Development of a sterile insect technique as a control strategy for the Asian citrus psyllid: establishing the effect of sterilizing X-rays on fecundity, fertility, and survival.

The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is a major pest of citrus due to its role as the vector of the bacterium that causes huanglongbing. In commercial citrus, ACP control currently relies on the application of insecticides, which may not be sustainable long-term, nor practical in urban areas. The sterile insect technique (SIT) is an alternative strategy in which large numbers of pests are reared, sterilized using radiation, and then released into the field to compete with wild individuals for matings, suppressing population growth. As a fundamental step toward the development of SIT for ACP, this study sought to identify the optimum radiation dose required to sterilize ACP without affecting their survival and mating capacity. Virgin adult ACP of both sexes were subjected to doses of X-ray irradiation ranging from 40 to 480 Gy, then paired with a nonirradiated mate and allowed to produce offspring. Fecundity was estimated as the number of eggs laid, and fertility as the proportion of those eggs that hatched. Females were more radio-sensitive than males, exhibiting a major drop in fecundity at even the lowest dose and 100% sterility at 80 Gy. In contrast, a fivefold higher dose (400 Gy) did not achieve complete sterility in males, with around 5% offspring survival. However, F1 progeny of males exposed to 320 Gy or higher were subsequently found to be 100% sterile. This confirmation of inherited sterility suggests that balancing the sterilizing effects of radiation against its mortality-inducing effects may warrant further evaluation.

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.

Re-collection and identity of Ooencyrtus californicus (Hymenoptera: Encyrtidae), and its new synonym, Ooencyrtus lucidus.

Girault (1917) very briefly described and diagnosed the encyrtid wasp species Ooencyrtus californicus Girault (Hymenoptera: Encyrtidae) with the following limited data (p. 22): "Sacramento, California, from bug eggs on Pinus sabiniana, September". The first author examined its two syntypes, poorly mounted on a slide, of which only parts of the four antennae and a slightly damaged fore wing remain (Triapitsyn et al. 2020). They concluded that this species was better considered a nomen dubium until fresh specimens could be collected from the same host plant in or near the type locality. In July 2019, an effort was made to re-collect O. californicus from foothill (or gray) pine, Pinus sabiniana (Pinaceae), in the Oakhurst area of Madera County, California, USA, where this pine is abundant, but no specimens were captured. At the same time, a very similar insect was collected from sentinel eggs of the invasive stink bug Bagrada hilaris (Burmeister) in Riverside, California. This insect was cultured in the laboratory as part of a B. hilaris biological control program. Due to the poor preservation of the type specimens and in the absence of genetic evidence, it was impossible to positively and properly attribute specimens of this newly collected parasitoid to O. californicus. Therefore, to provide a much needed scientific name for this native egg parasitoid, it was described as Ooencyrtus lucidus Triapitsyn Ganjisaffar in Triapitsyn et al. (2020). The two nominal species were separated based on minor differences in the proportions of the scape, pedicel, and first funicular segment of the female antenna (Triapitsyn et al. 2020). John S. Noyes, world expert on Encyrtidae and one of the reviewers of Triapitsyn et al. (2020), disagreed with the erection of O. lucidus as a new taxon based on the morphological similarities of the female antenna with that of O. californicus, so the description was published with the understanding that the two might be conspecific. But to settle this one way or another new specimens of O. californicus had to be collected to allow morphological and molecular analyses.

Anagrus avalae Soyka, 1956, a new synonym of A. bakkendorfi Soyka, 1946 (Hymenoptera: Mymaridae).

