ABSTRACT
AGRICULTURAL LANDSCAPE ECOLOGY OF SPOTTED WING DROSOPHILA IN THE CENTRAL VALLEY OF CALIFORNIA
The spotted wing drosophila, Drosophila suzukii Matsumura (Diptera: Drosophilidae), has established in North America and become an economic concern for a variety of fruit crops. In this thesis, the pest’s seasonal dynamics in various fruit orchards were monitored, and the potential of two major crop fruits (peach and citrus) as alternative hosts as well as the pest’s overwintering biology were investigated. D. suzukii adults were monitored in four regions in the Central Valley of California, from April 2013 to July 2014. Results showed significant changes in adult D. suzukii populations in time and migration among different fruit crops. Adult populations highly aggregated in citrus orchards and non-crop habitats during the winter season, suggesting that citrus orchards and non-crop habitats are possible overwintering sites for the pest. It was found that female D. suzukii was unable to lay eggs in intact fuzzy sections of peach fruit, but readily laid eggs in sections without fuzz or with insect damage. The results indicate that intact, pre-harvest peach fruit are unlikely to be infested by the fly, but any surface damage could render the fruit susceptible to the fly. The overwintering survival and development of D. suzukii were investigated in field-cages in a citrus orchard in California’s interior fruit growing region. Results suggested that D. suzukii may be able to overwinter as adults. Further laboratory tests showed that D. suzukii can successfully develop in damaged fresh or rotting citrus fruit.
Thomas James Stewart August 2015
AGRICULTURAL LANDSCAPE ECOLOGY OF SPOTTED WING DROSOPHILA IN THE CENTRAL VALLEY OF CALIFORNIA
by Thomas James Stewart
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Plant Science in the Jordan College of Agricultural Sciences and Technology California State University, Fresno August 2015 APPROVED For the Department of Plant Science:
We, the undersigned, certify that the thesis of the following student meets the required standards of scholarship, format, and style of the university and the student's graduate degree program for the awarding of the master's degree.
Thomas James Stewart Thesis Author
John Bushoven (Chair) Plant Science
Andrew Lawson Science and Mathematics
Kent Daane UC Cooperative Extension
For the University Graduate Committee:
Dean, Division of Graduate Studies AUTHORIZATION FOR REPRODUCTION OF MASTER’S THESIS
X I grant permission for the reproduction of this thesis in part or in its entirety without further authorization from me, on the condition that the person or agency requesting reproduction absorbs the cost and provides proper acknowledgment of authorship.
Permission to reproduce this thesis in part or in its entirety must be obtained from me.
Signature of thesis author: ACKNOWLEDGMENTS I am grateful to Kent Daane and Xing-geng Wang (University of California, Berkeley), Andrew Lawson and John Bushoven (California State University, Fresno) for their guidance, time and support on this study, Andy Molinar, Glenn Yokota, Gülay Kaçar, Rachael Muradian and Sean Tomajan (University of California Berkeley) at the Daane laboratory for their support and assistance with this study. I also thank Carlos Crisosto (University of California, Davis), Marshall Johnson and James Sievert (University of California, Riverside) for providing some experimental materials, equipment, and space; Ferrari Farms and Steve Chinchiolo for allowing me to use their orchards; and David Bellamy (U.S. Department of Agriculture - Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center) for providing rearing methods for D. suzukii. Funding was provided by the California Cherry Board (to the Daane Laboratory) and the Harvey Fellowship Grant through the Fresno State Jordan College of Agricultural Science and Technology. TABLE OF CONTENTS Page
INTRODUCTION ...... 1
Morphological Description ...... 1
Biology of the Pest ...... 1
Temperature Thresholds and Dispersal ...... 2
Overwintering ...... 3
Pest Damage and Economic Impact ...... 4
Current Control of D. suzukii ...... 6
Biological Control ...... 8
Landscape Ecology ...... 10
Summary ...... 12
HYPOTHESES ...... 13
GENERAL OBJECTIVES ...... 14 STUDY 1: POPULATION DYNAMICS OF DROSOPHILA SUZUKII IN THE FRUIT GROWING REGIONS OF THE CENTRAL VALLEY OF CALIFORNIA ...... 15
Abstract ...... 15
Introduction ...... 15
Materials and Methods ...... 18
Results ...... 22
Discussion ...... 26 STUDY 2: FACTORS LIMITING PEACH FRUIT (PRUNUS PERSICA) AS A POTENTIAL HOST FOR DROSOPHILA SUZUKII ...... 33
Abstract ...... 33
Introduction ...... 34
Materials and Methods ...... 36 vi vi Page
Results ...... 42
Discussion ...... 46 STUDY 3: ORANGE (CITRUS SINENSIS) AS A POTENTIAL OVERWINTERING HOST: ADULT SURVIVORSHIP AND FECUNDITY IN A CITRUS ORCHARD ...... 51
Abstract ...... 51
Introduction ...... 51
Materials and Methods ...... 54
Results ...... 57
Discussion ...... 60
GENERAL CONCLUSIONS ...... 64
LITERATURE CITED ...... 66
APPENDICES ...... 74
APPENDIX A: STUDY 1 TABLES AND FIGURES ...... 75
APPENDIX B: STUDY 2 TABLES AND FIGURES ...... 91
APPENDIX C: STUDY 3 TABLES AND FIGURES ...... 99
LIST OF TABLES
