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Laboratory and Field Susceptibility of Drosophilasuzukii Matsumura (Diptera: Drosophilidae) to

Entomopathogenic Fungal Mycoses1

Gabriel Alnajjar,2,4 Francis A. Drummond,2,3 and Eleanor Groden2

J. Agric. Urban Entomol. 33: 111–132 (2017)ABSTRACT Spotted wing drosophila (Drosophila suzukii Matsumura;Diptera: Drosophilidae) is a recently established invasive insect pest of berriesand stone fruits. We tested the susceptibility of adult D. suzukii to infec-tion by four species of entomopathogenic fungi: Beauveria bassiana (Bals.)Vuill., strains GHA and HF-23; Isaria fumosorosea Wize, strains FE-9901 andApopka 97;Metarhizium anisopliae var anisopliae (Metschn.), strain F-52; andMetarhizium robertsii, strain DW-346; in laboratory bioassays. Scanning elec-tron micrographs confirmed germination of conidia on D. suzukii integument.Exposure to conidia of all fungal isolates significantly increased mortality ratesin comparison to untreated flies. In a subsequent bioassay of the two mostvirulent isolates, increasing pathogen dose from 0–16,000 conidia mm−2 of B.bassiana strain GHA increased fly mortality and proportion of sporulating ca-davers. While fly inoculations with M. anisopliae at 0–4000 conidia mm−2 didnot yield any measurable mortality response, a positive correlation was ob-served between dose and frequency of sporulating cadavers. In addition, wefound that oocyte maturation rates were curtailed through one week of adult-hood development after sub-lethal exposure to B. bassiana conidia. Despitepromising laboratory results, a field cage experiment demonstrated that ap-plication of B. bassiana did not protect fruit from infestation. Taken together,these results show that entomopathogenic fungi are virulent againstD. suzukiiin the laboratory, but application in the field did not show promise.

KEY WORDS Biocontrol, Diptera, Drosophilidae, spotted wing drosophila,blueberry

Drosophila suzukiiMatsumura (Diptera: Drosophilidae), commonly referred toas the spotted wing drosophila (SWD), is a polyphagous insect pest species nativeto Southeast Asia (Kanzawa 1939). Currently, the species’ distribution extends be-yond Asia. It has recently been described as an invasive agricultural pest in NorthAmerica, South America and Europe (Walsh et al. 2010, Cini et al. 2012, Depraet al. 2014). Its establishment and persistence in the invaded range is believedto be due to unintentional introduction of infested fruits in climatically conducivegeographic regions that support wild and/or cultivated host plants (Asplen et al.2015, Cini et al. 2012).

1Accepted for publication 24 October 2017.2 University of Maine, School of Biology and Ecology, Orono, ME3 University of Maine, Cooperative Extension, Orono, ME4 Corresponding author, E-mail: [email protected]

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112 J. Agric. Urban Entomol. Vol. 33, (2017)

Unlike most Drosophila spp. that oviposit in senescing overripe or physicallydamaged fruit, SWD females utilize a sclerotized and serrated ovipositor to di-rectly penetrate ripe or ripening berries and stone fruits (Isaacs et al. 2012). Theresulting oviposition stings leave fruit skins with scars and tears, further threat-ening fruit integrity through increased vulnerability to microbial invasion by bac-teria or yeasts, and secondary infestation of other insect pests with frugivorousjuvenile stages (Louise et al. 1996, Asplen et al. 2015). Under favorable climaticconditions, SWD population growth can proceed exponentially. Female flies liveup to 4 wk, during which individuals are capable of producing well over 350 eggswith peak laboratory oviposition rates exceeding 25 eggs day−1 at 25◦C (Walshet al. 2011, Gerdeman et al. 2013, Kinjo et al. 2014).

Wild or lowbush blueberry (Vaccinium angustifolium Aiton) is a native shrubmanaged as an agricultural crop in Maine. Currently 24,300 ha are managedand yields vary annually between 30 and 45 million kg/yr (Yarborough 2013).Its cultivation contributes significantly to the economy of Maine, the CanadianMaritimes, and Quebec. In October 2011, only 3 yr after its introduction to theWest coast of mainland North America, SWD was reported in lowbush blue-berry fruits (Drummond & Yarborough 2013). Given the concern about insecti-cide residues on fruit (Haviland & Beers 2012) and the late season attack bythis new invasive pest in wild blueberries, early harvesting has become a recom-mended option for both organic and conventional growers (F. A. D. unpublisheddata). However, it has been proposed that temperature shifts favoring warmerwinters will promote an increase in overwinter survival of SWD adults, and ashift toward earlier population increases in the summers that follow (Šeparovicet al. 2013, Drummond 2016). This may lead to a greater likelihood of crop loss inMaine.

The most common current integrated pest management (IPM) recommenda-tion for SWD involves insecticidal treatment immediately after adult fly detec-tion in baited traps emitting volatile semiochemicals (Isaacs et al. 2012, Drum-mond & Yarborough 2013, Burrack et al. 2015). Unfortunately, it is believed thatinsecticide applications have limited effect on SWD juvenile stages, which occurwithin host fruits for a majority of their development and so frequent applica-tions are necessary to minimize fruit infestation. It is worth noting that manyinsecticide classes lethal to SWD adults also display toxicity toward managedand wild native bee species (Johnson et al. 2010, Bunch et al. 2014) that are themajor pollinators of wild blueberry and small fruit crops. Even though floweringof lowbush blueberry occurs prior to SWD invasion, runoff and drift of pesticidetoxins into surrounding ecosystems, as well as the potential overlap of blueberryfruits with populations of flowering plant species, necessitate the investigation ofnon-insecticidal management tactics, including biological control (Alnajjar et al.2017).

