Pervasive heteroplasmy in an invasive ambrosia beetle (Scolytinae) in southern California.

Heteroplasmy, the presence of multiple mitochondrial genotypes (mitotypes) within an individual, has long been thought to be a rare aberrance that is quickly removed by selection or drift. However, heteroplasmy is being reported in natural populations of eukaryotes with increasing frequency, in part due to improved diagnostic methods. Here, we report a seemingly stable heteroplasmic state in California populations of the polyphagous shothole borer (PSHB), Euwallacea fornicatus; an invasive ambrosia beetle that is causing significant tree dieback. We develop and validate a qPCR assay utilizing locked nucleic acid probes to detect different mitotypes, and qualitatively assess heteroplasmy in individual PSHB. We prove the utility of this assay by: (1) mitotyping field-collected PSHB, documenting the prevalence of heteroplasmy across its range in California; and, (2) measuring relative titers of each mitotype across multiple generations of heteroplasmic laboratory colonies to assess the stability of transmission through the maternal germline. We show that our findings are unlikely to be explained by the existence of NUMTs by next generation sequencing of contiguous sections of mitochondrial DNA, where each of the observed heteroplasmic sites are found within fully functional coding regions of mtDNA. Subsequently, we find heteroplasmic individuals are common in Californian field populations, and that heteroplasmy persists for at least 10 generations in experimental colonies. We also looked for evidence of the common occurrence of paternal leakage, but found none. In light of our results, we discuss competing hypotheses as to how heteroplasmy may have arisen, and continues to perpetuate, in Californian PSHB populations.

Reproductive compatibility among sympatric and allopatric isofemale lines of Trichogramma pretiosum Riley, 1879.

The present study evaluated the reproductive compatibility of Trichogramma pretiosum Riley, 1879, through an integrative approach using biological data and morphometry of three isofemale lines (isolines) collected from two geographical areas. These isolines differed in sequences of mitochondrial DNA and reproductive performance in the laboratory. The wasps used to initiate the isolines were collected in different environments: two lines from a Mediterranean climate in Irvine, California, USA, and one line from a tropical climate in Piracicaba, São Paulo, Brazil. Reproductive compatibility was studied by evaluating the sex ratio and number of adult offspring produced of all mating combinations between adults from these isolines. Morphometry was studied by measuring 26 taxonomically useful characters, followed by a multivariate analysis. For the allopatric matings among Brazilian and North American isolines, a low level of crossing incompatibility was recorded, in only one direction of the crosses; whereas the sympatric North American isolines were incompatible in both directions. Multivariate analysis of the morphometric data indicated no distinct groups, suggesting that despite the genetic and biological differences, the isofemale lines are morphologically similar.

Seasonal Dynamics of Flight Phenology of the Euwallacea fornicatus Species Complex and an Associated Parasitoid Wasp in Avocado Groves in Taiwan.

The Euwallacea fornicatus species complex (Coleoptera: Curculionidae: Scolytinae: Xyleborini) is a group of four cryptic ambrosia beetle species. Native to Asia, several members of the complex have invaded other continents, where they cause significant economic losses to agricultural crops (e.g., avocado) and natural ecosystems. We were primarily interested in developing management strategies by focusing on the flight behavior of the beetles. Thus, seasonal differences in flight activity were assessed using panel traps baited with a commercial quercivorol lure, placed in infested avocado orchards in Danei, Tainan, Taiwan. Same traps were used to investigate the flight activity of a natural enemy, an undescribed species of the Braconid genus Eucosmophorus sp. Shothole borer species were identified using a DNA-based, high resolution melting assay. Trap data were compared to the predictions of a simple degree-day model, incorporating developmental data and several environmental parameters known to influence flight. Such as the time period representing most of flight activity in a day and temperature-dependent flight propensity. In stark contrast to the degree-day model which predicted the highest emergence, and by extension flight, of shothole borers during spring and summer (May to November), flight activity was actually lowest during these months, and instead, peaked during the winter (October to March). Abundance of the parasitoid wasp closely mirrored flight activity of the shothole borers. The mismatch of trapping and modeling data can have many causes, heavy precipitation and possibly cooperative brood care may suppress the dispersal behavior of the shothole borers during the summer.