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Table A1. Geographical locations and monitoring sites of D. suzukii adult in California Central Valley during 2013 and 2014 ...... 76 Table A2. Periods of ripening and fruit availability of monitored fruit crops in California Central Valley ...... 77 Table A3. Sum and average (± S.E.) number of adult Drosophila suzukii and other species of frugivorous fruit flies captured in vinegar traps placed in four locations in some of California’s San Joaquin Valley fruit growing regions ...... 77 Table A4. Average (± S.E.) number of adult Drosophila suzukii captured in vinegar traps placed in different plant ecosystems at four locations in some of California’s San Joaquin Valley fruit growing regions from April 2013 to May 2014. The number of traps used is provided in parenthesis and varied according to the number of ecosystem sites monitored each location or the number of traps used at each site ...... 78 Table A5. Generalized Linear Model analyzing the effects of presence of mature fruit (Fruit), accumulated degree-days (DD), as well as their interaction on weekly captures of adult D. suzukii in different geographical locations and host plants ...... 79 Table A6. Correlation coefficients in the relationship between trap captures of adult D. suzukii in two closely located sites in different geographical locations in California’s Central Valley ...... 81 Table A7. Results of Generalized Linear Model testing the effects of trapping site (different orchard or non-orchard habitats) and seasons on the number of mature eggs of captured female D. suzukii or the percentage of captured females without mature egg in different geographical locations in California’s Central Valley ...... 82
Table B1. Brix and firmness of various types of tested peach fruit ...... 92 Table B2. Relationship (linear regression) of fruit brix and fruit firmness with the number of D. suzukii eggs per intact or damaged area (shaved to remove peach fuzz, simulated harvest damage, insect damage from peach twig borer and fork-tailed katydid feeding, and simulated hemipteran damage with variable sized punctures) using Earlirich (Er), Babcock (Bc) or Spring Snow (Sp) cv. peaches ...... 93 Table B3. Ovipositional time by D. suzukii on different fruits or artificial diet media ...... 94 viii viii Page
Table C1. Maximum and mean (± SE) survival days and number of eggs reproduced per female D. suzukii under the different food provision conditions in field cages in 2013 to 2014 winter ...... 100
LIST OF FIGURES
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Fig. A1. Weekly mean D. suzukii trap captures in cherry and other commercial fruit crop orchards in (A) Courtland (Sacramento County) and (B) Stockton (San Joaquin County), CA. Data for the three cherry orchards (within 2 km) in Courtland were pooled...... 83 Fig. A2. Weekly mean D. suzukii trap captures in (A) cherry and other commercial fruit orchards, (B) a riparian habitat area adjacent to the commercial orchards, and (C) a mixed fruit garden in Brentwood (Contra Costa County), CA. Data from the three riparian sites were pooled...... 84 Fig. A3. Weekly mean D. suzukii trap captures in different crop orchards within a 330-arca research farm at the University of California Kearney Agricultural Research and Extension Center in Parlier (Fresno County), CA. Data are grouped based on the fruiting seasons of monitored crops (A-D)...... 85 Fig. A4. Shifting correlations between the trap captures of D. suzukii in two different crops in Parlier over a range of gap between fruit ripening seasons...... 86 Fig. A5. Weekly mean trap captures of other drosophilids (except D. suzukii) in (A) Courtland, (B) Stockton and Brentwood, and (C) Parlier, CA. Data were pooled from all traps in different fruit orchards or site for each location...... 87 Fig. A6. Weekly mean trap captures of drosophilid parasitoids (Leptopilina spp.) in cider vinegar traps for D. suzukii in (A) Courtland and (B) Parlier, California. Data were pooled from all traps in different fruit orchards for each location...... 88 Fig. A7. Number of mature eggs of D. suzukii females captured in different orchards orsites in (A) Courtland, (B) Brentwood, and (C) Parlier. Data were pooled for each of three seasons (November to March, April to July and August to October). Bars refer to mean ± SE...... 89 Fig. A8. Percentage of captured D. suzukii females without mature eggs in different orchards or sites in (A) Courtland, (B) Brentwood, and (C) Parlier. Data were pooled from all traps for each month. Lines refer to mean monthly minimum daily temperature...... 90
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Fig. B1. The mean (± SE) number of eggs per treated area for intact Earlirich (Er.) and Babcock (Bc) fruit (minus the stem-end area), shaved Spring Snow sections ( 3 cm2), stem-end harvest damaged Earlirich and Spring fruit, peach twig borer (PTB) and the fork-tailed katydid (katydid) damaged Earlirich fruit, and simulated hemipteran feeding damage with variable sized needle punctures (0.3, 0.5 and 1.0 mm). Different letter above each bar represent a significant difference between the intact treatments means (the entire fruit) and the manipulated shaved or damaged fruit sections, regardless of damage size (ANOVA, P < 0.05)...... 95 Fig. B2. Relationship between the number of eggs deposited by D. suzukii per maximum width of area damaged by (A) the peach twig borer feeding (y = 0.552 + 2.098x, r2 = 0.251, P < 0.001) and (B) the fork- 2 tailed katydid feeding (y = -1.070 + 1.569x, r = 0.247, P < 0.001)...... 96 Fig. B3. Relationship between peach firmness on the stem-end area and the average firmness on the other fruit sections (mean values from three readings between the shoulder and tip of the fruit) for Earlirich (y = 5.214 + 0.484x, n = 130, r2 = 0.715, P < 0.001), Spring Snow (y = – 94.054 + 0.949x, n = 15, r2 = 0.681, P < 0.001) and Babcock (y = 2 26.969 + 0.478x, n = 21, r = 0.541, P < 0.001)...... 