers under more natural conditions, we quantified oviposition on infested and uninfested sentinel pepper fruit within individually caged plants and on clean and infested plants caged together. Females consistently laid more eggs on clean fruit than on infested fruits and moved within and among pepper plants to search for more acceptable oviposition sites. We conclude that oviposition plugs, along with contaminated female, but not male, frass contain a deterrent that, in the absence of any other cue, is enough to alert a female that a patch is occupied.     

Yeast communities from host plants and associated Drosophila in southern arizona: new isolations and analysis of the relative importance of hosts and vectors on comunity composition

Summary The yeast communities from slime fluxes of three deciduous trees (Prosopis juliflora, Populus fremontii and Quercus emoryi) and the necroses of two cacti (Opuntia phaeacantha and Carnegiea gigantea) were surveyed in the region of Tucson, Arizona. In addition, the yeasts carried by dipterans associated with the fluxes or necroses (Drosophila carbonaria, D. brooksae, D. nigrospiracula, D. mettleri, and Aulacigaster leucopeza) were sampled. The results indicate that each host sampled had a distinct community of yeasts associated with it. The dipterans, which can act as vectors of the yeasts, deposited yeasts from other sources in addition to those found on their associated hosts. It is argued that host plant physiology is relatively more important than the activity of the vector in determining yeast community composition. Furthermore, the average number of yeast species per flux or necrosis is not different from the average number of yeast species per fly. It is hypothesized that the vector may affect the number of species per individual flux or not, and that the number is lower than the rot or necrosis could potentially support.     

Cryptococcus bestiolae and Cryptococcus dejecticola, two new yeast species isolated from frass of the litchi fruit borer Conopomorpha sinensis Bradley

Two new yeast species, Cryptococcus bestiolae and Cryptococcus dejecticola, were discovered in the frass of the litchi fruit borer Conopomorpha sinensis Bradley. The yeasts utilize inositol, hydrolyze urea, produce starch-like substance, and contain CoQ10. Phylogenetic analyses of D1/D2 26S rDNA and internal transcribed spacer (ITS) sequences indicate that the yeasts are closely related to Bullera dendrophila and an undescribed species of Cryptococcus (strain CBS 8507). The two new species differed from each other by 17 nucleotides in the D1/D2 region and by 68 nucleotides in the ITS region. Cryptococcus bestiolae is a sister species to Cryptococcus sp. CBS 8507, from which it differs by eight nucleotides in the D1/D2 region and 59 nucleotides in the ITS region. Cryptococcus dejecticola and B. dendrophila differed by 13 nucleotides in the D1/D2 and 57 nucleotides in the ITS region. Cryptococcus bestiolae and Cr. dejecticola formed with B. dendrophila a well defined clade consisting of insect associated species. The type strain of Cr. bestiolae is TH3.2.59 (=CBS 10118=NRRL Y-27894), and the type strain of Cr. dejecticola is Litch 17 (=CBS 10117=NRRL Y-27898).     

Yeast succession in the Amazon fruit Parahancornia amapa as resource partitioning among Drosophila spp.

The succession of yeasts colonizing the fallen ripe amapa fruit, from Parahancornia amapa, was examined. The occupation of the substrate depended on both the competitive interactions of yeast species, such as the production of killer toxins, and the selective dispersion by the drosophilid guild of the amapa fruit. The yeast community associated with this Amazon fruit differed from those isolated from other fruits in the same forest. The physiological profile of these yeasts was mostly restricted to the assimilation of a few simple carbon sources, mainly L-sorbose, D-glycerol, DL-lactate, cellobiose, and salicin. Common fruit-associated yeasts of the genera Kloeckera and Hanseniaspora, Candida guilliermondii, and Candida krusei colonized fruits during the first three days after the fruit fell. These yeasts were dispersed and served as food for the invader Drosophila malerkotliana. The resident flies of the Drosophila willistoni group fed selectively on patches of yeasts colonizing fruits 3 to 10 days after the fruit fell. The killer toxin-producing yeasts Pichia kluyveri var. kluyveri and Candida fructus were probably involved in the exclusion of some species during the intermediate stages of fruit deterioration. An increase in pH, inhibiting toxin activity and the depletion of simple sugars, may have promoted an increase in yeast diversity in the later stages of decomposition. The yeast succession provided a patchy environment for the drosophilids sharing this ephemeral substrate.     