Entomopathogenic fungi are biological control agents that have been success-fully utilized for management of various widespread and destructive insect pestsin North American agriculture including the Colorado potato beetle, Leptino-tarsa decemlineata (Say) (Coleoptera: Chrysomelidae), the gypsy moth, Lyman-tria dispar dispar (L.) (Lepidoptera: Erebidae), and the European corn borer, Os-trinia nubilalis (Hubner) (Lepidoptera: Crambida) (Hajek et al. 1996, Butt et al.2001, Wraight & Ramos 2002, Kaufman et al. 2005). An overwhelming major-ity of these commercially developed myco-insecticide products contained one of

Biological control of Drosophila suzukii 113

three fungal species from the taxonomic division Ascomycota:Beauveria bassiana(Bals.) Vuill., Isaria fumosorosea Wize (formerly Paecilomyces fumosoroseus), andMetarhizium anisopliae var. anisopliae (Metschn.) (Butt et al. 2001). Investiga-tions of the resulting mycoses have shown induction of both lethal and sub-lethalhost syndromes in species of the orders Coleoptera, Hemiptera, Blattodea, Or-thoptera, Thysanoptera, Lepidoptera and Diptera (Butt et al. 2001, Costa et al.2001, Lacey et al. 2001, Shah & Pell 2003, Scholte et al. 2004, Godonou et al.2009).

The goal of this study was to explore the potential of entomopathogenic fungias biocontrol agents of SWD in Maine wild blueberry production. A preliminarySWD adult lethality screening was accomplished through direct mass conidia in-oculations of six entomopathogenic fungal strains within the species B. bassiana,I. fumosorosea, M. anisopliae, and Metarhizium robertsii. We then investigatedthe dose-dependent mortality response of SWD adults to infection by B. bassianastrain GHA andM. anisopliae strain F-52. The effect of sub-lethal B. bassiana ex-posure of teneral SWD females on their subsequent oocyte maturation was alsoevaluated. In addition, a myco-insecticide efficacy was evaluated in a field cagetrial.

Materials and Methods

Isolation& viability estimations of entomopathogenic fungi. Beauveriabassiana strains GHA andHF-23, and I. fumosorosea strains FE-9901 and Apopka97 were isolated from the formulated myco-insecticides Botanigard

R©, Balance

R©,

NoflyR©

and PreferalR©, respectively. A small sample of each formulated material

was suspended in 300 μl aqueous 0.01% TweenR©

solution. Beauveria bassianadilutions were cultured on a dodine growth medium, which was prepared usingthe protocols outlined in Sneh (1991). Suspensions of I. fumosorosea were platedon Sabouraud dextrose agar (SDA) with added antibiotics streptomycin and peni-cillin each at 100 mg ml−1 media mixture. After approximately 10 d of incubationat 25 ± 1◦C, spores of individual I. fumosorosea and B. bassiana colony formingunits (CFU) were collected and vortexed in 0.01% Tween

R©and transferred to

cultures containing SDA or SDA with yeast (SDAy), respectively. Conidia of M.robertsii were provided by the USDA ARS Collection of Entomopathogenic Fungi(Ithaca,NY) and cultured directly on 1

4 strength SDA.Galleria melonella (L.) (Lep-idoptera: Pyralidae) were inoculated with M. anisopliae conidia from laboratorycultures.Metarhizium anisopliae conidia obtained from sporulating G. melonellacadavers were suspended in 0.01% Tween

R©and plated on 1

4 strength SDA. Allcultured fungi were incubated in a scotophase growth chamber at 25 ± 1◦C for10–14 d and stored in a non-illuminated laboratory refrigerator at 4 ± 1◦C. Vi-ability tests were conducted prior to each bioassay. This entailed suspension ofconidia samples obtained from cultured fungi in 300 μl 0.01% Tween

R©, plating

on the fungus’ respective growth medium, and incubation in a scotophase growthchamber at 25 ± 1◦C for 20 h. A sampling grid was then superimposed on the ex-terior surface of each plate. Random grid sections were magnified at 200× undera phase contrast microscope in order to quantify the relative proportion of germi-nating vs non-germinated conidia. A minimum of 200 conidia was sampled fromeach culture.


Acquisition of SWD for experimentation. All SWD utilized in laboratoryexperiments were taken from laboratory reared colonies originating from capturedadults in Maine lowbush blueberry fields in Washington Co., Maine in 2013 (seeBallman et al. 2017). The laboratory colonies were infused annually with capturedwild flies to maintain genetic diversity similar to field populations. Each SWDculture was provided with Carolina Formula 4–24

R©instant Drosophila media

in 50-ml rearing vials and maintained in the laboratory under growth chamberconditions set to 25 ± 1◦C and a 12:12 (L:D) h cycle.

Scanning electron microscopy. We were interested in documenting fly in-tegument penetration by germinating B. bassiana and M. anisopliae conidia.Based on preliminary specimen preparation trials of uninfected flies, the utiliza-tion of aldehyde tissue fixation techniques (Pekrul & Grula 1979) was deemedunnecessary for SEM imaging of the SWD integument. Specimens held in 70%ethanol directly underwent a sequential series of ethanol dehydrations in 70%,80%, 85%, 90%, 95% and 100% ethanol, entailing complete submersion in eachdilution for three separate 7-min cycles. Specimens were held in 100% ethanolafter final dehydration, and desiccated with a Tousimis Samdri

R©PVT-3 Critical

– Point Dryer. Each fly was then grounded to the base of a mounting stub withsilver conduction paint and coated with a 35-nm layer of Gold/Palladium (Au/Pd)in a Cressington

R©108 Auto/SE Sputter Coater. Specimens were examined and

images were obtained under an AMRayR©-1820 scanning electron microscope.

Qualitative high dose fungal inoculation of flies. An initial study wasdesigned to determine which fungal isolates would be the most promising for fur-ther assessments for biological control of SWD. Viability tests for cultures of B.bassiana strain GHA (GHA) and HF-23 (HF-23), I. fumosorosea strains FE-9901(FE) and Apopka 97 (AP), and M. anisopliae strain F-52 (F-52) and M. robertsiistrain DW-346 (DW) yielded roughly 95%, 93%, 92%, 95%, 90% and 84% spore vi-ability (germination), respectively. Four hundred and twenty adult SWD (1:1 sexratio) were immobilized with CO2 and distributed equally among 21 culture vials.An inoculating loop was utilized for delicate swabbing of cultured conidia untiluniform spore coverage was achieved on the loop’s tip. Conidia were then placedin sterile 1-ml centrifuge vials to create three inoculation chambers per fungalisolate. Three additional vials were not inoculated and served as the control treat-ment. One set of 20 flies was introduced to each inoculation chamber or the controland vortexed at low intensity for approximately 30 sec. These treated flies werethen transferred back to new culture vials and placed in a growth chamber for5 d (the period at which control mortality reached a maximum of 20%). Mortalityamong fungal species and strains were assessed with nominal logistic regressionand a subsequent series of binomial pairwise contrasts with a significance level ofα = 0.05 (JMP

R©, SAS Institute 2015).