Soyka (1946) described the European fairyfly species Anagrus bakkendorfi Soyka (Hymenoptera: Mymaridae) from a single female collected on the same day, 7.x.1931, together with several non-type specimens incorrectly labeled as paratypes (Chiappini Triapitsyn 1999), on a window at its type locality, the former St. Ignatius Jesuit College in Valkenburg, Limburg, the Netherlands. Soyka (1956) did not mention type specimens of any of his species of Anagrus Haliday. He described Anagrus avalae Soyka (Soyka 1956) based apparently on a specimen collected on Mt. Avala, Belgrade, Serbia, former Yugoslavia, along with several other nominal species (some also from Valkenburg) that were later synonymized with A. avalae and A. bakkendorfi by Chiappini (1989) and Chiappini Triapitsyn (1999). The only, minor, morphological difference between the two nominal species is the length of the ovipositor in females relative to the length of the protibia, being at least 2.6× in A. bakkendorfi and at most 2.3× in A. avalae (Triapitsyn 2015). Specimens identifiable as both species were captured by the second author in 2000 by a Malaise trap in Iratibizkar, Irati Forest (Selva de Irati), Navarra, Spain. Chiappini Triapitsyn (1999) discussed both nominal species but despite recognizing their apparent conspecificity, no formal synonymy was proposed. Triapitsyn Berezovskiy (2004) and Triapitsyn (2015) also did not propose synonymy because at that time genetic evidence for conspecificity was lacking. However, Triapitsyn et al. (2019) obtained DNA and sequenced selected mitochondrial and nuclear ribosomal gene regions (COI and ITS2) from a specimen unambiguously identified by the first author as A. avalae that was collected in Sevenoaks, Kent County, England, United Kingdom. Because of this we decided to collect fresh specimens identifiable as A. bakkendorfi for molecular analysis and genetic comparison with those identifiable as A. avalae. Based on the previous record (Triapitsyn 2015), several females of A. bakkendorfi were successfully captured by the second author on 4.ix.2020 in Irati Forest, preserved in ethanol, and shipped to the first and third authors for morphological and molecular identification, respectively.

Establishment of a non-native xyleborine ambrosia beetle, Xyleborus monographus (Fabricius) (Coleoptera: Curculionidae: Scolytinae), new to North America in California.

Specimens of an ambrosia beetle, Xyleborus monographus (Fabricius), were found infesting oak trees in California. This is the first record of this species established in North America. Based on collection information, this species most likely has been established in the Napa County area for several years. A modified key to Xyleborus in North America, and diagnosis of the species is provided.

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.

Egg parasitoids of Arboridia apicalis (Nawa, 1913) (Hemiptera, Cicadellidae), a leafhopper pest of grapevines in Japan, with description of a new species of Anagrus Haliday, 1833 (Hymenoptera, Mymaridae).

Several species of egg parasitoids (Hymenoptera: Mymaridae and Trichogrammatidae) of the leafhopper pest of grapevines in Japan, Arboridia (Arboridia) apicalis (Nawa) (Hemiptera, Cicadellidae), were reared and identified for the first time. Using a combination of genetic and morphological evidence, Anagrus (Anagrus) arboridiae Triapitsyn & Adachi-Hagimori, sp. nov. (Mymaridae) is described and illustrated from Honshu Island (Shimane Prefecture) and Kyushu Island (Miyazaki Prefecture). It is shown to be different from Anagrus (Anagrus) japonicus Sahad and A. flaviapex Chiappini & Lin, to which it is most similar; the latter species was originally described from China and is newly recorded here from Okinawa Island, Japan. Mitochondrial and nuclear ribosomal DNA sequence data provide clear evidence for the separation of A. arboridiae from A. flaviapex, A. japonicus, and some other members of the Anagrus (Anagrus) atomus (L.) species group. Two other species of Anagrus Haliday, A. (Anagrus) avalae Soyka and A. atomus, are also identified in Japan from eggs of the leafhoppers Edwardsiana ishidae (Matsumura) and Eurhadina ? betularia Anufriev, respectively. An updated key to females of the Japanese species of Anagrus is given. Oligosita pallida Kryger (a new record for Japan), Oligosita sp., and an Aphelinoidea (Aphelinoidea) sp. (Trichogrammatidae) were the other, although much less abundant, apparent egg parasitoids of A. apicalis in Shimane Prefecture, mainly in non-organic vineyards.