High temperature mortality of Wolbachia impacts the sex ratio of the parasitoid Ooencyrtus mirus (Hymenoptera: Encyrtidae).

Background: Wolbachia bacteria are estimated to occur in more than half of all insect species. In Hymenoptera, Wolbachia often manipulates its host's reproduction to its own advantage. Wolbachia is likely the reason that males are rare in the uniparental Ooencyrtus mirus Triapitsyn & Power (Hymenoptera: Encyrtidae). The likelihood of producing male offspring can be increased by giving mothers a continuous supply of Bagrada hilaris (Burmeister) (Heteroptera: Pentatomidae) host eggs to parasitize for 2-3 weeks, by feeding the parents antibiotics, or by rearing parent wasps at high temperatures; all variables that have been shown to correlate with depleting Wolbachia titers in other organisms. The purpose of the current study was to determine whether thelytoky in O. mirus is due to Wolbachia, and if so, at what time in development the sex change occurs. We also wished to determine if Wolbachia removal results in the production of intersexes, as in some other hymenopterans. Finally, mating behavior was observed to see if and where it breaks down as a result of the species becoming thelytokous. Methods: Females were collected from parental lines of O. mirus reared at 26, 30, 31, 32, 33, 34, and 36 °C. The offspring of these females were reared at 26 °C, and their sex-ratio was determined. In a subsequent experiment, the parental generation was switched between 26 °C and 36 °C during development to narrow down the critical period at which changes occurred that subsequently affected the sex-ratio of their offspring. Results: The sex ratio was male biased in the offspring of O. mirus parents reared at 34 °C and 36 °C (high temperatures), even if the offspring themselves were reared at 26 °C. The constant temperature at which the percentage of males started to increase after two generations was 31 °C (10% males), rising to 39% males at 33 °C, and 100% males at 34 °C and 36 °C. Lasting more than 2 days, the critical period for the change toward a male biased sex ratio was during the second half of the parent's development. Molecular diagnostic assays confirmed that O. mirus females contain Wolbachia and males do not. Examination of preserved males and male-female pairs under a dissecting microscope showed no signs of intersex characters. Observation of the mating behavior of live O. mirus showed that males initiate courtship by drumming their antennae on a female's antennae, but after a few seconds, the females typically turn and walk away. However, a few instances of possible copulation were noted. Conclusions: As hypothesized, the results indicated that thelytoky in O. mirus is likely mediated by Wolbachia bacteria. To maximize the population growth rate without generating males, the best temperature for mass rearing this species is 30 °C.

The Attractiveness of α-Copaene to Members of the Euwallacea fornicatus (Coleoptera: Curculionidae) Species Complex in California and Taiwan.

Species belonging to the Euwallacea fornicatus Eichhoff (Coleoptera: Scolytinae) species complex have invaded the continental U.S. since at least 2003. Three species of this complex are known to have established, two in California (E. fornicatus; and Euwallacea kuroshio), and a third in Florida (Euwallacea perbrevis). Their native ranges are spread across southern and southeast Asia. In Taiwan, all three species occur in sympatry. They attack healthy trees of widely varied species and cause severe damage and death to the trees. The attractant quercivorol is commonly used to promote their detection by passive trapping. Recent studies in Florida have shown that trapping of E. perbrevis can be further improved by adding a synergist, α-copaene, alongside the quercivorol lure. Thus, we were interested in testing the effectiveness of α-copaene for trapping the other invasive members of the complex in California and in an area of Taiwan where all three species co-occur. We found that α-copaene marginally enhanced the trapping of E. perbrevis in Taiwan, but had no effect on the trapping of E. fornicatus or E. kuroshio in either California or Taiwan. We conclude that any enhancing effect of α-copaene is specific to E. perbrevis. This highlights the economic importance of accurate species identification in developing and implementing an efficient, and yet cost-effective, monitoring program for the management of E. fornicatus and E. kuroshio in California and elsewhere.