Conidia concentration mortality assays. Dose-mortality assays with B.bassiana strain GHA (95% viability) and M. anisopliae strain F-52 (92% viabil-ity) conidia were conducted sequentially on 0–3 d old adult flies. Each experimentwas initiated by removing all existing adults from thriving colonies of SWD. After3 d of colony development in a growth chamber, 300 0–3 d old adults (1:1 sex ratio)were collected, immobilized with CO2 and divided evenly into 30 culture tubes of10 flies each.

One layer of standard grade filter paper was placed in each of 30, 4.5-cmPetri dishes. Suspensions of 1.2 × 105–109 GHA conidia ml−1 and 2.1 × 104–108


F-52 conidia ml−1 0.01% TweenR©

were prepared within 90 min of the applica-tion. To allow for mortality comparisons between the two fungal pathogens, anadditional suspension of 1.9 × 108 B. bassiana conidia ml−1 0.01% Tween

R©was

included as a treatment during the M. anisopliae assay. Prior to spraying, filterpaper surfaces were lightly misted with dH2O and covered with one layer of 0.22-μm GV Millipore

R©filter paper. Conidia suspensions were homogenized using a

vortex mixer before being applied to the pre-moistened MilliporeR©

filters usinga Burkard

R©computer controlled sprayer (Rickmansworth, England) with a click

setting of 6 and 69 kPa (10 psi) of pressure. Five replicate dishes with MilliporeR©

filters plus a dish of water agar were treated with each concentration of each fun-gus (approximately 0.25 μl applied per treatment dish). Control groups were ex-posed to an aqueous 0.01% Tween

R©solution.

After spraying, water droplets were allowed to evaporate for roughly 15 minto prevent drowning of immobilized SWD. Ten male and ten female adults werethen released onto each treated surface. Dishes were wrapped with rubber bands,placed in plastic bags with a moist paper towel, and incubated in the dark at 25± 1◦C for 24 h. Flies were then immobilized with CO2, transferred to a culturevial and placed back into the 25◦C growth chamber with a 12:12 (L:D) h cycle.Sprayed water agar plates were observed at 400× under a phase contrast mi-croscope equipped with a grid reticle to estimate the density of conidia per mm2

deposited on the filters.Starting 24 h after initial conidia contact, dead flies were collected daily for 6 d.

Cadavers removed from the culture tubes were surface sterilized in 10% benza-lkonium chloride, followed by two rinses in dH2O, and allowed to dry by blottingon filter paper. Cadavers were then placed in individual wells in 48-well microtiterplates. Plates were held in plastic bags with a moistened paper towel to encour-age sporulation of infected individuals. An extra set of eight adults was exposedto both fungal pathogens at the corresponding dosage of 1 × 108 conidia ml−1 ap-plication. Approximately 24 h after inoculation, these specimens were preservedin 70% ethanol for qualitative scanning electron microscopy examination of sporegermination on the fly integument.

The dose-mortality response of flies to these entomopathogenic fungi was quan-tified with nominal logistic regression (JMP

R©, SAS Institute 2015). Designating

a control mortality threshold of 20%, both logit models were constructed with 3 dof mortality and sporulation data. Greater ratios of males were observed in somereplicates. This was attributed to the visual confirmation of individuals as be-ing male based on the presence of wing spots. Given that newly emerged adultmale SWD require additional post-eclosion maturation time for the pigmentationof wing spots, their absence during fly collections likely led to the disproportionatecollection of individuals that were incorrectly identified as female. For the pur-poses of collecting sporulation data, manipulation of cadavers was minimized topreserve specimen integrity. Since handling of cadavers was minimized, the gen-der of dead flies that happened to become covered with Drosophila media couldnot be determined and, unfortunately, the intended gender-specific response as-sessment could not be conducted.

SWD oocyte maturation during B. bassiana mycosis. An experimentwas conducted to assess sublethal dose effects of B. bassiana on SWD fecundity.Fungal culture viability was estimated to be approximately 95% using the meth-ods previously outlined. Thriving SWD cultures were immobilized with CO2 and


all live adults were removed. Immature SWD in the cultures were then allowed tocontinue development for 20 h in a growth chamber set at 25 ± 1◦C and a 12:12(L:D) h cycle. Newly emerged flies were anaesthetized and examined to ascertaingender by applying slight pressure to the mediolateral abdominal region of fliesin order to force protrusion of copulatory organs for accurate gender identificationof individual flies. A suspension of 1 × 108 conidia ml−1 0.01% Tween

R©and a

0.01% TweenR©

only control were applied to 18 4.5-cm Petri dishes with Milliporefilters on top of moistened filter papers using the protocols described above fordose-response bioassays.

After spraying, a small amount Drosophila media (about 0.2 ml) was placedon the periphery of each dish such that there was minimal hindrance of conidia-treated surfaces from contacting fly appendages. Flies were then introduced tosprayed Millipore filters, and each dish was sealed with Parafilm

R©to maintain

moisture. Sexual maturation was allowed to progress under growth chamber con-ditions at 25 ± 1◦C with a 12:12 (L:D) h cycle. An OMEGA

R©OM-90 series tem-

perature/humidity data logger was placed directly in the growth chamber and ina sprayed and sealed Petri dish without flies to monitor relative humidity duringthe incubation period. Prior to dissections, any mortality was noted but cadaverswere allowed to remain in Petri dishes. Three replicated SWD dissections wereconducted on each of 5, 6 and 7 d of post-eclosion as adults. To accomplish this,live flies were euthanized in 70% ethanol and ovaries were carefully extracted inorder to count the number of fully developed oocytes held by each female, withtreatment samples divided up by replicate and dissection day. The media con-tained within each chamber was also examined for the presence of SWD eggs.Overall, six total dishes (three of each treatment) were processed at any givendissection time. At this time, cadavers were removed from dishes, surface ster-ilized, placed in individual wells of 48-well microtiter plates, maintained witha moist paper towel, and monitored for sporulation. An additional set of eightadult SWD was exposed to the experimental B. bassiana conidia dosage. About20 h after inoculation, these individuals were euthanized in 70% ethanol so in-fection could be qualitatively confirmed in individuals via scanning electron mi-croscopy. A generalized linear model was utilized under the assumption of expo-nentially distributed response data with Firth bias adjusted estimates (JMP

R©,

SAS Institute 2015). Time (fly post-eclosion age in days) and treatment (exposuredose of conidia) and their interaction were included in the model as independentvariables.