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.

Egg parasitoids of the tea green leafhopper Empoascaonukii (Hemiptera, Cicadellidae) in Japan, with description of a new species of Anagrus (Hymenoptera, Mymaridae).

Fairyfly (Hymenoptera, Mymaridae) egg parasitoids of the tea green leafhopper Empoasca (Matsumurasca) onukii Matsuda (Hemiptera, Cicadellidae), an economically important pest in Asia of the tea plant, Camelliasinensis, were identified from specimens reared in Japan. Using a combination of genetic and morphological evidence, Anagrus (Anagrus) rugmanjonesi Triapitsyn & Adachi-Hagimori, sp. n., is described and illustrated. It is shown to be different from the most similar A.turpanicus Triapitsyn & Hu, an egg parasitoid of a leafhopper pest of cultivated grapes which is known from Xinjiang Uyghur Autonomous Region in China. Mitochondrial and nuclear ribosomal DNA sequence data provide clear evidence for the separation of A.rugmanjonesi from A.turpanicus and other members of the Anagrusincarnatus Haliday species complex. A key to females of the Japanese species of Anagrus Haliday is given. Two other species of Mymaridae, Aresconenocki (Subba Rao & Kaur) and Stethyniumempoascae Subba Rao, are also identified, albeit the latter one only tentatively. Both latter taxa are newly recorded from Japan, and E.onukii represents their new host association.

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.

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.

High-resolution melt analysis without DNA extraction affords rapid genotype resolution and species identification.

Extracting and sequencing DNA from specimens can impose major time and monetary costs to studies requiring genotyping, or identification to species, of large numbers of individuals. As such, so-called direct PCR methods have been developed enabling significant savings at the DNA extraction step. Similarly, real-time quantitative PCR techniques (qPCR) offer very cost-effective alternatives to sequencing. High-resolution melt analysis (HRM) is a qPCR method that incorporates an intercalating dye into a double-stranded PCR amplicon. The dye fluoresces brightly, but only when it is bound. Thus, after PCR, raising the temperature of the amplicon while measuring the fluorescence of the reaction results in the generation of a sequence-specific melt curve, allowing discrimination of genotypes. Methods combining HRM (or other qPCR methods) and direct PCR have not previously been reported, most likely due to concerns that any tissue in the reaction tube would interfere with detection of the fluorescent signal. Here, we couple direct PCR with HRM and, by way of three examples, demonstrate a very quick and cost-effective method for genotyping large numbers of specimens, using Rotor-Gene HRM instruments (QIAGEN). In contrast to the heated-block design of most qPCR/HRM instruments, the Rotor-Gene's centrifugal rotor and air-based temperature-regulation system facilitate our method by depositing tissues away from the pathway of the machine's fluorescence detection optics.

Mitochondrial DNA Variation Among Populations of Rhynchophorus ferrugineus (Coleoptera: Curculionidae) From Pakistan.

The Red Palm Weevil (RPW) Rhynchophorus ferrugineus (Olivier) is a voracious pest of palm species. In recent decades its range has expanded greatly, particularly impacting the date palm industry in the Middle East. This has led to conjecture regarding the origins of invasive RPW populations. For example, in parts of the Middle East, RPW is commonly referred to as the "Pakistani weevil" in the belief that it originated there. We sought evidence to support or refute this belief. First reports of RPW in Pakistan were from the Punjab region in 1918, but it is unknown whether it is native or invasive there. We estimated genetic variation across five populations of RPW from two provinces of Pakistan, using sequences of the mitochondrial cytochrome oxidase subunit I gene. Four haplotypes were detected; two (H1 and H5) were abundant, accounting for 88% of specimens across the sampled populations, and were previously known from the Middle East. The remaining haplotypes (H51 and H52) were newly detected (in global terms) and there was no geographic overlap in their distribution within Pakistan. Levels of haplotype diversity were much lower than those previously recorded in accepted parts of the native range of RPW, suggesting that the weevil may be invasive in Pakistan. The affinity of Pakistani haplotypes to those reported from India (and the geographical proximity of the two countries), make the latter a likely "native" source. With regards the validity of the name "Pakistani weevil", we found little genetic evidence to justify it.