One becomes two: second species of the Euwallacea fornicatus (Coleoptera: Curculionidae: Scolytinae) species complex is established on two Hawaiian Islands.

The cryptic species that make up the Euwallacea fornicatus species complex can be readily distinguished via their DNA sequences. Until recently, it was believed that the Hawaiian Islands had been invaded by only one of these cryptic species, E. perbrevis (tea shot hole borer; TSHB). However, following the 2016 deposition of a DNA sequence in the public repository GenBank, it became evident that another species, E. fornicatus (polyphagous shot hole borer; PSHB), had been detected in macadamia orchards on Hawai'i Island (the Big Island). We surveyed the two most-populous islands of Hawai'i, Big Island and O'ahu, and herein confirm that populations of TSHB and PSHB are established on both. Beetles were collected using a variety of techniques in macadamia orchards and natural areas. Individual specimens were identified to species using a high-resolution melt assay, described herein and validated by subsequent sequencing of specimens. It remains unclear how long each species has been present in the state, and while neither is currently recognized as causing serious economic or ecological damage in Hawai'i, the similarity of the newly-confirmed PSHB population to other damaging invasive PSHB populations around the world is discussed. Although the invasive PSHB populations in Hawai'i and California likely have different geographic origins within the beetle's native range, they share identical Fusarium and Graphium fungal symbionts, neither of which have been isolated from PSHB in that native range.

Evaluation of δ 15N analysis to trace the origin of Diaphorina citri (Hemiptera: Liviidae) to citrus orchard fertilization management.

We investigated the variability of nitrogen stable isotope ratios 15N/14N (expressed as δ15N) on citrus orchards with different fertilization management practices (organic versus conventional) and its correlation with the δ15N values of the key citrus pest Diaphorina citri Kuwayama (Hemiptera: Liviidae) feeding on such plant material. Tracing the origin of this pest in open field is crucial since the insect is a vector of the incurable and devastating citrus disease known as Huanglongbing. We hypothesized that the origin (natal tree) of the pest may be deduced by correlating the δ15N values obtained from the young citrus leaves and from adults of D. citri raised on them. First, laboratory experiments were performed to understand the acquisition and incorportation of the δ15N values by D. citri. Second, we confirmed the positive correlation between the δ15N values of the young citrus leaves and D. citri. Finally, field sampling was carried out in 21 citrus orchards from Southern California to study the variability on the δ15N values on organic and conventional commercial citrus orchards. Laboratory results suggest that the analyses of the δ15N values can be regarded as a useful method to trace the origin of the pest. However, the high variability in nitrogen resource used in both fertilization management practices (especially in organic orchards) by growers makes the application of this technique unfeasible to pinpoint the origin of D. citri in the citrus agroecosystem.

Distinct epigenomic and transcriptomic modifications associated with Wolbachia-mediated asexuality.