Myco-insecticide field efficacy trial. A field-cage study was conducted inthe summer of 2015 to assess the biological control potential of B. bassiana coni-dia applied on lowbush blueberry for SWD control. Nine nylon mesh cages (cus-tom sewn by Young’s Canvas Shop

R©in Hampden,ME) were erected over lowbush

blueberry plants on 30 July 2015 at University of Maine Blueberry Hill Experi-ment Farm in Jonesboro, Maine. The nine mesh cages consisted of three tan-meshcages enclosing approximately 15 m2 (6 × 2.5 × 3 m) with 121 holes per cm2,and six black-mesh cages enclosing approximately 16 m2 (6 × 2.7 × 3 m) withabout 81 holes per cm2. Three statistical blocks (replicates) with each of threetreatments consisted of one set of tan cages, and two sets of black cages. Onered Solo

R©cup baited with sugar water and yeast (Burrack et al. 2015) was de-

ployed on a metallic 76-cm plant support post in each cage for 1 wk in order todetect the presence of any adults prior to initiating the experiment. No SWD were


detected prematurely, before the initiation of the experiment, in any of the ninecages.

On 12 August 2015, the day prior to SWD release, flies from laboratory-reared colonies were aggregated in culture vials containing Drosophila media.Estimations of SWD abundance in each culture vial were qualitatively obtainedvia visual comparison with colonies of the following known fly densities: 50,100, 150 and 200 flies. Colonies were distributed evenly among six sets total-ing 2000 flies each, and held in an air conditioned room until transport to thefield site at University of Maine Blueberry Hill Experiment Farm on the followingday.

Beauveria bassiana spray applications and conidia sampling. Preceding SWDintroduction into cages on 13 August 2015, the formulated B. bassiana (strainGHA) product Mycotrol

R©was applied at the recommended application rate of

2.3 L ha−1 (2.5×1013 conidia ha−1) to one randomly selected release cage ineach of the three blocks of cages using a CO2 powered backpack sprayer (R &D Sprayers

R©, Opelousas, LA) fitted with a hollow-cone nozzle (TeeJet

R©8002VS).

In order to assess the effect of cage shading on conidia longevity on foliage andfruit, a non-caged 43-m2 section of adjacent field was divided into three sam-pling plots and sprayed with Mycotrol

R©at the same recommended rate and

application date as the enclosure cage applications. At 0 (about 10 min), 24,48 and 96 h after application, six blueberry leaves were collected from the B.bassiana treated plants in each of the treated cages and non-caged plots. In eachcage and plot, two leaves were sampled from each of three areas: high, mediumand low stem positions. Disks (11 mm in diameter) were cut from each leaf us-ing a copper cork-borer, and the six disks cut from the same cage or plot werepooled in one sterile centrifuge vial and stored in a chilled cooler containing icepacks.

Immediately upon arrival at the laboratory, each of the pooled leaf disks sam-pled was homogenized in 30 ml 0.01% Tween

R©for 1 min. Conidia suspensions

from these samples were serially diluted to 0.1× the stock concentration, andthree 0.5-ml aliquots were each cultured on individual Petri dishes containingDodine

R©wheat germ growth medium prepared utilizing the protocols outlined in

Sneh (1991). These cultures were then incubated in the dark at 25 ± 1◦C untilgrowth progressed sufficiently for conidia and conidiophore observations via phasecontrast microscopy. Plates containing morphological analogues of B. bassianawere divided into equal quadrants numbered 1 to 4. A random number genera-tor was utilized to randomly designate a quadrant from each plate, from whichthe number of suspected B. bassiana colony forming units (CFU) was counted andmultiplied by four. This number represents the approximate relative abundanceof B. bassiana conidia occupying leaf disks sampled from cage-shaded and non-shaded study areas throughout the experiment. The main effects and interactionof time and shading were analyzed by ANOVA (Randomized Complete Block De-sign) on log-transformed 0.1× dilution conidia counts. An inverse-prediction wasthen utilized to estimate the half-life expectancy of conidia based on the samplingdata (JMP


Spotted wing drosophila release. Immediately following the initial leaf sam-pling bout at 0 h, 2,000 flies were introduced into each of six of the nine cages.The treatment cages for the experiment were as follows: 1) Mycotrol

R©spray

application and 2000 flies released (S), 2) no MycotrolR©

spray application and


2000 flies released (NS), and 3) an exclusion control cage with no MycotrolR©

ap-plication and no flies released (C). Each vial was opened by hand and allowed toremain undisturbed overnight. Flies still localized on media were forcibly tappedonto a clean Petri dish containing noDrosophilamedia.After 1 h, flies still remain-ing in the Petri dish were euthanized in 70% ethanol, removed from the study areaand subtracted from the release total.