Host Location of Hyssopus pallidus, a Larval Parasitoid of the Codling Moth, Cydia pomonella

The effectiveness of parasitoids as biological control agents depends largely on their host location behavior. In this study we describe the host-searching behavior of Hyssopus pallidus (Askew), a larval parasitoid of the codling moth Cydia pomonella L. We observed parasitoid behavior on mature apples (a potential host patch) and elucidated some of the stimuli which determine host location. Female wasps showed more complex and intensive searching on infested apples than on mechanically damaged or noninfested apples. Wasps were able to enter infested apples through the calyx or the tunnel made by the host larvae and parasitize them. Area-restricted searching was observed on infested apples, in particular on areas contaminated with host frass. In a further bioassay, we confirmed that host frass contains a host location kairomone. The kairomone appears to be produced by the host independently from its diet, even though frass produced by hosts fed on an apple are more attractive than frass produced by hosts fed on a fruit-devoid artificial diet. The capability of H. pallidus to locate its host inside apples makes the foraging strategy of this species potentially useful as a biological control agent.     

Candida kazuoi sp. nov. and Candida hasegawae sp. nov., two new species of ascomycetous anamorphic yeasts isolated from insect frass in Thailand

Two strains of anamorphic yeasts isolated from insect frass collected in southern Thailand were assigned to the genus Candida based on the conventional taxonomic criteria used for yeast classification. In the phylogenetic tree based on the D1/D2 domain of the 26S rDNA, these strains are distant from the known species of yeasts and considered to represent two different new species. They are named Candida kazuoi sp. nov. and Candida hasegawae sp. nov.     

Drosophila regulate yeast density and increase yeast community similarity in a natural substrate

Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in 'niche construction', facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop.     

Host finding and discrimination in Diglyphus isaea,a parasitoid of the chrysanthemum leaf miner,Chromatomyia syngenesiae

Naive and experienced Diglyphus isaea were attracted by host plant odours of lettuce and chrysanthemum to search and probe on infested and uninfested leaves. A slight preference was shown for leaves infested with Chromatomyia syngenesiae. At close range, visual stimuli were unnecessary for oviposition and host‐feeding. Contact with uninfested lettuce and chrysanthemum elicited searching and probing behaviour whereas host frass did not. Host larval movement appeared to aid host detection at close range. The number of ovipositor probes increased with proximity to live hosts but not for stationary, dead hosts. Dead hosts were frequently walked over or missed when D. isaea passed within 0.5 cm of the stationary larvae. Dead hosts were also rejected for oviposition but not for host feeding. Both naive and experienced females discriminated between healthy hosts and those which had been attacked by conspecifics or encountered previously.     

Candida arabinofermentans, a new L-arabinose fermenting yeast

Candida arabinofermentans (type strain NRRL YB-2248, CBS 8468), a new yeast that ferments the pentose L-arabinose, is described. The three known strains of this new species were isolated from insect frass of pine and larch trees in the U.S. Phylogenetic analysis of nucleotide sequences from the D1/D2 domain of large subunit (26S) ribosomal DNA places C. arabinofermentans among the methanol-assimilating yeasts and most closely related to Candida ovalis. Strains of the new species produce 0.7-1.9 g/l ethanol from L-arabinose.     

