A Whole-Genome Scan for Association with Invasion Success in the Fruit Fly Drosophila suzukii Using Contrasts of Allele Frequencies Corrected for Population Structure.

Evidence is accumulating that evolutionary changes are not only common during biological invasions but may also contribute directly to invasion success. The genomic basis of such changes is still largely unexplored. Yet, understanding the genomic response to invasion may help to predict the conditions under which invasiveness can be enhanced or suppressed. Here, we characterized the genome response of the spotted wing drosophila Drosophila suzukii during the worldwide invasion of this pest insect species, by conducting a genome-wide association study to identify genes involved in adaptive processes during invasion. Genomic data from 22 population samples were analyzed to detect genetic variants associated with the status (invasive versus native) of the sampled populations based on a newly developed statistic, we called C2, that contrasts allele frequencies corrected for population structure. We evaluated this new statistical framework using simulated data sets and implemented it in an upgraded version of the program BayPass. We identified a relatively small set of single-nucleotide polymorphisms that show a highly significant association with the invasive status of D. suzukii populations. In particular, two genes, RhoGEF64C and cpo, contained single-nucleotide polymorphisms significantly associated with the invasive status in the two separate main invasion routes of D. suzukii. Our methodological approaches can be applied to any other invasive species, and more generally to any evolutionary model for species characterized by nonequilibrium demographic conditions for which binary covariables of interest can be defined at the population level.

Laboratory evaluation of the life history of hibiscus mealybug, Nipaecocus viridis (Hemiptera: Pseudococcidae), on selected citrus and potential non-citrus hosts in Florida.

Nipaecoccus viridis (Newstead) (Hemiptera: Pseudococcidae) is an emerging invasive pest of citrus. There is no information on the life history of N. viridis in relation to citrus cultivars produced in Florida. Here, we quantified the life-history parameters of N. viridis on 6 citrus and 3 non-citrus host plant species under laboratory conditions. We also investigated the effect of mode of reproduction (sexual vs. parthenogenic) on the fecundity of N. viridis. Nipaecoccus viridis was able to develop to maturity and reproduce on all host plant taxa tested. The individual host plants affected the developmental and survival rate, adult longevity, and offspring production of N. viridis. All citrus genotypes commercially grown in Florida were susceptible to N. viridis, with the exception of Citrus aurantium L., which suggests that there is need for management of this invasive pest in Florida citrus groves. Murraya paniculata L., Hibiscus rosa-sinensis L., and Punica granatum L. were also acceptable hosts for N. viridis. Sexually reproducing female N. viridis produced significantly more eggs than those reproducing asexually. Based on our results, propagation of M. paniculata and other citrus relatives should be discouraged around citrus groves in Florida. Given the fitness benefit of sexual reproduction in this pest, pheromone-based tools like mating disruption and mass trapping of adult males could be considered as part of an integrated pest management program with insecticides.

Feeding behavior and hormoligosis associated with imidacloprid resistance in Asian citrus psyllid, Diaphorina citri.

Imidacloprid is a neonicotinoid insecticide used for managing the Asian citrus psyllid, Diaphorina citri Kuwayama, which serves as vector of phytopathogens causing citrus greening. However, development of resistance to neonicotinoids among populations of D. citri has coincided with occasional control failures in the field. The objectives of this research were to (1) survey current levels of imidacloprid resistance in Florida citrus; (2) compare feeding behavior between imidacloprid-resistant and susceptible D. citri using electrical penetration graph recordings, and (3) investigate the possible amplification of insecticide hormoligosis associated with resistance. Field surveys confirmed that the susceptibility of D. citri populations to imidacloprid has decreased in commercial Florida citrus groves compared with a laboratory-susceptible population. Following 12 generations of selection, resistance to imidacloprid increased by 438 fold compared with the susceptible strain. Imidacloprid-susceptible D. citri feeding on citrus exhibited significantly more bouts associated with intercellular pathway (C), phloem penetration (D), phloem salivation (E1), and nonprobing (Np) activities than imidacloprid-resistant counterparts. However, there were no differences observed in the frequency or duration of phloem ingestion or xylem feeding between susceptible and resistant D. citri. There was no statistical difference in fecundity between resistant and susceptible strains. However, the fecundity of imidacloprid-susceptible female D. citri treated with a sublethal concentration of imidacloprid (LC25 ) increased significantly compared with controls, while such hormoligosis was less pronounced among imidacloprid-resistant psyllids. Our results suggest that imidacloprid-resistant psyllids may cease feeding sooner than susceptible counterparts following sublethal exposure to this insecticide, indicative of a behavioral resistance mechanism.