Laboratory Performance Predicts the Success of Field Releases in Inbred Lines of the Egg Parasitoid Trichogramma pretiosum (Hymenoptera: Trichogrammatidae).

In this study we assessed the relationship between the laboratory and field performance of different isofemale lines of Trichogramma pretiosum Riley. In comparative assays, we used three rare mitochondrial haplotypes as genetic markers of the isofemale lines, and by introgressing these mitochondrial haplotypes into each of 15 genetically different nuclear lines, also tested the assumption that mitochondria are neutral markers. In a laboratory trial, 45 isofemale lines (15 nuclear genotypes x three mitochondrial haplotypes) were ranked in three categories (best, intermediate and worst) according to the mean offspring production and the proportion of female offspring. Subsequently, lines from each of the three categories were selected for field releases to quantify field parasitism on Ephestia kuehniella. Temporally separate releases were done in a transgenic Bt cornfield, with four plots, each with 50 points of recapture. The points of recapture consisted of trap cards with eggs of E. kuehniella collected daily. The trap cards were maintained in the laboratory at 25°C until the adult wasps emerged, and the maternal identity of the wasps was determined using qPCR and high-resolution melt curve analysis to determine the mitochondrial haplotype. The results showed that these measures of laboratory performance (fecundity and offspring sex ratio) were good predictors of field success in T. pretiosum. We also report strong evidence discrediting the assumption that mitochondria are neutral, in view of the correlation between performance and mitochondrial haplotype.

Identification, pathogenicity and abundance of Paracremonium pembeum sp. nov. and Graphium euwallaceae sp. nov.--two newly discovered mycangial associates of the polyphagous shot hole borer (Euwallacea sp.) in California.

Fusarium euwallaceae is a well-characterized fungal symbiont of the exotic ambrosia beetle Euwallacea sp. (polyphagous shot hole borer [PSHB]), together inciting Fusarium dieback on many host plants in Israel and California. Recent discoveries of additional fungal symbionts within ambrosia beetle mycangia suggest these fungi occur as communities. Colony-forming units of Graphium euwallaceae sp. nov. and Paracremonium pembeum sp. nov., two novel fungal associates of PSHB from California, grew from 36 macerated female heads and 36 gallery walls collected from Platanus racemosa, Acer negundo, Persea americana and Ricinus communis. Fungi were identified based on micromorphology and phylogenetic analyses of the combined internal transcribed spacer region (nuc rDNA ITS1-5.8S-ITS2 [ITS barcode]), elongation factor (EF 1-α), small subunit (18S rDNA) sequences for Graphium spp., ITS, EF 1-α, calmodulin (cmdA), large subunit of the ATP citrate lyase (acl1), ß-tubulin (tub2), RNA polymerase II second largest subunit (rpb2) and large subunit (28S rDNA) sequences for Paracremonium spp. Other Graphium spp. recovered from PSHB in Vietnam, Euwallacea fornicatus in Thailand, E. validus in Pennsylvania and Paracremonium sp. recovered from PSHB in Vietnam were identified. F. euwallaceae was recovered from mycangia at higher frequencies and abundances in all hosts except R. communis, in which those of F. euwallaceae and P. pembeum were equal. P. pembeum was relatively more abundant within gallery walls of A. negundo and R. communis. In all hosts combined F. euwallaceae was relatively more abundant within PSHB heads than gallery walls. All three fungi grew at different rates and colonized inoculated excised stems of P. americana and A. negundo. P. pembeum produced longer lesions than F. euwallaceae and G. euwallaceae on inoculated avocado shoots. Results indicate PSHB is associated with a dynamic assemblage of mycangial fungal associates that pose additional risk to native and nonnative hosts in California.