Field cages for SWD adults and larvae. The Oregon State University SWDdegree-day phenologymodel (Oregon StateUniversity 2017) was utilized to projectpopulation growth rates so fruit sampling bouts could be conducted when a highproportion of offspring from introduced flies would have developed to the thirdlarval instar. On 25 August 2015, approximately 131 degree-days (◦C) after SWDrelease, study plot fruits were sampled in order determine the impact of treatmenton maggot infestations. Five total samples (about 473 ml each) of blueberries occu-pying high, medium and low stem positions were collected from various randomlyselected clones. An additional sample of fallen berries was obtained to assess SWDlarvae that may have caused premature fruit drop. All samples were held in a lab-oratory refrigerator and processed within one week of the collection date. Fruitswere gently crushed and mixed with 10% saline solution to facilitate dissociationof insect larvae from the fruit pulp. The control treatment cage fruit was assessedto determine whether wild SWD or blueberry maggot flies, Rhagoletis mendax(Curran) (Diptera: Tephritidae), not released might have entered into the sealedcages and confounded the results. Samples were strained into a black tray after30 min and SWD larvae were counted. Weighted averages for stem and groundedlarval abundances were transformed into ordinal ranks with 0 = 0 larvae, 1 = 1–10 larvae, 2 = 11–100 larvae, 3 = ≥100 larvae. Ranked data were then analyzedvia ordinal logistic regression (JMP


To quantify adult abundance after the collection of fruit samples within studyenclosures, one red Solo

R©cup baited with sugar water and yeast was deployed on

a metallic 76-cm plant support post in each of the nine cages. Traps were allowedto capture flies undisturbed for 6 d, after which the contents were filtered andexamined under a dissecting microscope for both male and female adult SWD.The total number of captured SWD was divided by the predicted quantity of SWDintroduced to cages in order to assess the capture efficacy of individual traps. AnANOVA (RCB) was then conducted along with a subsequent Tukey post-hoc test(JMP


Results

Scanning electron microscopy. Scanning electron micrographs of inocu-lated flies show germination of both B. bassiana and M. anisopliae conidia on flyexoskeletons (Figure 1). Hyphae of both fungal strains are seen directly penetrat-ing the cuticle and do not appear constrained to less direct invasion mechanismsthrough natural openings of the exoskeleton such as spiracles, or thinner less scle-rotized areas such as intersegmental membranes in the abdomen.

Qualitative high dose inoculation of flies. Single dose inoculation of adultSWD with fungal conidia resulted in an overall significant difference in mortal-ity of treated vs control groups of flies at 5 d post exposure (X2

(6, n = 20)=88.19,P < 0.0001) for all pathogen isolates (Figure 2). Mortality as a result of exposure


Fig. 1. Scanning electron micrographs of germinating B. bassiana conidia onthe femur (A) and pronotum (B), and M. anisopliae conidia on the tibia(C) and femur (D) of Drosophila suzukii. Arrows in B indicate conidiawith germ tubes; black arrow in C indicates indirect hyphal penetrationthrough a seta follicle; white arrows in C and D indicate appressorial(ap) formation and direct penetration through the cuticle. E. The host’sshows the host’s terminally located tarsal segments after surface inoc-ulation withM. anisopliae conidia. (Bar = 10 μm for A; bar = 20 μm forB and E; bar = 5 μm for the rest.)

to M. anisopliae F-52 was greater than all other fungal species and strains. Thetwo strains of B. bassiana (GHA and HF-23), and I. fumosorosea (FE-9901) didnot differ from one another, but resulted in greater mortality than I. fumosorosea(Apopka 97) and M. robertsii (DW-346).

Conidia concentration mortality assays. Fly mortality exceeded 20% inone control replicate of each pathogen assay. Each dataset was analyzed with andwithout the outliers, with logit model outputs indicating no notable difference inB. bassiana mortality, but an outlier effect with the M. anisopliae experiment.With the exclusion of these outliers, evidence for a positive relationship betweenB. bassiana conidia exposure concentration and adult mortality was provided bylogistic regression (X2

(1, N = 480) = 65.4, P < 0.0001; Table 1). Based on the predic-tive model (Figure 3), the pathogen’s predicted log-transformed lethal dose (LD)10, 25, 50, 75, 90 and 99% values were as follows: 1.1, 2.7, 4.2, 5.8, 7.3, and 10.7conidia mm−2, respectively. A mortality increase was not observed in responseto increasing levels of M. anisopliae conidia (P = 0.693; Table 1). A significantdifference in fly mortality was observed between the two pathogens at the dose


Fig. 2. Average proportion of dead SWD adults five days after mass conidia in-oculation. Treatments tested include a control (Tween only, C),M.aniso-plae strain F-52 (F),B. bassiana strains GHA (GHA) and HF-23 (HF), I.fumosorosea strains FE9901 (FE) and Apopka 97 (AP), andM. robertsiistrain DW-346 (DW). Bars displaying dissimilar letters denote a signif-icant difference in fly mortality among treatments.

of 1.9×108 conidia ml−1 (X2(1, N = 160) = 5.03, P = 0.025), with greater mortality

observed in flies exposed to B. bassiana. Both fungi were able to utilize SWDfor growth, development and asexual reproduction, as mycelia were observed ondead flies three weeks after surface sterilization (Figure 4). Furthermore, in thecase of both entomopathogens, cadaver sporulation frequencies increased in re-sponse to higher conidia dosages [B. bassiana: (X2

(1, N=122) = 67.39, P < 0.0001;

Table 1. Fly mortality and sporulation logit results after indirect topicalexposure to varying concentrations of B. bassiana (strain GHA)orM. anisopliae (strain F-52) conidia mm−2. Critical dose valuesfor mortality and sporulation are provided for the threshold atwhich 50% of the population would have died (LC50) three daysafter conidia exposure, or where 50% of fly cadavers will havesporulated (SC50) three weeks after death.

Fungal Isolate & Slope LC50 95% CI& Response Variable n ± SE 95% CI or SC50 for LC50 X2

GHA Mortality 480 0.70 ± 0.10 0.52–0.91 4.2 3.8–4.8 50.06F-52 Mortalitya 480 0.05 ± 0.12 - - - 0.15GHA Sporulation 122 2.39 ± 0.45 1.62–3.39 3.4 - 28.88F-52 Sporulation 58 1.78 ± 0.47 2.32–3.35 2.8 - 14.06

alogistic regression not significant


Fig. 3. Proportion mortality of SWD flies three days after exposure to vary-ing surface concentrations of B. bassiana strain GHA conidia (conidiamm−2). The log (dose) impact on mortality is represented as an expo-nential relationship. The highest log (dose) tested in the bioassay was4.2, which corresponds with the predicted LD50 based on these data.

Figure 5);M. anisopliae: (X2(1, N=58) = 25.01, P < 0.0001; Figure 5)]. No sporulation

was observed on dead flies from the control replicates during either fungal speciesassay.