97 Fig. B4. Effects of different treatments substrates for (A) unsuccessful oviposition attempts the cessation or giving-up time with intact or shaved peach sections before the female fly left the arena (log-rank test, 2 = 6.0, P = 0.014) and (B) successful ovipositional time with substrates of shaved peach, intact cherry, damaged peach, and 2 artificial diet (log-rank test, =166.2, P < 0.001)...... 98 Fig. C1. Survival and development of immature (egg, larva and pupa) D. suzukii in field-cage test in Parlier, CA. (A) Mean daily air and soil temperature during the winter of 2013 to 2014 near the field study site. (B) Mean (± SE) percentage of each immature stage successfully developed into adults when the tests were launched at the 8 different dates from 22 November 2013 to 28 March 2014. Climatic data were obtained from a nearest (300 m) weather station. . 101 Fig. C2. Survival of adult D. suzukii under different food provision conditions in field cages launched on (A) 19 November, (B) 27 December, (C) 22 January, (D) 24 February, and (E) 28 March during the winter of 2013 to 2014. W = water, HW = honey water, HWO = honey water + orange, F = female, M = male. Different letters to the nearest survival curves indicate significant difference between different food treatments (Survival Analysis, log-rank test, with the significance of paired comparisons adjusted to a treatment-wide level of alpha = 0.05 using the sequential Bonferroni adjustment)...... 102
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Fig. C3. Comparison of adult female D. suzukii longevity in the field cage tests during the winter of 2013 to 2014 when honey water and orange as a food and/or ovipositional medium were provided for the adults. Dates on the top of the curves indicate the launching date of the field tests while different letters to the right of the nearest survival curves indicate significant difference among monthly releases (Survival Analysis, log-rank test, with the significance of paired comparisons adjusted to a treatment-wide level of alpha = 0.05 using the sequential Bonferroni adjustment)...... 103 Fig. C4. Oviposition by D. suzukii on post-harvest orange fruit with different conditions. Bars refer to mean ± SE and different letters over the bars indicate significant difference (One-way ANOVA and Tukey’s HSD, P < 0.05)...... 104
INTRODUCTION
Morphological Description The spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a member of the melanogaster species group within the Drosophilidae (Hauser 2011). Adult flies are generally 2-3 mm in length with red eyes and a brown abdomen and thorax (Vlach 2010, Walsh et al. 2011). The abdomen is marked at the ends of abdominal segments with unbroken bands of darkened pigment (Vlach 2010). Males are easily identified by a single spot on the first vein of each wing and a set of black sex combs on each foreleg (Hauser 2011, Walsh et al. 2011). The females have a serrated ovipositor with many darkened and sharp teeth which is six to eight times the length of the spermatheca (Hauser 2011, Walsh et al. 2011). D. suzukii eggs are white and glossy with two white filaments the length of the egg protruding from the head end which function as breathing spiracles (Kanzawa 1939, Lee et al. 2011, Walsh et al., 2011). After the eggs are deposited, the developing larvae become visible as they near the time to emergence (Kanzawa 1939, Walsh et al. 2011). The larvae emerge in the fruit, and are white and cylindrical with visible mouthparts and respiratory organs (Walsh et al. 2011). The larvae develop into pupae, which start grayish yellow and harden and darken as development progresses (Walsh et al. 2011).
Biology of the Pest Drosophila suzukii was first described in Japan in 1916 as a pest of cherries (Kanzawa 1939). It is believed to be native to eastern Asia, and is now widely distributed in China, Japan, North and South Korea, Thailand, Myanmar, Pakistan, India, and the Russian Far East (Kanzawa 1939, Oku 2003, Wu et al. 2007), being found from the cooler northern to warmer southern islands in Japan (Kanzawa 2 2
1939, Kimura 2004) and many coastal and inland provinces in China (Wu et al. 2007, Yang 2011). More recently, D. suzukii concurrently invaded North America and Europe (Hauser 2011, Lee et al. 2011b, Walsh et al. 2011, Calabria et al. 2012, Cini et al. 2012) and South America (Deprá et al. 2014). In the continental United States, it was first detected in Santa Cruz County in 2008 (Bolda et al. 2010), and since then the fly has rapidly expanded its range throughout the United States, including all western and eastern coastal states as well as some inland states, several Canadian provinces, and Mexico (NAPIS 2014). In Europe, D. suzukii has been found in nine countries since it was first reported in Spain and Italy in 2008 (Cini et al. 2012). Typical of the vinegar fly family, D. suzukii are attracted to sweet, fermented materials. Decomposing fruit, wines, and vinegars have all been shown to attract adult D. suzukii. Earlier and more recent studies have shown that adult D. suzukii utilize olfactory queues for food resource allocation (Kanzawa 1939, Cha et al. 2012). D. suzukii attacks various soft- and thin-skinned fruits, such as cherries, strawberries, blackberries, blueberries, grapes and stone fruits (Kanzawa 1939, Mitsui et al. 2010, Lee et al. 2011a, Walsh et al. 2011, Burrack et al. 2013). Its wide host range and penchant for attacking fresh and ripening fruit can cause significant economic impact to this wide variety of soft- and thin-skinned fruit crops (Bolda et al. 2010, Beers et al. 2011, Goodhue et al. 2011, Lee et al. 2011a, Walsh et al. 2011, Burrack et al. 2013).