Gut-associated yeast in bark beetles of the genus Dendroctonus Erichson (Coleoptera: Curculionidae: Scolytinae)

Scolytine bark beetles are the most destructive pests of conifers; they sometimes aggregate in such large numbers that they actually kill their hosts. They maintain close relationships with yeasts and fungi, in particular those that are assumed to aid in digestive, detoxification processes and pheromone production. In this study, 403 yeast strains were isolated from the guts, ovaries, eggs and frass of nine bark beetle species in the genus Dendroctonus Erichson. The beetles were collected from 10 conifer species at 34 locations in Mexico, Guatemala and the USA. Yeast identification was based on partial DNA sequences from 18S rDNA, 26S rDNA and internal transcribed spacer (ITS1), as well as morphological and physiological characteristics. A combined phylogenetic analysis delimited 11 clades with sequences similar to Candida arabinofermentans , C. ernobii , C. membranifaciens (including C. lessepsii , Pichia mexicana and P. scolyti ), C. oregonensis , C. piceae , Kuraishia capsulata (including K. capsulata and K. cf. molischiana ), Pichia americana , P. canadensis , P. glucozyma , P. guilliermondii and an undescribed species of Candida . Nucleotide divergences between the major clades were at least 5% while, with the exception of 30 isolates, yeasts within clades differed from named reference species at fewer than 1% of the nucleotide sites. There do not appear to be obligate relationships between particular yeasts and specific anatomical partitions, nor between particular yeasts and bark beetle species. Some yeasts do appear to be preferentially associated with bark beetles feeding on different conifer genera and therefore host plant defences may limit yeast community diversity in Dendroctonus .  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 325–342.     

Successive Microbial Populations in Calimyrna Figs

Smyrna-type (Calimyrna) figs have essentially sterile internal tissue until visited by the pollinating fig wasp, Blastophaga psenes, which introduces a specific microflora consisting of Candida guilliermondii var. carpophila and Serratia plymuthica. This flora persists and develops in numbers throughout the ripening period until maturity of the fruit. These organisms do not cause spoilage. The presence of C. guilliermondii var. carpophila appears to increase the attractiveness of the fruit to drosophilae. Drosophila (mainly D. melanogaster) carry spoilage yeasts and bacteria on their exterior body parts, and introduce these organisms during ovipositing in the fruit cavity. The spoilage yeasts consist almost entirely of apiculate yeasts (Hanseniaspora valbyensis, H. uvarum, and Kloeckera apiculata) and of Torulopsis stellata, which cause active fermentative spoilage. Spoilage bacteria (primarily Acetobacter melanogenus) are also introduced with the yeasts. Organic acids are produced by these yeasts as well as by the Acetobacter. A number of minor spoilage yeasts were also identified.     

Do yeasts and Drosophila interact just by chance?

The fruit fly Drosophila melanogaster and the baker's yeast Saccharomyces cerevisiae are classic research model organisms that are also associated in nature, at least around vineyards. Sharing the same ephemeral fruit niche, winged Drosophila feed on immotile yeasts. That a yeast diet is essential for larval development, and that saprophagous fruit flies are attracted to a suite of yeast volatiles, has been well established over the last century. Recently, research has focussed on the potential mutual benefit of this interaction hypothesising yeasts also benefit via dispersal from ephemeral fruits. It now appears that the concept of a co-evolved mutualism between yeasts and Drosophila has permeated the literature. However, until robust evidence regarding the evolution and maintenance of this yeast-fly association has been provided, we suggest there is no compelling evidence to reject the more simplistic null hypothesis that these interactions are due to exaptation, and not a mutualism driven by natural selection.     

Midgut and fat body bacteriocytes in neotropical cerambycid beetles (Coleoptera: Cerambycidae)

Xylophagous insects derive nutrients from intractable substrates by producing or ingesting cellulolytic enzymes, or by maintaining associations with symbiotic microbes. Wood-boring cerambycid beetle larvae sometimes house maternally-transmitted endosymbiotic yeasts that are presumed to provide their hosts with nutritional benefits. These are thought to be absent from species in the large subfamily Lamiinae; nevertheless yeasts have been repeatedly isolated from the guts of neotropical lamiines. The objective of this study was to conduct transmission electron microscopy (TEM) studies of cerambycid larval midgut tissues to determine if gut yeasts were intracellular, or simply present in the gut lumen. Nine cerambycid larvae were harvested from two trees in the Brazil nut family (Lecythidaceae) in the rain forest of SE Peru; seven were identified using mtDNA sequence data and processed for TEM. Yeasts cultured from larval frass or exuvia, and identified with rDNA sequence data, were identical or similar to yeasts previously isolated from beetles. In TEM analyses yeast cells were found only in the gut lumens, sometimes associated with fragments of thick-walled xylem cells. Apparent bacteriocytes were found in either midgut or fat body tissue of three larval specimens, including two lamiines. This is the first report of a potential fat body symbiosis in a cerambycid beetle. Future studies of cerambycid symbiosis should distinguish the identities and potential roles of free-living organisms in the gut lumen from those of organisms harbored within gut epithelial or fat body tissue.     

