Dietmar Schwarz

Differences in performance and transcriptome‐wide gene expression associated with Rhagoletis (Diptera: Tephritidae) larvae feeding in alternate host fruit environments

Host race formation, the establishment of new populations using novel resources, is a major hypothesized mechanism of ecological speciation, especially in plant‐feeding insects. The initial stages of host race formation will often involve phenotypic plasticity on the novel resource, with subsequent genetically based adaptations enhancing host‐associated fitness differences. Several studies have explored the physiology of the plastic responses of insects to novel host environments. However, the mechanisms underlying evolved differences among host races and species remain poorly understood. Here, we demonstrate a reciprocal larval performance difference between two closely related species of Rhagoletis flies, R. pomonella and R. zephyria, specialized for feeding in apple and snowberry fruit, respectively. Microarray analysis of fly larvae feeding in apples versus snowberries revealed patterns of transcriptome‐wide differential gene expression consistent with both plastic and evolved responses to the different fruit resources, most notably for detoxification‐related genes such as cytochrome p450s. Transcripts exhibiting evolved expression differences between species tended to also demonstrate plastic responses to fruit environment. The observed pattern suggests that Rhagoletis larvae exhibit extensive plasticity in gene expression in response to novel fruit that may potentiate shifts to new hosts. Subsequent selection, particularly selection to suppress initially costly plastic responses, could account for the evolved expression differences observed between R. pomonella and R. zephyria, creating specialized races and new fly species. Thus, genetically based ecological adaptations generating new biodiversity may often evolve from initial plastic responses in gene expression to the challenges posed by novel environments.

The hitchhiker's guide to Europe: the infection dynamics of an ongoing Wolbachia invasion and mitochondrial selective sweep in Rhagoletis cerasi

Wolbachia is a maternally inherited and ubiquitous endosymbiont of insects. It can hijack host reproduction by manipulations such as cytoplasmic incompatibility (CI) to enhance vertical transmission. Horizontal transmission of Wolbachia can also result in the colonization of new mitochondrial lineages. In this study, we present a 15‐year‐long survey of Wolbachia in the cherry fruit fly Rhagoletis cerasi across Europe and the spatiotemporal distribution of two prevalent strains, wCer1 and wCer2, and associated mitochondrial haplotypes in Germany. Across most of Europe, populations consisted of either 100% singly (wCer1) infected individuals with haplotype HT1, or 100% doubly (wCer1&2) infected individuals with haplotype HT2, differentiated only by a single nucleotide polymorphism. In central Germany, singly infected populations were surrounded by transitional populations, consisting of both singly and doubly infected individuals, sandwiched between populations fixed for wCer1&2. Populations with fixed infection status showed perfect association of infection and mitochondria, suggesting a recent CI‐driven selective sweep of wCer2 linked with HT2. Spatial analysis revealed a range expansion for wCer2 and a large transition zone in which wCer2 splashes appeared to coalesce into doubly infected populations. Unexpectedly, the transition zone contained a large proportion (22%) of wCer1&2 individuals with HT1, suggesting frequent intraspecific horizontal transmission. However, this horizontal transmission did not break the strict association between infection types and haplotypes in populations outside the transition zone, suggesting that this horizontally acquired Wolbachia infection may be transient. Our study provides new insights into the rarely studied Wolbachia invasion dynamics in field populations.

Hybridization and the spread of the apple maggot fly, Rhagoletis pomonella (Diptera: Tephritidae), in the northwestern United States.

Hybridization may be an important process interjecting variation into insect populations enabling host plant shifts and the origin of new economic pests. Here, we examine whether hybridization between the native snowberry‐infesting fruit fly Rhagoletis zephyria (Snow) and the introduced quarantine pest R. pomonella (Walsh) is occurring and may aid the spread of the latter into more arid commercial apple‐growing regions of central Washington state, USA. Results for 19 microsatellites implied hybridization occurring at a rate of 1.44% per generation between the species. However, there was no evidence for increased hybridization in central Washington. Allele frequencies for seven microsatellites in R. pomonella were more ‘R. zephyria‐like’ in central Washington, suggesting that genes conferring resistance to desiccation may be adaptively introgressing from R. zephyria. However, in only one case was the putatively introgressing allele from R. zephyria not found in R. pomonella in the eastern USA. Thus, many of the alleles changing in frequency may have been prestanding in the introduced R. pomonella population. The dynamics of hybridization are therefore complex and nuanced for R. pomonella, with various causes and factors, including introgression for a portion, but not all of the genome, potentially contributing to the pest insects spread.