Temperature Thresholds and Dispersal Climate can impede the dispersal of animals (Walters et al. 2006). D. suzukii are able to withstand a range of environmental conditions and this may 3 3 allow them to establish in regions where other herbivores with more narrow climatic tolerances and host ranges may not (Cini et al. 2012). The estimated lower and upper thresholds for the development of D. suzukii are 10 and 30°C, respectively (Tochen et al. 2014). Another study, conducted in Japan, found that D. suzukii has lower and upper lethal limits of –0.9 and 32 °C, respectively (Kimura 2004). The fly may complete 3 to 10 generations within its current range in the United States and Canada (Walsh et al. 2011). D. suzukii adults are most active between the temperatures of 20 to 25 °C, and their activity is reduced when temperatures exceed 30°C (Walsh et al. 2011). Adult D. suzukii also have a high dispersal potential to move and locate host resources (Mitsui et al. 2010). The dispersal ability combined with the thermal tolerance of D. suzukii has allowed this pest to rapidly disperse throughout the United States, Canada, and Europe once it established in these regions (Hauser, 2011). For example, Mitsui et al. (2010) suggests that in central Japan D. suzukii adults breed at low altitudes early in the summer, move to high altitudes later in the season, and then later adult generations of D. suzukii move back to low altitudes for breeding. These populations are mostly composed of reproductively immature adults that are likely in reproductive diapause (Mitsui et al. 2010). It is suggested, however, that the purpose of this dispersion pattern is to consume resources that are exploitable at the time, rather than to avoid unfavorable summer conditions (Mitsui et al. 2010).
Overwintering Because D. suzukii is most active during the summer, most studies on D. suzukii focus on the summer life cycle. However overwintering survival of insects directly dictate their summer population dynamics (Leather et al. 1993). There 4 4 have been many studies conducted on factors which influence Drosophila overwintering success, such as cold tolerance, however there is still a lack of literature on the actual overwintering biology of D. suzukii. It has been found that the egg, larval, and pupal stages of D. suzukii will not survive temperatures below freezing (CFIA 2011, Lee et al. 2011b). As stated previously, the lower lethal limit for D. suzukii is -0.9 °C and the upper lethal limit is 32 °C (Kimura, 2004). Dalton et al. (2011) performed studied the overwintering temperature response of D. suzukii in Oregon. The experiment subjected adults and pupae to an 84-day chilling period, with temperature treatments ranging from 1 to 10 °C and with an additional subset exposed to a 7-day freeze period (-2 °C). The study found that D. suzukii at 10 °C survived the longest period; with mortality increasing below this temperature. D. suzukii were unable to survive more than 17 d at 1 °C. These results create a conflict between what is observed in nature and in the laboratory. D. suzukii are established in Hokkaido, Japan, where winter temperatures average between -12 to -4 °C (CFIA 2011, Walsh et al. 2011). This discrepancy suggests that adults must overwinter in sheltered areas, such as under pebbles or leaves, and in colder areas may even seek shelter in man-made enclosures (Dalton et al. 2011, Lee et al. 2011b). Understanding how D. suzukii utilize the agricultural landscape of the Central Valley of California to successfully overwintering may be crucial for the advancement of D. suzukii control strategies.
Pest Damage and Economic Impact Unlike most other Drosophila flies that infest damaged or rotting fruit, the serrated ovipositor of D. suzukii enables females to lay eggs inside ripe and ripening fruit. In fact, ripening fruits are preferred over overripe ones (Mitsui et al. 5 5
2006, Lee et al. 2012). Most damage caused by D. suzukii is due to fly larvae feeding on fruit flesh, however ovipositor wounds can cause physical damage to the fruit and these wounds can provide access to secondary infection by both insects and pathogens, causing faster deterioration and further losses. Additional costs include production costs related to pest control (e.g., monitoring and chemical input costs, increased labor and fruit selection, reduction of the fruit shelf life) and a decreased foreign market (Goodhue et al. 2011). Kanzawa (1939) observed that in Japan D. suzukii oviposited most often on cherries, peaches, plums, persimmons, strawberries and grapes, but was also opportunistic in its host range and would feed on fruits that dropped on the ground, often those that spoiled or fermented. A partial list of the fly’s host range includes apricots, blackberries, blueberries, cherries, figs, grapes, hardy kiwis, mulberries, nectarines, overripe pears, peaches, persimmons, plums, pluots, raspberries, and strawberries; it has also been observed feeding upon injured or culled fruit including apples and oranges (Walsh et al. 2011). In many of China’s cherry and stone fruit regions, D. suzukii fruit damage is often found associated with two related fly species: Drosophila melanogaster and D. hydei (both belonging to the D. melanogaster group) (Wu et al. 2007, Yang et al. 2011). The majority of the United States specialty small fruit production occurs in the pacific coastal states. For example, in 2008, California, Oregon, and Washington accounted for virtually all commercial raspberry and blackberry production, as well as 84, 83, and 26% of the value of cherry, strawberry, and blueberry production, respectively (Walsh et al. 2011). Nationally, these crops have a $4.48 billion value (USDA NASS 2012). Assuming 20% yield loss, producers would annually lose $511 million in the pacific states and $1.04 billion, nationally (Bolda et al. 2010). Stakeholder feedback highlights increased annual 6 6 integrated pest management (IPM) costs, which approximates 7-14% of the total farm gate value of these crops (equates to costs ranging from $364-728 million annually). Furthermore, market loss due to quarantines, minimum residue restrictions, as well as increased costs due to postharvest cold treatments and fumigation also negatively impact grower profits (Walsh et al. 2011).