Sanitizing Equipment and Personnel to Prevent the Spread of Hibiscus Mealybug [Nipaecoccus viridis (Hemiptera: Pseudococcidae)] in Florida Citrus.

Hibiscus mealybug (Nipaecoccus viridis, Newstead) is an emerging and serious pest of citrus in Florida. Preventing the spread of hibiscus mealybug by sanitizing personnel and equipment such as individual protective covers can help limit new infestations. Two mealybug life stages, first instars (crawlers) and eggs inside ovisacs, were tested as they are the most likely to be accidentally transported on people and tools. Isopropanol sprays in 3 commercially available concentrations (50%, 70%, and 90%) were tested against crawlers and ovisacs, and steam treatments and hot water immersion were tested against ovisacs. All concentrations of isopropanol resulted in >90% incapacitation of mealybug crawlers, especially when two sprays of isopropanol were applied. However, no concentration of isopropanol reliably killed all mealybug eggs within ovisacs. Steam treatments and hot water immersion for 10 min or longer at 49°C and for 5 min or longer at 54.5°C resulted in 100% mortality of mealybug adults and eggs inside ovisacs. However, adults and ovisacs insulated inside of individual protective covers did not experience 100% mortality until 30 min or longer at 49°C, 10 min or longer at 54.5°C, and 5 min or longer at 60°C for steam treatments. Isopropanol sprays can effectively incapacitate the majority of hibiscus mealybug crawlers on personnel and tools, but are not effective against ovisacs. Steam treatments and hot water immersion can reliably lead to 100% mortality of ovisacs, but longer times and higher temperatures must be used for steam treatments if ovisacs are insulated by equipment.

Detectability of Hibiscus Mealybug, Nipaecoccus viridis (Hemiptera: Pseudoccocidae), DNA in the Mealybug Destroyer, Cryptolaemus montrouzieri (Coleoptera: Coccinellidae), and Survey of Its Predators in Florida Citrus Groves.

The Hibiscus mealybug, Nipaecoccus viridis (Newstead), has recently established in Florida citrus and become a pest of concern given secondary pest outbreaks associated with management of citrus greening disease. Chemical controls used to manage other citrus arthropod pests are not as effective against N. viridis due to its waxy secretions, clumping behavior, and induced cellular changes to host plant tissue which increase microhabitats. Populations of this mealybug pest are regulated by natural enemies in its native region, but it remains unclear if resident natural enemies in Florida citrus could similarly suppress N. viridis populations. This investigation: 1) established species-specific primers for N. viridis based on the mitochondrial gene Cytochrome-oxidase 1 (COI), 2) determined duration of N. viridis DNA detectability in a known predator, the mealybug destroyer (Cryptolaemus montrouzieri Mulsant), by using identified primers in molecular gut content analysis, and 3) screened field-collected predators for the presence of N. viridis DNA. The detection rate of N. viridis DNA was >50% at 36 h after adult C. montrouzieri feeding but DNA was no longer detectable by 72 h after feeding. Field-collected predators were largely comprised of spiders, lacewings, and C. montrouzieri. Spiders, beetles (primarily C. montrouzieri), and juvenile lacewings were the most abundant predators of N. viridis, with 17.8, 43.5, and 58.3 of field-collected samples testing positive for N. viridis DNA, respectively. Our results indicate that Florida citrus groves are hosts to abundant predators of N. viridis and encourage the incorporation of conservation or augmentative biological control for management of this pest.

Suitability of Formulated Entomopathogenic Fungi Against Hibiscus Mealybug, Nipaecoccus viridis (Hemiptera: Pseudococcidae), Deployed Within Mesh Covers Intended to Protect Citrus From Huanglongbing.