Wolbachia are maternally transmitted intracellular bacteria that induce a range of pathogenic and fitness-altering effects on insect and nematode hosts. In parasitoid wasps of the genus Trichogramma, Wolbachia infection induces asexual production of females, thus increasing transmission of Wolbachia. It has been hypothesized that Wolbachia infection accompanies a modification of the host epigenome. However, to date, data on genome-wide epigenomic changes associated with Wolbachia are limited, and are often confounded by background genetic differences. Here, we took sexually reproducing Trichogramma free of Wolbachia and introgressed their genome into a Wolbachia-infected cytoplasm, converting them to Wolbachia-mediated asexuality. Wolbachia was then cured from replicates of these introgressed lines, allowing us to examine the genome-wide effects of wasps newly converted to asexual reproduction while controlling for genetic background. We thus identified gene expression and DNA methylation changes associated with Wolbachia-infection. We found no overlaps between differentially expressed genes and differentially methylated genes, indicating that Wolbachia-infection associated DNA methylation change does not directly modulate levels of gene expression. Furthermore, genes affected by these mechanisms exhibit distinct evolutionary histories. Genes differentially methylated due to the infection tended to be evolutionarily conserved. In contrast, differentially expressed genes were significantly more likely to be unique to the Trichogramma lineage, suggesting host-specific transcriptomic responses to infection. Nevertheless, we identified several novel aspects of Wolbachia-associated DNA methylation changes. Differentially methylated genes included those involved in oocyte development and chromosome segregation. Interestingly, Wolbachia-infection was associated with higher levels of DNA methylation. Additionally, Wolbachia infection reduced overall variability in gene expression, even after accounting for the effect of DNA methylation. We also identified specific cases where alternative exon usage was associated with DNA methylation changes due to Wolbachia infection. These results begin to reveal distinct genes and molecular pathways subject to Wolbachia induced epigenetic modification and/or host responses to Wolbachia-infection.

Members of the Euwallacea fornicatus species complex exhibit promiscuous mutualism with ambrosia fungi in Taiwan.

Carrillo, J.D., Rugman-Jones, PF., Husein, D., Stajich, J.E., Kasson, M.T., Carrillo, D., Stouthamer, R., and Eskalen, A. 2019. Members of the Euwallacea fornicatus species complex exhibit promiscuous mutualism with ambrosia fungi in Taiwan. A number of ambrosia beetles have come to prominence in recent years because of the damage they inflict on a variety of trees within invaded habitats across the globe. Ambrosia beetles rely on symbiotic microorganisms, mainly fungi, as a dedicated food source and carry those microorganisms around with them within specialized organs termed mycangia. Investigation of members of the Euwallacea fornicatus species complex and their fungal symbionts in Taiwan revealed promiscuous symbioses with ambrosial Fusaria clade (AFC) members, Graphium spp., and Paracremonium spp. based on co-phylogenetic analyses. For AFC members, a novel diagnostic PCR assay targeting mating type genes MAT1-1-1 and MAT1-2-1 was developed and validated by amplicon size and sequencing. Mating type screening of AFC members revealed the isolates screened are all heterothallic (self-sterile), with both MAT types represented and recovered from fungi vectored by E. fornicatus (tea shot hole borer), E. kuroshio (Kuroshio shot hole borer), and E. whitfordiodendrus (polyphagous shot hole borer) in Taiwan. Members of the Euwallacea fornicatus species complex and the variety of ambrosia fungi they utilize further confirms that their relationship with these fungi are more likely promiscuous in native areas, as opposed to strictly obligate to a specific combination of fungi as observed in invaded areas.

Impact of the Temperature on the Phenology of Diaphorina citri (Hemiptera: Liviidae) and on the Establishment of Tamarixia radiata (Hymenoptera: Eulophidae) in Urban Areas in the Lower Colorado Desert in Arizona.

The invasive pest Diaphorina citri Kuwayama was first detected in Arizona in 2009. Since late 2013, the parasitoid Tamarixia radiata (Waterson), the main biocontrol agent of D. citri, has been released as part of a biological control program on citrus grown in urban areas of two western Arizona counties in the lower Colorado desert environment. Here we report a 3-yr survey aimed at evaluation of T. radiata releases on D. citri populations and assess the impact of the climate conditions on the phenology of D. citri and on the establishment success of T. radiata. We also monitored the phenology of D. citri as part of this assessment on different citrus host species. We show that the high summer temperatures in the Arizona desert halt the development of D. citri for about 3 mo every year which appears to have limited the establishment and impact of T. radiata. At survey sites distant from release areas the parasitism rates over the season ranged from 0 to 75% and on average peaked around 50% in 2016 but it was low or absent in 2015 and 2017, respectively. We discuss the consequences of this phenology of D. citri in the desert areas for the prospects of long-term establishment of T. radiata and the management of this key citrus pest.