Current Control of D. suzukii There are no registered insecticides that will control maggots within fruit, and current control efforts rely heavily on the use of insecticides against adult D. suzukii in crops (Beers et al. 2011, Bruck et al. 2011). Therefore, most control methods rely on frequent applications of insecticides in fruit fields in an attempt to kill adult D. suzukii before they successfully oviposit (deposit eggs) in the fruit. The success of this tactic depends on the timing of the application, as the insecticide must contact the adult female between the time that she enters the crop field and when she successfully oviposits because once inside the fruit the fly’s larvae is protected from most insecticides. This is made more difficult by the high mobility and fecundity of adult D. suzukii (Mitsui et al. 2010), hence the need for high frequency applications. This insecticide-based approach is necessary to protect fruit crops in regions where D. suzukii has established, but can also cause issues concerning pesticide residues, health risks for laborers, and negative impacts for beneficial organisms in the treated fields (Walsh et al. 2011). In particular, organic production is seriously threatened because few effective insecticides are available (Walsh et al. 2011). The pest’s high fecundity (around 400 eggs per female) and short generation time can allow it to complete several generations in a single cropping cycle and an explosive population growth (Kanzawa 1939, Tochen and Walton in prep). The fast generation turnover 7 7 requires up to two chemical applications per week during the ripening stage. Multiple pesticide use increases the risk of residues in fruits, promote insect resistance and negatively affect pollinators and other beneficial species. Several exporters to Asia lost consignments due to unacceptably high residue levels needed to control D. suzukii close to harvest. It also presents timing difficulties with respect to insecticide applications because of pre-harvest interval concerns with many of the insecticides (Walsh et al. 2011). There are some cultural controls that help reduce pest densities, such as an early harvest to avoid infestation, costly repeated harvests to remove fruit before it reaches peak susceptibility to D. suzukii, and removal of residual fruit which can reduce quality of fruit and increase labor costs (Lee et al. 2012). However, these management techniques result in increased production costs and, therefore, insecticides remain the best and most commonly used control tool. A wide range of soft-skinned ornamental or landscape plants have fruit that can serve as hosts for D. suzukii, creating untreated refuges that can be a source of flies that re-infest the cash crop or form overwintering habitats. It is thus crucial to suppress those outside source populations on non-crop hosts to prevent re- infestations in treated crops, and this may reduce the pest pressure to a manageable level in crops. Current controls strategies target only the cash crop, with little regard to the landscape management of this polyphagous pest. From this perspective, biological control may hold more promise than insecticides if natural enemies can track the movement of the pest, and target not only pest populations on crops but also those source populations in unmanaged habitats. Any reduction in the sizes of source populations surrounding the crop fields would greatly improve the efficiency of other control strategies. 8 8 Biological Control Most work on D. suzukii biology and control has focused on crop damage, chemical, and cultural controls (Beers et al. 2011, Bruck et al. 2011, Dalton et al. 2011, Goodhue et al. 2011, Landolt et al. 2011, Lee et al. 2011a, 2011b, 2012, Walsh et al. 2011). These programs are needed, especially in the short term, but there is a surprising lack of information about the natural enemies or efforts to develop biological control of D. suzukii. Numerous parasitoid species attack Drosophilids, most belong to four hymenopteran families: Braconidae, Figitidae, Diapriidae and Pteromalidae. Most biological control research has targeted those Drosophila species living in fermenting substrates, and by far most studies have been done in Europe and the information centers mainly on three frugivorous larval drosophila parasitoid species: Leptopilina heterotoma (Thompson), L. boulardi Barbotin, Carton & Kelner, and Asobara tabida (Nees) (Prévost 2009). However, none of these larval parasitoids (L. heterotoma, L. boulardi and A. tabida) seems to develop from D. suzukii in the United States. At this time, only two generalist pupal parasitoid species (Pachycrepoideus vindemmiae Rondani and Trichopria sp.) are commonly found attacking D. suzukii in North America (Chabert et al. 2012). Mitsui et al. (2007) conducted surveys on parasitoid species attacking frugivorous Drosophilidae in Japan, which is the only published survey from Southeast Asia. They reported 15 parasitoid species on various frugivorous Drosophilidae. The dominant larval parasitoids were in the genera Asobara, Leptopilina and Ganaspis. The species had slightly different geographic ranges in the Japanese island chain: A, japonica Belokobylskij was found throughout island chain; whereas A. tabida, A. rossica Belokobylskij, A. rufescens (Förster), and L. heterotoma occurred primarily from northern to central parts of the main islands; 9 9