Mesh exclusion bags are increasingly being adopted by Florida citrus growers to protect young citrus trees from Asian citrus psyllid, Diaphorina citri Kuwayama, and Huanglongbing disease. These mesh bags exclude larger insects such as D. citri but may allow entry of minute insects. Hibiscus mealybugs, Nipaecoccus viridis (Newstead), have been observed thriving in the micro-habitat created by these covers on trees. Entomopathogenic fungi (EPF) and insect growth regulators (IGRs) are effective against several mealybug species under various growing conditions, but their efficacy against N. viridis or within the microclimate within exclusion bags is unknown. Therefore, we evaluated various formulations of entomopathogenic fungi with and without IGR against N. viridis using laboratory bioassays. We then conducted semifield bioassays to determine effectiveness of EPF formulations alone and in combination with an IGR applied to citrus trees enveloped within mesh bags under field conditions. Survival probabilities of N. viridis nymphs exposed to all Beauveria bassiana-based products tested were comparable to malathion under laboratory conditions and reduced survival as compared to controls (water only). Under field conditions, mortality of N. viridis nymphs on leaves sprayed with each fungal formulation tested was significantly greater than on control treatments (Water, Suffoil X, 435 oil) up to four weeks post application. There were no differences in the colony forming units per leaf area amongst all fungal treatments. Formulated B. bassiana-based products applied alone or combined with an IGR should be effective tools for managing N. viridis populations on young citrus trees protected with mesh exclusion bags.

Laboratory and Field Evaluation of Host-Related Foraging Odor-Cue Combinations to Attract Drosophila suzukii (Diptera: Drosophilidae).

The invasive spotted-wing drosophila, Drosophila suzukii (Matsumura), is a major pest of soft-skinned fruits. Since its introduction into North America and Europe, significant progress has been made in understanding the volatile cues used by this fly during food, oviposition site, and mate finding. Despite this progress, commercially available lures are non-selective. Here, we tested two Hanseniaspora uvarum (Niehaus) yeast compounds (isoamyl acetate and isobutyl acetate) and a leaf compound ß-cyclocitral alone and in combination with a blend of four fermentation compounds ('Fermentation lure': acetic acid, ethanol, methionol, and acetoin) to improve D. suzukii attraction and selectivity. In laboratory assays, males and females were attracted to all seven individual compounds, although in electrophysiological assays, their antennae exhibited a dose-dependent response to only four of these compounds. In two-choice cage studies, the Fermentation lure was more attractive to D. suzukii than water controls, whereas ß-cyclocitral and the mixture of isoamyl acetate and isobutyl acetate were not attractive in this larger-cage study. Moreover, adding the two-component H. uvarum compound blend to the Fermentation lure reduced D. suzukii attraction to the Fermentation blend. When these experiments were repeated in blueberry, raspberry, blackberry, and cherry orchards across several states in the United States over 2 yr, similar outcomes were observed: ß-cyclocitral or the mixture of the H. uvarum blend did not improve the attractiveness of the Fermentation lure or its selectivity. This study demonstrates that cues from different sources may interfere with each other and reduce D. suzukii attraction to otherwise attractive odor combinations.

Detecting Specific Resource Use by Drosophila suzukii (Diptera: Drosophilidae) Using Gut Content Analysis.

Drosophila suzukii Matsumura (Diptera: Drosophilidae) is an invasive, highly polyphagous pest of soft-skinned fruits throughout much of the world. A better understanding of the ecology of adult flies, including their nutritional resources, is needed to advance ecologically based management approaches. In this study, we evaluate the capability of polymerase chain reaction-based gut content analysis to detect a known food resource from DNA extracted from laboratory-reared flies. Using strawberry as a focal host and available DNA primers, we validated that DNA from this host could be detected for up to 7 d post-consumption. With the development of specific primers for additional hosts, we expect that this technique will enable researchers to better understand how D. suzukii adults use, and move between, nutritional resources.

The Influence of Exotic Lady Beetle (Coleoptera: Coccinellidae) Establishment on the Species Composition of the Native Lady Beetle Community in Missouri.

The diversity and abundance of native lady beetles (Coccinellidae) in North America has declined in recent decades. This decline is often correlated with the introduction and establishment of exotic lady beetle species, including Coccinella septempunctata L. and Harmonia axyridis Pallas, suggesting that exotic species precipitated the decline of native lady beetles. We examined species records of native coccinellids in Missouri over 118 yr and asked whether the species composition of the community experienced a shift following the establishment of the exotic species. We found that the contemporary native coccinellid community is different from the community that was present nearly a century ago. However, there was no evidence for a recent abrupt shift in composition triggered by the establishment of exotic species. Instead, our data suggest that the native lady beetle community has been undergoing consistent and gradual change over time, with some species decreasing in abundance and others increasing. While not excluding exotic species as a factor contributing to the decline of native lady beetle species, our findings suggest that other continuous factors, like land use change, may have played a more influential role in determining the composition of the native coccinellid communities within our region.