Molecular Species Identification of Cryptic Apple and Snowberry Maggots (Diptera: Tephritidae) in Western and Central Washington

ABSTRACT In Washington state, identification of the quarantine apple pest Rhagoletis pomonella (Walsh) is complicated by the presence of the cryptic species Rhagoletis zephyria Snow (Diptera: Tephritidae). Distinguishing the two flies is important because there is a zero tolerance policy for R. pomonella in apple production for export. Here, we attempt to distinguish the two species by scoring R. pomonella and R. zephyria populations from western and south—central Washington for a set of 11 nuclear markers, including four single nucleotide polymorphisms (SNPs) developed for rapid and inexpensive genotyping using Taqman real-time quantitative—polymerase chain reaction. We show that the four SNPs maybe adequate in most cases for distinguishing whether a fly originated from apple or black hawthorn (the two major host plants for R. pomonella representing an economic risk) versus snowberry (the major host for R. zephyria, and not a commercial threat). However, directional introgression of R. zephyria alleles into R. pomonella can complicate the identification of flies of mixed ancestry based only on the four SNPs. Moreover, this problem is more acute in the sensitive apple-growing regions of central Washington where our results imply hybridization is common. Consequently, application of the four SNP quantitative—polymerase chain reaction assay can immediately assist ongoing apple maggot monitoring, while the development of additional genetic markers through next-generation sequencing would be valuable for increasing confidence in species identification and for assessing the threat posed by hybridization as R. pomonella further spreads into the more arid apple-growing regions of central Washington.

wisepair: a computer program for individual matching in genetic tracking studies

Individual‐based data sets tracking organisms over space and time are fundamental to answering broad questions in ecology and evolution. A ‘permanent’ genetic tag circumvents a need to invasively mark or tag animals, especially if there are little phenotypic differences among individuals. However, genetic tracking of individuals does not come without its limits; correctly matching genotypes and error rates associated with laboratory work can make it difficult to parse out matched individuals. In addition, defining a sampling design that effectively matches individuals in the wild can be a challenge for researchers. Here, we combine the two objectives of defining sampling design and reducing genotyping error through an efficient Python‐based computer‐modelling program, wisepair. We describe the methods used to develop the computer program and assess its effectiveness through three empirical data sets, with and without reference genotypes. Our results show that wisepair outperformed similar genotype matching programs using previously published from reference genotype data of diurnal poison frogs (Allobates femoralis) and without‐reference (faecal) genotype sample data sets of harbour seals (Phoca vitulina) and Eurasian otters (Lutra lutra). In addition, due to limited sampling effort in the harbour seal data, we present optimal sampling designs for future projects. wisepair allows for minimal sacrifice in the available methods as it incorporates sample rerun error data, allelic pairwise comparisons and probabilistic simulations to determine matching thresholds. Our program is the lone tool available to researchers to define parameters a priori for genetic tracking studies.

Identification of Host Fruit Volatiles from Snowberry (Symphoricarpos albus), Attractive to Rhagoletis zephyria Flies from the Western United States

A mixture of behaviorally active volatiles was identified from the fruit of snowberry, Symphoricarpos albus laevigatus, for Rhagoletis zephyria flies reared from snowberry fruit. A nine-component blend containing 3-methylbutan-1-ol (3%), dimethyl trisulfide (1%), 1-octen-3-ol (40%), myrcene (8%), nonanal (9%), linalool (13%), (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT, 6%), decanal (15%), and β-caryophyllene (5%) was identified that gave consistent electroantennogram activity and was behaviorally active in flight tunnel tests. In other flight tunnel assays, snowberry flies from two sites in Washington state, USA, displayed significantly greater levels of upwind oriented flight to sources with the snowberry volatile blend compared with previously identified volatile blends from domestic apple (Malus domestica) and downy hawthorn (Crataegus mollis) fruit from the eastern USA, and domestic apple, black hawthorn (C. douglasii) and ornamental hawthorn (C. monogyna) from Washington state. Selected subtraction assays showed that whereas removal of DMNT or 1-octen-3-ol significantly reduced the level of upwind flight, removal of myrcene and β-caryophyllene, or dimethyl trisulfide alone did not significantly affect the proportion of upwind flights. Our findings add to previous studies showing that populations of Rhagoletis flies infesting different host fruit are attracted to unique mixtures of volatile compounds specific to their respective host plants. Taken together, the results support the hypothesis that differences among flies in their behavioral responses to host fruit odors represent key adaptations involved in sympatric host plant shifts, contributing to host specific mating and generating prezygotic reproductive isolation among members of the R. pomonella sibling species complex.

Type-I Prenyl Protease Function Is Required in the Male Germline of Drosophila melanogaster

Many proteins require the addition of a hydrophobic prenyl anchor (prenylation) for proper trafficking and localization in the cell. Prenyl proteases play critical roles in modifying proteins for membrane anchorage. The type I prenyl protease has a defined function in yeast (Ste24p/Afc1p) where it modifies a mating pheromone, and in humans (Zmpste24) where it has been implicated in a disease of premature aging. Despite these apparently very different biological processes, the type I prenyl protease gene is highly conserved, encoded by a single gene in a wide range of animal and plant groups. A notable exception is Drosophila melanogaster, where the gene encoding the type I prenyl protease has undergone an unprecedented series of duplications in the genome, resulting in five distinct paralogs, three of which are organized in a tandem array, and demonstrate high conservation, particularly in the vicinity of the active site of the enzyme. We have undertaken targeted deletion to remove the three tandem paralogs from the genome. The result is a male fertility defect, manifesting late in spermatogenesis. Our results also show that the ancestral type I prenyl protease gene in Drosophila is under strong purifying selection, while the more recent replicates are evolving rapidly. Our rescue data support a role for the rapidly evolving tandem paralogs in the male germline. We propose that potential targets for the male-specific type I prenyl proteases include proteins involved in the very dramatic cytoskeletal remodeling events required for spermatid maturation.