Ganaspis xanthopoda (Ashmead) was found from central to southern parts; A. leveri was found in the southern part; and A. pleuralis Ashmead, L. victoriae Nordlander and Ganaspis sp. were found primarily in the subtropical islands. These parasitoids are not true specialists, and attacked numerous fly species in the D. melanogaster group and D. polychaeta group in the subtropical islands. Nevertheless, among these parasitoids, A. japonica, L. heterotoma, and G. xanthopoda were found to attack D. suzukii larvae (Ideo et al. 2008) and Trichopria sp. and P. vindemmiae were reported to attack D. suzukii pupae Mitsui et al. (2007). Moreover, some species of Ganaspis were reported to have a high level of specificity for D. suzukii, attacking D. suzukii larvae feeding inside fresh fruit on trees (Kasuya et al. 2013). In contrast, the more generalist A. japonica and L. heterotoma were only able to attack fruit fly larvae after the fruit had fallen from the tree and were decaying on the ground (Mitsui and Kimura, 2010). In North America, some common drosophila parasitoid species occur, including A. tabida, L. heterotoma, L. boulardi, Ganaspis sp., Trichopria sp., and P. vindemiae (Hertlein 1986, Kacsoh and Schlenke 2012). However, the species composition, geographic distribution, and host range of drosophilid parasitoids are poorly documented in North America. Kacsoh and Schlenke (2012) tested 24 parasitoid strains representing 15 species from four families and widely ranging geographical origins, and found that only 7 parasitoid strains were able to develop from D. suzukii. The failure of parasitoid development presumably resulted from resistance – an immune response – by D. suzukii against the parasitoid eggs placed inside the host, whereas these same parasitoids could successfully develop in D. melanogaster. The Japanese species A. japonica had the highest successful development (79%) from D. suzukii. The only other larval parasitoid able to develop from D. suzukii was a Ganaspis sp. collected in Florida and Hawaii. 10 10
However, two G. xanthopoda strains, one from Hawaii and the other from Uganda, were not effective against D. suzukii (Kacsoh and Schlenke 2012), suggesting that there may be genetic variation in the flies’ immune resistance against parasitoids or in the parasitoids’ virulence against the flies. In Oregon, two parasitoid species (Asobara sp. and P. vindemmiae) were reared from field-placed traps baited with D. suzukii and D. melanogaster, and P. vindemmiae was recently found to readily attack D. suzukii (Brown et al. 2011). Some studies strongly suggest that only co-adapted, specialized parasitoids may be able to develop from D. suzukii (Chabert et al. 2012, Kacsoh and Schlenke 2012). The unique ecological niche of D. suzukii (attacking fresh fruit) may further limit access by some parasitoid species to fly larvae feeding in the fruit. Therefore, parasitoid species able to attack and develop on D. suzukii may have evolved both ecological specialization on and physiological adaptation to the host. However, there is relatively little information on this pest’s host plant use, seasonal occurrence and spatial patterns, landscape movement, or overwriting biology. Understanding D. suzukii’s landscape ecology is critical for the developing sustainable or area-wide management strategies such as biological.
Landscape Ecology The agricultural landscape in California Central Valley fruit-growing regions is often dominated by a mosaic of susceptible cultivated crops and unmanaged non-crop host plants. For example, adult D. suzukii may colonize some favorable crops such as cherries during their fruiting period and, after this crop is harvested, the fly population may largely move to other crops or unmanaged habitats that include ornamental or wild fruits. Dispersal among susceptible hosts may persist throughout the year and, therefore, immigration from 11 11 unmanaged host plants may form the population of D. suzukii that attacks cash crops that would otherwise be eliminated by recurrent insecticide applications. This present difficulty in timing insecticide applications and has resulted in the need for better monitoring of the adult populations and improved control programs for D. suzukii as a landscape pest rather than a single crop program. Biological controls may play a role in this pest’s suppression on the landscape level by reducing pest densities in the non-crop habitats. Host plant species in unmanaged habitats might not directly affect the abundance of D. suzukii in the neighboring commercial orchards, i.e. ornamental host fruits may act as a sink that would indirectly affect the D. suzukii populations in commercial orchards by providing refuge for natural enemies. For example, landscape-based management to reduce D. suzukii populations in cherries may include reducing the source potential of the fly in nearby crops and/or uncultivated habitats, thereby manipulating the spatial arrangements of source and sink habitats. Again, from this perspective, biological control could also hold promise if natural enemies can establish in those unmanaged habitats, and reduce the sizes of source populations surrounding the crop fields. However, the manner by which D. suzukii populations move among host plants and their interactions with parasitoids across orchard boundaries is largely unknown. Also, the source or sink potential of unmanaged habitats can vary dramatically in space and time, and the consequences of this variation on the dynamics of pest populations also remain largely unknown. The efficacy of these approaches would be greatly enhanced as sufficient knowledge of the biology of the pest and its enemies is determined. The long-term goal for improved integrated pest management of D. suzukii is to develop and add sustainable management options to the current pesticide programs, and especially 12 12 to improve natural regulation of D. suzukii source populations at the landscape level, thereby reducing pest pressure to the cash crop. This will in turn reduce reliance on insecticides while still protecting the marketable crop. Understanding the seasonal occurrence and distribution as well as population dynamics is crucial for the development of sustainable and efficient management strategies for this highly mobile pest (Cini et al. 2012).