Three new ascomycetous yeasts from insect-associated arboreal habitats

A new species of Pichia and two new species of Candida are described and were determined to be genetically isolated from all other currently recognized ascomycetous yeasts from their sequence divergence in the species-variable D1/D2 domain of large subunit (26S) ribosomal DNA. The three species were primarily isolated from the frass of wood-boring insects living in pine and spruce trees. The new species and their type strains are the following: Pichia ramenticola NRRL YB-1985 (CBS 8699), mating type alpha (NRRL YB-3835, CBS 8700, mating type a), Candida piceae NRRL YB-2107 (CBS 8701), and Candida wyomingensis NRRL YB-2152 (CBS 8703). Pichia ramenticola and C. piceae assimilate methanol as a carbon source; P. ramenticola is the first known heterothallic ascomycetous yeast to utilize this compound.     

铃木氏果蝇不同地理种群中Wolbachia的检测和系统发育分析

铃木氏果蝇Drosophila suzukii是原产于东南亚地区的重要果树害虫, 近年来传入北美和欧洲等地区造成严重的危害。本研究利用Wolbachia的16S rDNA和wsp基因特异引物（分别为16S-F/16S-R和81F/691R）对铃木氏果蝇7个地理种群（中国的5个种群、 韩国的1个种群和美国的1个种群）的Wolbachia进行了PCR检测并对检测结果进行了比较; 对感染个体体内Wolbachia的16S rDNA基因片段进行测序, 确定了我国铃木氏果蝇体内Wolbachia的分类地位。基于Wolbachia的16S rDNA基因特异引物检测结果发现, 我国5个铃木氏果蝇种群广泛感染Wolbachia（感染率36.7%~80.0%）, 而韩国和美国2个种群均未检测到该菌的感染。而利用wsp基因特异引物无法检测到该菌。基于Wolbachia的16S rDNA基因构建系统发育树表明, 我国铃木氏果蝇种群感染的Wolbachia全部属于A组。这些结果为研究Wolbachia感染对铃木氏果蝇生物学及生态学的影响奠定了基础。     

Are Drosophila preferences for yeasts stable or contextual?

Whether there are general mechanisms, driving interspecific chemical communication is uncertain. Saccharomycetaceae yeast and Drosophila fruit flies, both extensively studied research models, share the same fruit habitat, and it has been suggested their interaction comprises a facultative mutualism that is instigated and maintained by yeast volatiles. Using choice tests, experimental evolution, and volatile analyses, we investigate the maintenance of this relationship and reveal little consistency between behavioral responses of two isolates of sympatric Drosophila species. While D. melanogaster was attracted to a range of different Saccharomycetaceae yeasts and this was independent of fruit type, D. simulans preference appeared specific to a particular S. cerevisiae genotype isolated from a vineyard fly population. This response, however, was not consistent across fruit types and is therefore context‐dependent. In addition, D. simulans attraction to an individual S. cerevisiae isolate was pliable over ecological timescales. Volatile candidates were analyzed to identify a common signal for yeast attraction, and while D. melanogaster generally responded to fermentation profiles, D. simulans preference was more discerning and likely threshold‐dependent. Overall, there is no strong evidence to support the idea of bespoke interactions with specific yeasts for either of these Drosophila genotypes. Rather the data support the idea Drosophila are generally adapted to sense and locate fruits infested by a range of fungal microbes and/or that yeast–Drosophila interactions may evolve rapidly.