Large-scale molecular diet analysis in a generalist marine mammal reveals male preference for prey of conservation concern

Abstract Sex‐specific diet information is important in the determination of predator impacts on prey populations. Unfortunately, the diet of males and females can be difficult to describe, particularly when they are marine predators. We combined two molecular techniques to describe haul‐out use and prey preferences of male and female harbor seals (Phoca vitulina) from Comox and Cowichan Bay (Canada) during 2012–2013. DNA metabarcoding quantified the diet proportions comprised of prey species in harbor seal scat, and qPCR determined the sex of the individual that deposited each scat. Using 287 female and 260 male samples, we compared the monthly sex ratio with GLMs and analyzed prey consumption relative to sex, season, site, and year with PERMANOVA. The sex ratio between monthly samples differed widely in both years (range = 12%–79% males) and showed different patterns at each haul‐out site. Male and female diet differed across both years and sites: Females consumed a high proportion of demersal fish species while males consumed more salmonid species. Diet composition was related to both sex and season (PERMANOVA: R 2 = 27%, p < 0.001; R 2 = 24%, p < 0.001, respectively) and their interaction (PERMANOVA: R 2 = 11%, p < 0.001). Diet differences between males and females were consistent across site and year, suggesting fundamental foraging differences, including that males may have a larger impact on salmonids than females. Our novel combination of techniques allowed for both prey taxonomic and spatiotemporal resolution unprecedented in marine predators.

Can sex-specific consumption of prey be determined from DNA in predator scat?

Sex-biased predation, a predator’s bias for one prey sex over the other, can have important demographic impacts on prey species of conservation concern. Yet, it is difficult to measure in the wild. Molecular scatology has been used to indirectly determine the proportion of prey items consumed in the diet, and it may be possible to apply similar approaches to determine the sex-biased consumption of prey items. We developed a molecular method to indirectly examine sex-specific predation employing scat, focusing on predator–prey interactions between Chinook salmon (Oncorhynchus tshawytscha) and harbor seals (Phoca vitulina richardii). We established that the proportions of male and female Chinook DNA can be determined in a controlled sample by measuring a Y-linked marker, growth hormone pseudogene, using qPCR. We then applied the assay to harbor seal scat samples. Although the assay amplified in 83% of scat samples, 27% of samples quantified had an estimated male proportion > 1, which may have been due to a lack of robustness of the PCR assay in samples. Lastly, we constructed a biomass calibration curve to determine whether DNA measurements could estimate the proportions of male and female biomass consumed. The calibration curve was skewed by high male DNA density precluding our ability to quantify the relative amounts. We demonstrated that nuclear prey markers can be amplified in predator scat, however, contamination and extreme DNA density differences between the prey sexes may pose practical difficulties to estimate the relative amounts of male to female biomass consumed.

Genetic Evidence for the Introduction of Rhagoletis pomonella (Diptera: Tephritidae) into the Northwestern United States

Abstract The apple maggot fly, Rhagoletis pomonella Walsh (Diptera: Tephritidae), is a serious quarantine pest in the apple-growing regions of central Washington and Oregon. The fly is believed to have been introduced into the Pacific Northwest via the transport of larval-infested apples near Portland, Oregon, within the last 40 yr. However, R. pomonella also attacks native black hawthorn, Crataegus douglasii Lindley (Rosales: Rosaceae), and introduced ornamental hawthorn, Crataegus monogyna Jacquin, in the region. It is, therefore, possible that R. pomonella was not introduced but has always been present on black hawthorn. If true, then the fly may have independently shifted from hawthorn onto apple in the Pacific Northwest within the last 40 yr after apples were introduced. Here, we test the introduction hypothesis through a microsatellite genetic survey of 10 R. pomonella sites in Washington and 5 in the eastern United States, as well as a comparison to patterns of genetic variation between populations of Rhagoletis cingulata Loew and Rhagoletis indifferens Curran, two sister species of cherry-infesting flies known to be native to the eastern and western United States, respectively. We report results based on genetic distance networks, patterns of allelic variation, and estimated times of population divergence that are consistent with the introduction hypothesis for R. pomonella. The results have important implications for R. pomonella management, suggesting that black hawthorn-infesting flies near commercial apple-growing regions of central Washington may harbor sufficient variation to utilize apple as an alternate host, urging careful monitoring, and possible removal of hawthorn trees near orchards.