Summary In conclusion, D. suzukii is a serious pest of many crops and has already caused substantial economic damage for farmers in agricultural regions in Southeast Asia, Europe, Canada, and now the eastern and western United States. The agricultural systems of California that are susceptible to D. suzukii vary geographically, in crop host plants, and in environmental conditions. Therefore, it is important to assess the life history traits of local D. suzukii and evaluate possible patterns in movement and host suitability for the fly in different agricultural landscapes in order to understand their impact on our fruit crop system and aid in the development of future control programs.
HYPOTHESES
1. Population densities of adult D. suzukii will vary across different fruit crop systems in time and space. 2. Adult D. suzukii females will be incapable of oviposition in some host plants, such as intact peach fruit, but can utilize host materials under certain circumstances. 3. California citrus will serve as a sufficient overwintering host.
GENERAL OBJECTIVES
1. To monitor population densities of adult D. suzukii among 15 different San Joaquin Valley fruit crops over two growing seasons, and examine factors influencing landscape movement among these crops. 2. To evaluate the suitability and developmental performance of D. suzukii in suspected host fruits that are grown in the San Joaquin Valley. 2.2. Peach (Prunus persica) as a potential host: factors limiting and facilitating ovipositional success. 2.3. Orange (Citrus sinensis) as a potential overwintering host: adult survivorship and fecundity in a citrus orchard during winter months, reproductive success in citrus as a host.
STUDY 1: POPULATION DYNAMICS OF DROSOPHILA SUZUKII IN THE FRUIT GROWING REGIONS OF THE CENTRAL VALLEY OF CALIFORNIA
Abstract Drosophila suzukii represents a serious pest of several fruit crop systems in California’s Central Valley, which is one of the world’s major fruit growing regions. This study followed D. suzukii seasonal population dynamics in multiple cropping and riparian systems in four cherry-producing counties California’s San Joaquin Valley. Apple cider vinegar baited traps were used to weekly monitor D. suzukii adults, from April 2013 to April or July 2014, in crop and non-crop sites. Results show peak captures were in the spring and fall seasons. In cherry orchards, adult trap counts were highest near harvest-time (June) and declined thereafter as fly populations moved to other crop (e.g., citrus) or non-crop riparian habitats. The number of captured flies was significantly affected by the availability of suitable host fruits, accumulated degree-days (10-30 °C), and the host × fruit interaction). Mature egg load per female was higher earlier than later in the fruit. The results suggest fly populations move among crop and/or non-crop habitats during the year. We also report the presence of trapped D. suzukii adults bearing melanized and encapsulated parasitoids, and a small number (3%) of dissected females contained hatched eggs, suggesting ovoviviparous larvae.
Introduction The spotted wing drosophila, Drosophila suzukii Matsumura, is native to eastern Asia (Kanzawa 1939), but has widely invaded North America and Europe (Walsh et al. 2011, Cini et al. 2012, 2014), and was more recently found in South America (Deprà et al. 2014). In the continental United States, the fly now occurs in most fruit production regions since being first detected in California in 2008 16 16
(Bolda et al. 2010, Walsh et al. 2011, Burrack et al. 2013, NAPIS 2014). D. suzukii is unique among Drosophilidae in that adult females have serrated ovipositors (Atallah et al. 2014), allowing them to oviposit in intact (ripening) soft- and thin-skin fruits such as cherries, blueberries, blackberries, mulberries, raspberries, and strawberries (Kanzawa 1939, Mitsui et al. 2006, Lee et al. 2011, Burrack et al. 2013, Yu et al. 2013, Hamby et al. 2014). Although it is unlikely for the fly to oviposit into fruits with thick and hard skin or fuzzy surfaces such as apples, grapes, loquats, oranges, pears, peaches, persimmons, figs and pomegranates, it can complete development when these ‘suboptimal’ hosts are damaged or because rotted or overripe (e.g., Bellamy et al. 2013, Stewart et al. 2014). Therefore, numerous cultivated and uncultivated fruits and vegetables can be utilized by D. suzukii (Mitsui et al. 2010, Berry 2012, Klick et al. 2012). Because of the fly’s short generation time, high reproductive capacity, wide host range and penchant for attacking ripening fruit, it can cause significant economic losses to susceptible crops (Goodhue et al. 2011, Walsh et al. 2011, Emiljanowicz et al. 2014, Tochen et al. 2014, Wiman et al. 2014). Pesticide applications have been the primary control tactic against D. suzukii in North America (Beers et al. 2011, Bruck et al. 2011). However, the efficacy of insecticide-based programs could be limited by the abundant non-crop hosts that may act as reservoirs for the fly’s reinvasion into the treated commercial crop. D. suzukii is a highly mobile pest. For example, Mitsui et al. (2010) reported that D. suzukii migrated from low altitudes to high altitudes during the summer seasons likely seeking for better host sources, but returned to the low altitudes during the winter seasons for favorable overwintering conditions. Immigration from unmanaged hosts may support the persistence of D. suzukii in commercial orchards, which would otherwise be eliminated by recurrent insecticide spraying 17 17