Laboratory Hybridization Between the Green Lacewings Chrysoperla comanche (Neuroptera: Chrysopidae) and Chrysoperla rufilabris, Predators of the Asian Citrus Psyllid.

Chrysoperla comanche (Banks) and its sibling species Chrysoperla rufilabris (Burmeister) are voracious predators of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), an invasive pest that vectors a bacterium responsible for the lethal and incurable citrus greening disease. The Comanche lacewing naturally occurs in Southern California, whereas C. rufilabris is currently one of only two commercially available green lacewing in the United States. These sister species can be separated by larval morphological traits, by differences in courtship songs, and possibly by three nuclear genes wingless, PepCK, ATPase, yet they are not distinguishable based on the mitochondrial barcode gene (COI). Releasing in a new area a biological control agent capable of hybridizing with a resident species may pose risks that range from local displacement to irreversible loss of genetic identity. Therefore, we performed no-choice laboratory crosses to assess pre- and postzygotic isolation. We show that fertile and viable hybrid progeny could be readily obtained in interspecific crosses and backcrosses and, although there is a trend toward lower hybrid fitness, postzygotic isolation is overall weak and might not prevent loss of genetic identity under natural conditions. It remains to be determined if differences in courtship songs will prevent hybridization in the wild, as shown for other green lacewings. We also report a low prevalence of Rickettsia infection in both species.

Genome-wide analyses of single nucleotide polymorphisms reveal the consequences of traditional mass-rearing on genetic variation in Aphytis melinus (Hymenoptera: Aphelinidae): the danger of putting all eggs in one basket.

BACKGROUND: Aphytis melinus DeBach (Hymenoptera: Aphelinidae) is a highly effective biocontrol agent of the California red scale Aonidiella aurantii (Maskell) (Hemiptera: Diaspididae). It is commercially reared and used for augmentative releases within integrated pest management programs. However, mass rearing of biocontrol agents can result in population bottlenecks and high levels of inbreeding and/or adaptation to the factitious rearing conditions. Although these factors can all negatively impact field performance of biocontrol agents, few empirical studies have examined the genetic consequences of mass rearing. We used double-digest RAD sequencing (ddRADseq) to investigate the effect of traditional mass rearing on genetic variation among insectary colonies of A. melinus relative to wild populations in native (Pakistan) and introduced (California) ranges. RESULTS: Analyses of up to 9700 single nucleotide polymorphisms (SNPs) revealed that insectary populations had less genomic variation than introduced populations. This was evidenced by fewer private alleles, reduced heterozygosity, and greater missing data in the insectary populations. Further, California insectaries formed a distinct genomic cluster relative to the other samples, a surprising result given that the insectary colonies were putatively established at different times and from different source populations. These differences were evident across most data sets also after we filtered out contaminant DNA from the most common host species (Aspidiotus nerii Bouché and A. aurantii). CONCLUSION: We hypothesize that this pattern would only result if: (i) directional selection for 'captive' phenotypes produces convergent patterns of genomic variation across insectaries; or (ii) the California insectary colonies were all founded from a unifying source population and/or that the insectaries regularly exchange 'genetic' stocks. We show that RADseq is an effective method to investigate the effects of mass rearing on genetics of biocontrol agents. © 2019 Society of Chemical Industry.

An Experimental Test of the Host-Plant Range of Nonrecombinant Strains of North American Xylella fastidiosa subsp. multiplex.