Oocyte maturation during B. bassiana mycosis. The histograms in Fig-ure 6 depict an exponential distribution of egg count data. We estimate that asurface dosage of 2,900 conidia mm−2 was administered to the treated flies. In-teguments of euthanized specimens exposed to fungal treatments for qualitativeSEM examination supported the assumption of positive infection byB. bassiana in

Fig. 4. Entomopathogenic fungi sporulating on SWD cadavers. Photographstaken ca. three weeks after indirect topical exposure toB. bassiana (left)or M. anisopliae (right) conidia. (Bar = 0.75 mm for both images).


Fig. 5. Proportion of dead SWDflies sporulating following exposure to 0–16,000or 0–4000 conidia mm−2 ofB. bassiana (A) andM.anisopliae (B), respec-tively. Sporulation was evaluated three weeks after mortality of inocu-lated flies.

individuals exposed to this conidia concentration. Upon inspection of media sam-ples, no eggs were found in any of the replicates. Therefore, these data show thatB.bassiana exposure and likely mycosis suppressed oocyte maturation rates in im-mature females (X2

(1, n = 13) = 5.34, P = 0.02; Figure 7). Flies sampled at 5 and 6 dpost exposure displayed similar oocyte maturation rates. However, by Day 7, therewere significantly fewermature oocytes in female flies exposed toB. bassiana com-pared with the control flies.

Myco-insecticide field efficacy trial. The decay of B. bassiana conidiasampled on lowbush blueberry foliage was not impacted by shading from thecages (F7, 16 = 9.02, P = 0.54). However, the density of viable conidia did de-cline significantly with time post-application (F7, 16 = 9.02, P = 0.0001). The totalcounts at each collection interval were plotted over time and fitted with a logistic


Fig. 6. Histograms of egg maturation data in female SWD after sub-lethal B.bassiana exposure. Data were analyzed under the assumption of expo-nentially distributed data of both non-treated (top) and treated (bottom)flies. The total number of mature oocytes counted in each individual isrepresented here.

decay function (Figure 8), and the inverse prediction approximated a B. bassianaconidia half-life of about 3.4 h after initial application. Based on the measuredquantities of conidia on the leaves sampled at 0 h post-spray, the actual applica-tion rate tested in this study is estimated to be roughly 3500 conidia per mm2.This corresponds to conidia coverage roughly one order of magnitude less thanthe predicted lethal concentration expected to kill 50% of the population (LC50)derived from concentration assays.

The ordinal logistic model suggests that a significant treatment effect existedwith ranked SWD larval infestations (X2

(2, N = 9) = 14.23, P = 0.0008; Figure 9).


Fig. 7. Oocyte maturation in teneral SWD females exposed to control (O) or B.bassiana (X) inoculated surfaces. Treatment exposure was initiated nolater than 21 h after pupal eclosion for any given individual. Dissec-tions were conducted on sets of females after 5, 6 and 7 d of adulthooddevelopment.

Fig. 8. Decay in B. bassiana conidia viability from MycotrolR©

treated foliageover time, fitted by logarithmic regression. Sample collections were ob-tained 0 (ca. 10 min), 24, 48 and 96 h after application. Each data pointat a given time interval represents the mean B. bassiana cfu abundancefrom three replicate Petri dishes inoculated with leaf suspensions sam-pled from a single study plot. All averages are plotted together since acage shading effect on conidia longevity was not observed.


Fig. 9. Average abundance (N) of ranked SWD larvae and captured flies / trapsampled in each of the three control cages (control), non-sprayed releasecages (not sprayed), and B. bassiana protected release cages (sprayed).Error bars were constructed using one standard error of the mean. Barsdisplaying dissimilar letters (a and b for adults; A and B for larvae)denote a significant difference in mortality among treatments.

Ranked larval infestation in fruits were lower in the control cages (C) than cageswhere flies were released, but there was no difference in larval infestation betweenthe Mycotrol

R©treated cages (S) and cages that did not receive a spray applica-

tion (NS). Similarly, analysis of the adult abundance (# captured flies / trap) in thecages suggests a significant treatment effect (F2, 6 = 5.46, P = 0.045), which again,is due to the lower quantity of adults observed in the control cages compared withthe cages containing SWD, with no significant difference in adult SWD countsbetween cages receiving the B. bassiana application and cages not treated withthe myco-insecticide. The mean (±SD) SWD capture rate of individual traps wasfound to be 15.7 ± 7.6% of the release total. Lastly, including an estimate of suc-cessful release density as a covariate did not explain any additional variation inmeasurements of adult abundance among sprayed and unsprayed cages (F2, 6 =0.15, P = 0.86).

Discussion

Collectively, the results of this investigation suggest that, while the ento-mopathogens tested are virulent against SWD flies following laboratory inocu-lation, their utilization as biocontrol agents in SWDmanagement does not appearpromising, at least for a single spray application directed at adult flies. Laboratoryinduced mycoses resulted in both lethal and sub-lethal effects in SWD flies. After


exposure to pathogen inoculated surfaces with concentrations ranging from 0–16,000 B. bassiana conidia/mm2, a positive dose-mortality response was observed.While no dose-response assessment was conducted for comparison, Cuthbertsonet al. (2014) also reported significant increases in adult SWDmortality after directtopical contact with B. bassiana conidia.