Developmental succession of the microbiome of Culex mosquitoes.

BACKGROUND: The native microflora associated with mosquitoes have important roles in mosquito development and vector competence. Sequencing of bacterial V3 region from 16S rRNA genes across the developmental stages of Culex mosquitoes (early and late larval instars, pupae and adults) was used to test the hypothesis that bacteria found in the larval stage of Culex are transstadially transmitted to the adult stage, and to compare the microbiomes of field-collected versus laboratory-reared mosquitoes. RESULTS: Beta diversity analysis revealed that bacterial community structure differed among three life stages (larvae, pupae and adults) of Culex tarsalis. Although only ~2% of the total number of bacterial OTUs were found in all stages, sequences from these OTUs accounted for nearly 82% of the total bacterial sequences recovered from all stages. Thorsellia (Gammaproteobacteria) was the most abundant bacterial taxon found across all developmental stages of field-collected Culex mosquitoes, but was rare in mosquitoes from laboratory-reared colonies. The proportion of Thorsellia sequences in the microbiomes of mosquito life stages varied ontogenetically with the greatest proportions recovered from the pupae of C. tarsalis and the lowest from newly emerged adults. The microbiome of field-collected late instar larvae was not influenced significantly by differences in the microbiota of the habitat due to habitat age or biopesticide treatments. The microbiome diversity was the greatest in the early instar larvae and the lowest in laboratory-reared mosquitoes. CONCLUSIONS: Bacterial communities in early instar C. tarsalis larvae were significantly more diverse when compared to late instar larvae, pupae and newly emerged adults. Some of the bacterial OTUs found in the early instar larvae were also found across developmental stages. Thorsellia dominated the bacterial communities in field-collected immature stages but occurred at much lower relative abundance in adults. Differences in microbiota observed in larval habitats did not influence bacterial community profiles of late instar larvae or adults. However, bacterial communities in laboratory-reared C. tarsalis larvae differed significantly from the field. Determining the role of Thorsellia in mosquitoes and its distribution across different species of mosquitoes warrants further investigation.

Phylogeography of the walnut twig beetle, Pityophthorus juglandis, the vector of thousand cankers disease in North American walnut trees.

Thousand cankers disease (TCD) of walnut trees (Juglans spp.) results from aggressive feeding in the phloem by the walnut twig beetle (WTB), Pityophthorus juglandis, accompanied by inoculation of its galleries with a pathogenic fungus, Geosmithia morbida. In 1960, WTB was only known from four U.S. counties (in Arizona, California, and New Mexico), but the species has now (2014) invaded over 115 counties, representing much of the western USA, and at least six states in the eastern USA. The eastern expansion places TCD in direct proximity to highly valuable (> $500 billion) native timber stands of eastern black walnut, Juglans nigra. Using mitochondrial DNA sequences, from nearly 1100 individuals, we examined variation among 77 samples of WTB populations across its extended range in the USA, revealing high levels of polymorphism and evidence of two divergent lineages. The highest level of genetic diversity for the different lineages was found in the neighboring Madrean Sky Island and Western New Mexico regions, respectively. Despite their proximity, there was little evidence of mixing between these regions, with only a single migrant detected among 179 beetles tested. Indeed, geographic overlap of the two lineages was only common in parts of Colorado and Utah. Just two haplotypes, from the same lineage, predominated over the vast majority of the recently expanded range. Tests for Wolbachia proved negative suggesting it plays no role in "driving" the spread of particular haplotypes, or in maintaining deep levels of intraspecific divergence in WTB. Genotyping of ribosomal RNA corroborated the mitochondrial lineages, but also revealed evidence of hybridization between them. Hybridization was particularly prevalent in the sympatric areas, also apparent in all invaded areas, but absent from the most haplotype-rich area of each mitochondrial lineage. Hypotheses about the specific status of WTB, its recent expansion, and potential evolutionary origins of TCD are discussed.