Nonrecombinant strains of Xylella fastidiosa subsp. multiplex (those lacking evidence of significant intersubspecific homologous recombination) infect the xylem of a wide range of native and nonnative trees in North America. However, the degree to which different strains have a specialized host range remains poorly understood. We tested eight strains isolated from five different tree species (almond, olive, sweetgum, and plum in California and oak in Washington, DC). Experiments were conducted in greenhouses in Riverside, CA, and each strain was tested on 11 to 15 of the 17 plant species tested. Hosts infected by the most strains were plum (5 of 8 strains) and almond (4 of 8), while their congener peach was only infected by 1 of 8. No strains infected oleander or mulberry. All strains successfully infected their original host, with peach, olive (1 of 7), and sweetgum (2 of 6) only infected by such strains. Of the 90 total strain-novel-host combinations tested, 11 resulted in unambiguous infection, 2 gave ambiguous results, and the remaining 77 failed to result in symptoms or bacterial spread. All eight strains had a unique host range, including two pairs of strains with the same multilocus sequence typing sequence type, providing strong evidence of extensive plant-host specialization. There was little evidence that host relatedness was driving host specificity.

Genomic Signals of Adaptation towards Mutualism and Sociality in Two Ambrosia Beetle Complexes.

Mutualistic symbiosis and eusociality have developed through gradual evolutionary processes at different times in specific lineages. Like some species of termites and ants, ambrosia beetles have independently evolved a mutualistic nutritional symbiosis with fungi, which has been associated with the evolution of complex social behaviors in some members of this group. We sequenced the transcriptomes of two ambrosia complexes (Euwallacea sp. near fornicatus⁻Fusarium euwallaceae and Xyleborus glabratus⁻Raffaelea lauricola) to find evolutionary signatures associated with mutualism and behavior evolution. We identified signatures of positive selection in genes related to nutrient homeostasis; regulation of gene expression; development and function of the nervous system, which may be involved in diet specialization; behavioral changes; and social evolution in this lineage. Finally, we found convergent changes in evolutionary rates of proteins across lineages with phylogenetically independent origins of sociality and mutualism, suggesting a constrained evolution of conserved genes in social species, and an evolutionary rate acceleration related to changes in selective pressures in mutualistic lineages.

Comparative genomics of the miniature wasp and pest control agent Trichogramma pretiosum.

BACKGROUND: Trichogrammatids are minute parasitoid wasps that develop within other insect eggs. They are less than half a millimeter long, smaller than some protozoans. The Trichogrammatidae are one of the earliest branching families of Chalcidoidea: a diverse superfamily of approximately half a million species of parasitoid wasps, proposed to have evolved from a miniaturized ancestor. Trichogramma are frequently used in agriculture, released as biological control agents against major moth and butterfly pests. Additionally, Trichogramma are well known for their symbiotic bacteria that induce asexual reproduction in infected females. Knowledge of the genome sequence of Trichogramma is a major step towards further understanding its biology and potential applications in pest control. RESULTS: We report the 195-Mb genome sequence of Trichogramma pretiosum and uncover signatures of miniaturization and adaptation in Trichogramma and related parasitoids. Comparative analyses reveal relatively rapid evolution of proteins involved in ribosome biogenesis and function, transcriptional regulation, and ploidy regulation. Chalcids also show loss or especially rapid evolution of 285 gene clusters conserved in other Hymenoptera, including many that are involved in signal transduction and embryonic development. Comparisons between sexual and asexual lineages of Trichogramma pretiosum reveal that there is no strong evidence for genome degradation (e.g., gene loss) in the asexual lineage, although it does contain a lower repeat content than the sexual lineage. Trichogramma shows particularly rapid genome evolution compared to other hymenopterans. We speculate these changes reflect adaptations to miniaturization, and to life as a specialized egg parasitoid. CONCLUSIONS: The genomes of Trichogramma and related parasitoids are a valuable resource for future studies of these diverse and economically important insects, including explorations of parasitoid biology, symbiosis, asexuality, biological control, and the evolution of miniaturization. Understanding the molecular determinants of parasitism can also inform mass rearing of Trichogramma and other parasitoids for biological control.