It is important to note that all six fungal strains increased SWD mortality af-ter amass lethal conidia exposure. Interestingly,M.anisopliae strain F-52 inducedthe greatest mortality of the six strains tested, but failed to produce a significantfly mortality effect during the follow up dose-response assay. Despite this incon-sistency, conidia of both pathogens were observed germinating on flies euthanizedno more than 24 h after physical contact with the insect exoskeleton. Investiga-tions on the contrastingmechanics of direct and indirect host integument invasionby entomopathogenic fungi have been conducted previously on other insect taxa,including pest species in the orders Coleoptera and Lepidoptera (Pekrul & Grula1979, Fernandez et al. 2001, Talaei-hassanloui et al. 2007, Guerri-Agullo et al.2009). The formation of appressoria at the distal end of a conidia germ tube isgenerally regarded as evidence for direct enzymatic degradation and hyphal in-vasion of the host cuticle, due in part to the high mitochondrial composition andmetabolic activity of cells forming the structure. On SWD flies, appressoria forma-tion and integument penetration was observed (Figure 1). Furthermore, the fre-quency of sporulation, i.e., observed formation of mycelium on cadavers, increasedwith conidia dose with both entomopathogenic fungi (Figure 5). Collectively, theseresults provide complimentary evidence for positive laboratory infection, even inthe absence of a significant dose-mortality response toM. anisopliae induced my-cosis. Given our understanding of the differing mechanisms by which activity ofB. bassiana and M. anisopliae mycotoxins affect host longevity (Sowjanya Sreeet al. 2008, Qadri et al. 2011), it is possible that over a longer time period thanexamined here, infection by M. anisopliae at similar concentrations would havesubstantially elevated the proportional mortality of exposed SWD flies.

Sublethal effects and particularly impacts on host fecundity have been demon-strated for a number of insects exposed to entomopathogenic fungi (Gindin et al.2006, Quesada-Moraga et al. 2006, Darbro et al. 2012). Insect fecundity reductionshave been documented in red palm weevils [Rhynchophorus ferrugineus (Olivier);Coleoptera: Curculionidae] treated with sub lethal doses of M. anisopliae inocu-lum, with reduced oviposition rates and reduced egg hatch over time (Gindin et al.2006). Exposure of adult yellow fever mosquitoes (Aedes aegypti L.; Diptera: Culi-cidae) to B. bassiana conidia significantly altered the reproductive output of indi-viduals. On average, control flies laid a greater quantity of eggs throughout theirlifetime in comparison to pathogen inoculatedA.aegypti (Darbro et al. 2012). Inter-estingly, the opposite trend was observed in adult A. aegypti through the first 48 hpost-exposure. During this time period, the mean oviposition rate of B. bassianatreated females exceeded that of individuals exposed to the control treatment. Noincrease in the oviposition rates ofB. bassiana infected SWD females was observedduring this study, and may have been due to the absence of copulation with maleflies. However, in response to this sub-lethal B. bassiana inoculation, virgin fe-male oocyte development rates were similar in control vs diseased individuals un-til seven days of adultmaturationwhen the ovaries of pathogen inoculated femalescontained significantly fewer mature oocytes than those of control flies. Giventhe practical limitations of this study, it is not clear whether oocyte development


becomes delayed and the same number of eggs will be produced over a longer pe-riod of time, or if total egg production and oviposition will be suppressed duringthe sub-lethal phase of fungal infection in SWD females.

The capacity for various entomopathogenic fungi to significantly suppress in-sect population growth has been described previously for a number of insects, in-cluding SWD (Gindin et al. 2006, Cuthbertson et al. 2014, Moorthi et al. 2015).In addition to reducing host fecundity, immature host growth and developmentrates have been reduced following laboratory inoculations with entomopathogenicfungi. Direct topical exposure of Spodoptera litura (F.) (Lepidoptera: Noctuidae) tosub-lethal concentrations of I. fumosorosea or B. bassianamycotoxins persistentlysuppressed development and consumption rates of individuals throughout subse-quent life stages (Moorthi et al. 2015).While fungal pathogenicity of juvenile SWDwas not examined in this study, similar pathogen effects could restrict the recruit-ment of reproductively vigorous adults to invading populations during the earlystage of infestations.

Preliminary testing of the fungal isolate utilized by BalanceR©, B. bassiana

strain HF-23, yielded a significant mortality response in adult SWD. In relationto mortality data and available literature of the other entomopathogenic fungitested,M. anisopliae and B. bassiana strain GHA were selected for further biocon-trol evaluation in this assessment.However, despite strong evidence for laboratoryinfection of flies by B. bassiana, no results were obtained that justify the imple-mentation of these fungal pathogens in SWD pest management. Direct sprayingof caged lowbush blueberry crops with the product Mycotrol

R©failed to reduce the

relative abundance of larvae and flies sampled in study enclosures.With the implemented application methodology of spraying the foliage, SWD

adults needed to contact and pick up conidia on their legs, mouthparts or ab-domen. In complimentary laboratory assessments, SWD adults displayed acuteand chronic vulnerability to mycoses in response to a surface inoculation approachthought to represent a practical pathogen-insect contact mechanism for wild adultSWD. The SEMmicrographs ofM. anisopliae inoculated flies illustrate that a con-siderable quantity of sensilla and setae are found on the species’ tarsal segments.These structures protrude from the exoskeleton and substantially influence thesurface area found on this body region, seemingly resulting in a considerable con-centration of conidia on this body region. Once picked up, conidia may then bephysically transferred to other body regions through grooming or copulatory be-haviors. Based on the lowbush blueberry foliage sampled immediately after spray-ing, the predicted coverage tested in the field study (3500 conidia mm−2) corre-sponded in magnitude with the second highest dose tested during the B. bassianalaboratory assay (2500 conidia mm−2). This conidia concentration significantly in-creased mortality rates of flies under laboratory conditions utilizing an analogous,indirect surface exposure protocol to that implemented in the field. Given thatB. bassiana conidia obtained from leaf samples were successfully cultured on aselective growth medium, and that there was no significant impact of spraying onthe abundance of adult SWD captured in release cages, it is plausible to suspectthat uncontrolled factors may have impeded the germination of virulent sporesafter their attachment to the insect cuticle.

Insufficient relative humidity (RH) has been shown to limit infection and con-trol efficacy of B. bassiana toward arthropod hosts (Shipp et al. 2003). While nocritical RH thresholds were derived from the experiment, diet inoculation with


1×108 B. bassiana strain HQ917687 conidia ml−1 supplied to M. domestica fliesand larvae at varying laboratory RH levels generally resulted in greater mortalityrates inmore humid laboratory climates with temperatures ranging from 20–35◦C(Mishra et al. 2013). Temperatures outside of this range were shown to negate thevirulence of fungal conidia in both M. domestica life stages, regardless of climaticRH composition.