Host and symbiont genetic contributions to fitness in a Trichogramma-Wolbachia symbiosis.

The fitness effects associated with Wolbachia infection have wide-ranging ecological and evolutionary consequences for host species. How these effects are modulated by the relative influence of host and Wolbachia genomes has been described as a balancing act of genomic cooperation and conflict. For vertically transmitted symbionts, like cytoplasmic Wolbachia, concordant host-symbiont fitness interests would seem to select for genomic cooperation. However, Wolbachia's ability to manipulate host reproductive systems and distort offspring sex ratios presents an evolutionary conflict of interest with infected hosts. In the parthenogenesis-inducing (PI) form of Wolbachia found in many haplodiploid insects, Wolbachia fitness is realized through females and is enhanced by their feminization of male embryos and subsequent parthenogenetic reproduction. In contrast, as long as Wolbachia is not fixed in a population and sexual reproduction persists, fitness for the host species is realized through both male and female offspring production. How these cooperating and competing interests interact and the relative influence of host and Wolbachia genomes were investigated in the egg parasitoid Trichogramma kaykai, where Wolbachia infection has remained at a low frequency in the field. A factorial design in which laboratory cultures of Wolbachia-infected T. kaykai were cured and re-infected with alternative Wolbachia strains was used to determine the relative influence of host and Wolbachia genomes on host fitness values. Our results suggest fitness variation is largely a function of host genetic background, except in the case of offspring sex ratio where a significant interaction between host and Wolbachia genomes was found. We also find a significant effect associated with the horizontal transfer of Wolbachia strains, which we discuss in terms of the potential for coadaptation in PI-Wolbachia symbioses.

Two Novel Fungal Symbionts Fusarium kuroshium sp. nov. and Graphium kuroshium sp. nov. of Kuroshio Shot Hole Borer (Euwallacea sp. nr. fornicatus) Cause Fusarium Dieback on Woody Host Species in California.

Shot hole borer (SHB)-Fusarium dieback (FD) is a new pest-disease complex affecting numerous tree species in California and is vectored by two distinct, but related ambrosia beetles (Euwallacea sp. nr. fornicatus) called polyphagous shot hole borer (PSHB) and Kuroshio shot hole borer (KSHB). These pest-disease complexes cause branch dieback and tree mortality on numerous wildland and landscape tree species, as well as agricultural tree species, primarily avocado. The recent discovery of KSHB in California initiated an investigation of fungal symbionts associated with the KSHB vector. Ten isolates of Fusarium sp. and Graphium sp., respectively, were recovered from the mycangia of adult KSHB females captured in three different locations within San Diego County and compared with the known symbiotic fungi of PSHB. Multigene phylogenetic analyses of the internal transcribed spacer region (ITS), translation elongation factor-1 alpha (TEF1-α), and RNA polymerase II subunit (RPB1, RPB2) regions as well as morphological comparisons revealed that two novel fungal associates Fusarium kuroshium sp. nov. and Graphium kuroshium sp. nov. obtained from KSHB were related to, but distinct from the fungal symbionts F. euwallaceae and G. euwallaceae associated with PSHB in California. Pathogenicity tests on healthy, young avocado plants revealed F. kuroshium and G. kuroshium to be pathogenic. Lesion lengths from inoculation of F. kuroshium were found to be significantly shorter compared with those caused by F. euwallaceae, while no difference in symptom severity was detected between Graphium spp. associated with KSHB and PSHB. These findings highlight the pest disease complexes of KSHB-FD and PSHB-FD as distinct, but collective threats adversely impacting woody hosts throughout California.

Pheromones of three ambrosia beetles in the Euwallacea fornicatus species complex: ratios and preferences.