Throughout the duration of the August 2015 field study in Jonesboro, ME, pre-cipitation events were extremely sparse. According to logged weather data takenat the field site, from initiation of the experiment until the collection of fruit sam-ples a total rainfall of 8.6 mm is estimated to have fallen under daily temperatureextremes ranging from 12.7–29.6◦C.Moreover, from myco-insecticide applicationsup through the 96 h leaf sampling, a mere 0.25 mm of precipitation was detectedat the field site. Given approximated conidia viability half-life of about 3.4 h, theprecipitation experienced over this time interval may have been inadequate forsustaining relative humidity conditions needed for successful germination. Thiswould effectively prevent substantial rates of SWD infection and enabled flies toinflict the severe fruit infestations observed. Unfortunately, no SEM micrographswere obtained from fly integuments of this experiment to provide evidence in sup-port of this hypothesis. For further consideration as biocontrol agents of SWD,future myco-insecticidal efficacy experiments against SWD should contemplatelaboratory or greenhouse assessments in order to obtain information on the in-oculation efficacy of entomopathogenic fungal conidia on fruits and leaves. It willalso be worth exploring the necessity for multiple vs single myco-insecticide appli-cations in an attempt to increase the probability of pathogen-host contact duringperiods of sufficient RH.

Future assessments. One necessary consideration of insect pest biocontrol isthe stage-specific physiological and phenological vulnerability of a pest species toa given management technique. In the context of lowbush blueberry infestationswith SWD,Ballman et al. (2017) show that the localization of pupating individualsmay be highly dependent on the type of fruit host with a greater frequency of pupaemetamorphosed in soil substrates as opposed to within lowbush blueberry fruits.These findings are contrary to the purported pupation sites proposed by Walshet al. (2011), but appear to be similar with the pupation strategies described byAsplen et al. (2015). Given the management goal of sustaining low densities of re-productively active adult SWD (Walsh et al. 2011), it is possible that the targetingof viable soil-dwelling pupae could aid in achieving this goal.

Formulations containing entomopathogenic fungi may be incorporated into theirrigation systems for the localization of virulent conidia in the soil. Given the ap-parent pupation strategy of SWD in the lowbush blueberry agroecosystem, thismethod of delivery could substantially impede the recruitment of reproductivelyactive adults into an invading population. Although not tested here, pathogenicitytoward juvenile SWD could retard the propagation of gametes and maturation ofreproductive organs.We observed this in ovarian development rates ofB. bassianainfected female SWD throughout first week of adult maturation in virgin females.Coupled with the post-eclosion time requirements for completing reproductivedevelopment in both sexes of many Drosophila spp. (Markow & O’Grady 2008),expanding myco-insecticidal exposures to include juvenile SWD might enhancethe degree of control obtained beyond that achieved through exclusive targetingof fly populations. Beris et al. (2012) have shown that laboratory induction of


pupal mycoses in Mediterranean fruit fly [Ceratitis capitata (Wiedemann);Diptera: Tephritidae] incurred a slight mortality response, and consequent re-ductions in adult longevity. Therefore, the capacity for entomopathogenic fungito disrupt juvenile as well as adult maturation, and curtail the average longevityof infected individuals, should be acknowledged during future evaluations on thepotential application of myco-insecticides in crops susceptible to SWD invasion.

The association of Hymenopteran parasitoids with SWD pupal hosts has beendescribed in some regions of Europe, as has predation by soil dwelling insects(Gabarra et al. 2014, Ballman et al. 2017). Further laboratory evaluations have fa-cilitated the consideration of these natural enemies in biological control for SWD.Concomitant treatments are often recommended in pest management practicesfor sustained efficacy (Pedigo and Rice 2006), and the cumulative effects of ento-mopathogenic fungal applications into agroecosystems harboring predators andparasitoids could further restrict pest population growth rates during early stageinfestations. Despite evidence supporting the compatibility of predator and para-sitoid release in conjunction with entomopathogenic fungi to combat pestiferousinsect populations in the field (Labbé et al. 2009), specific targeting of SWD pupaewill inevitably bring the pathogen in close proximity with beneficial insects, natu-rally occurring or otherwise. Evaluations might therefore consider looking at thepotential conflicts, if any, of this myco-insecticide treatment protocol on the abun-dance of antagonistic parasitoids and predators as pertains to biocontrol effortsfor novel pests such as SWD.

One alternative to irrigation or spray delivery involves the auto-disseminationof conidia in reservoirs deployed within no-kill traps emitting attractive, volatilesemiochemicals.Mycoses are then able to propagate throughout an invading insectpopulation via horizontal transmission mechanisms during copulatory and/or ag-gregation behaviors. El-Sufty et al. (2011) designed a dry, mass inoculation devicefor red palm weevil adults. The investigation included an aggregation pheromoneand found that trap visitors frequently experienced mortality increases in the lab-oratory. Deploying devices in date plantations not only resulted in a significantmortality response over a 2-yr sampling period, but also appears to have mani-fested in successful and extensive dispersal of the fungus via horizontal contactwith control populations. The demonstrated capacity for fungal pathogens to re-produce within a SWD host, coupled with sustained efforts in the identificationof species-specific symbioses of SWD with microbial yeasts (Hamby et al. 2012),could aid in the development of an analogous inoculation trap for utilization inSWD management as a medium for selective infection.

Acknowledgements

We want to thank Dr. Andrei Alyokhin at the University of Maine for providing a reviewof this manuscript. We would also like to thank two anonymous reviewers and the editorfor valuable suggestions for improving this manuscript. In addition, we thank Judy Collins,Tamara Levitsky, Elissa Ballman, Jen Lund, Michael Hahn and Andrew Wilson for their re-search assistance in the laboratory and field.Mr. Kelly Edwards and Dr. Seth Tyler providedthe technical assistance for producing the electron microscopy images. We would also liketo acknowledge the reviews from two anonymous reviewers that led to the improvement ofthis paper. This is the Maine Agricultural and Forestry Experiment Station journal articlenumber 3562. Partial funding for this project was provided by the National Institute of Foodand Agriculture, U.S. Department of Agriculture Specialty Crops Research Initiative under


Agreement No. 2015-51181-24252, and the University of Maine Agricultural and ForestryResearch Station Hatch Project ME021505.

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