Three cryptic species in the Euwallacea fornicatus species complex were reared in laboratory colonies and investigated for the presence of pheromones. Collections of volatiles from combinations of diet, fungus, beetles, and galleries from polyphagous shot hole borer (Euwallacea sp. #1) revealed the presence of 2-heneicosanone and 2-tricosanone only in the presence of beetles, regardless of sex. Subsequent examination of volatiles from the other two species, tea shot hole borer (Euwallacea sp. #2) and Kuroshio shot hole borer (Euwallacea sp. #5), revealed these two ketones were present in all three species but in different ratios. In dual choice olfactometer behavioral bioassays, mature mated females were strongly attracted to a synthetic binary blend of ketones matching their own natural ratios. However, females in each species were repelled by ketone blends in ratios corresponding to the other two species. Males of each species responded similarly to females when presented with ratios matching their own or the other two species. The presence of these compounds in the three beetle species, in ratios unique to each species, and their strong species-specific attraction and repellency, suggests they are pheromones. The ecological function of these pheromones is discussed. In addition to the pheromones, the previously known attractant (1S,4R)-p-menth-2-en-1-ol (also known as quercivorol) was discovered in the presence of the fungal symbionts, but not in association with the beetles. Quercivorol was tested in a dual-choice olfactometer and was strongly attractive to all three species. This evidence suggests quercivorol functions as a kairomone for members of the E. fornicatus species complex, likely produced by the symbiotic fungi.

Quercivorol as a lure for the polyphagous and Kuroshio shot hole borers, Euwallacea spp. nr. fornicatus (Coleoptera: Scolytinae), vectors of Fusarium dieback.

The polyphagous shot hole borer and Kuroshio shot hole borer, two members of the Euwallacea fornicatus species complex (Coleoptera: Curculionidae: Scolytinae), are invasive ambrosia beetles that harbor distinct species of Fusarium fungal symbionts. Together with the damage caused by gallery construction, these two phytopathogenic Fusarium species are responsible for the emerging tree disease Fusarium dieback, which affects over 50 common tree species in Southern California. Host trees suffer branch dieback as the xylem is blocked by invading beetles and fungi, forcing the costly removal of dead and dying trees in urban areas. The beetles are also threatening natural riparian habitats, and avocado is susceptible to Fusarium dieback as well, resulting in damage to the avocado industries in California and Israel. Currently there are no adequate control mechanisms for shot hole borers. This paper summarizes efforts to find a suitable lure to monitor shot hole borer invasions and dispersal. Field trials were conducted in two counties in Southern California over a span of two years. We find that the chemical quercivorol is highly attractive to these beetles, and perform subsequent field experiments attempting to optimize this lure. We also explore other methods of increasing trap catch and effects of other potential attractants, as well as the deterrents verbenone and piperitone.

Penetrance of symbiont-mediated parthenogenesis is driven by reproductive rate in a parasitoid wasp.

Trichogramma wasps are tiny parasitoids of lepidopteran eggs, used extensively for biological control. They are often infected with the bacterial symbiont Wolbachia, which converts Trichogramma to an asexual mode of reproduction, whereby females develop from unfertilized eggs. However, this Wolbachia-induced parthenogenesis is not always complete, and previous studies have noted that infected females will produce occasional males in the lab. The conditions that reduce penetrance of the parthenogenesis phenotype are not well understood. We hypothesized that more ecologically relevant conditions of limited host access will sustain female-biased sex ratios. After restricting access to host eggs, we found a strong relationship between reproductive rate and sex ratio. By limiting reproduction to one hour a day, wasps could sustain up to 100% effective parthenogenesis for one week, with no significant impact on total fecundity. Reproductive output in the first 24-hours appears to be critical to the total sex ratio of the entire brood. Limiting oviposition in that period resulted in more effective parthenogenesis after one week, again without any significant impact on total fecundity. Our data suggest that this phenomenon may be due to the depletion of Wolbachia when oviposition occurs continuously, whereas Wolbachia titers may recover when offspring production is limited. In addition to the potential to improve mass rearing of Trichogramma for biological control, findings from this study help elucidate the context-dependent nature of a pervasive symbiotic relationship.