(Klick et al. 2012). Host plants in unmanaged habitats could act as sinks or sources of D. suzukii populations in commercial crops. It is therefore critical to understand the pest’s seasonal phenology and factors triggering dispersal and persistence of pest population in the agricultural landscape in order to develop landscape-based, risk-reduced strategies for highly mobile and polyphagous pests such as D. suzukii (Carriére et al. 2012, Mazzi and Dorn 2012). Risk of crop infestation from D. suzukii adults that originated in non-crop hosts may depend in part, on the degree and timing of movement between crop and non-crop habitats, and these traits may largely depend on the local climatic conditions, the distribution and availability of host species and landscape traits. The Central Valley of California is a major fruit growing region, and its agricultural landscape can be a mosaic of various cultivated crops – including fruit trees – with scattered unmanaged vegetation, as well as by various urban plantations. Such diverse landscapes present a management challenge for this highly polyphagous and mobile pest because of the availability of ample alternative hosts outside the commercial crop. Two recent studies monitored the fly’s population dynamics in northern and southern regions of the San Joaquin Valley. Harris et al. (2014) reported trap captures of adult D. suzukii in several crop blocks as well as citrus trees and wild bushes in a nearby home yard. They showed two capture peaks of adult D. suzukii in spring through midsummer and fall, but low trap captures during the hottest summer months and coldest winter months, and found that a higher number of flies were captured in the traps that were hung in citrus trees during the winter seasons. However, the seasonal occurrence patterns of D. suzukii in multi–crop agricultural landscapes is still not fully understood, especially what factors might trigger the local dispersal or occurrence dynamics are unclear. 18 18
In this framework, the aim of this study was to understand the seasonal phenology of D. suzukii in different landscapes; this was accomplished by monitoring adult fly population dynamics in different crops and geographical regions. Specifically, we monitored adult fly population dynamics in four major cherry producing regions in California and in various major fruit crops and several non-crop sites. We analyzed the effects of environmental factors and landscape traits (host availability) on the fly’s spatial and temporal population dynamics. We also dissected captured female flies to determine their reproductive status and analyzed the factors influencing adult egg load.
Materials and Methods
Monitoring Sites Studies were conducted in four cherry production regions in the Central Valley of California: Courtland, CA (Sacramento County), Stockton, CA (San Joaquin County), Brentwood, CA (Contra Costa County) and Parlier, CA (Fresno County) (Table A1, see Appendix A for Study 1 tables). Courtland is located at the Sacramento River Delta, and cherry and pear are the two major fruits grown in this area. Near Courtland, 10 monitoring traps for D. suzukii were placed in three organic cherry orchards, one kiwi orchard and one pear orchard. The three traps placed in cherry orchards were 1.0–1.7 km apart, whereas the kiwi and pear orchards were adjacent to the monitored cherry orchards. The agricultural landscape in the Stockton area is dominated by various nut and stone fruit crops. Nine traps were placed in two organic cherry orchards and one peach orchard. The peach orchard was about 1.3 km from one cherry orchard and 30 km from the other orchard. Near Brentwood, nine monitoring traps were placed in four organic fruit orchards (a single trap each in a cherry, apricot, 19 19 pear and mixed stone fruit orchard), three traps spread along one riparian area, and two traps in a mixed 4.5 ha organic fruit crop farm. The riparian area was located at the edge of the fruit orchard and about 0.3 and 0.8 km to the nearest plum and cherry orchards, respectively; there was a small orchard of mixed fruits located about 5 km away from the riparian area and about 1.1 and 1.3 km to the nearest apricot and pear orchards, respectively. Potential host plants including Klamath plum (Prunus subcordata Benth.), cherry plum P. cerasifera Ehrh.), and a prickly pear cactus Opuntia sp. were present in the riparian area, while various other fruits (apple, fig, loquat, lemon and persimmon) were grown in the fruit garden. Near Parlier, CA (Fresno County), traps were placed within the University of California’s Kearney Agricultural Research and Extension Center that had multiple small fruit plots. Fourteen different orchard crops were used: apple, apricot, blackberry, blueberry, cherry, citrus, fig, grape, kiwi, mixed cherry and stone fruit, peach, pomegranate, plum and persimmon, with 42 monitoring traps placed in these fields (3 traps in each of the 14 fields). There was no replication of the different types of crops monitored. The fields did not receive any insecticides for D. suzukii, although pesticides were used for other pests. The stone fruit (peach and nectarine) fields were eventually dropped from the study because they were pulled out and the mixed cherry site was dropped because of insecticide sprays. All monitoring orchards were 0.2 to 3 km apart.
Field Monitoring Adult D. suzukii were monitored weekly from April 2013 to May 2014 in Courtland, Stockton or Brentwood, and from April 2013 to July 2014 in Parlier using traps baited with apple cider vinegar at 5% acidity (Great Value Apple Cider Vinegar®, Wal-Mart Stores, Inc., Bentonville, AR). The trap design was similar to 20 20 the “Haviland Trap” (Lee et al. 2012) with each trap made of a 750 ml Rubbermaid plastic container (Huntersville, NC) with a 7.5 cm diameter hole cut in the lid that was covered with 0.6 cm hardware cloth. Each trap was covered with a Pherocon trap cover (TRÉCÉ Inc. Adair, OK) to block direct sunlight or rain. In the field, each trap was filled with 300 ml of apple cider vinegar. The vinegar bait had one tablespoon of Bon-Ami Free and Clear® unscented soap (Bon-Ami Company, Kansas City, MO) added to each gallon (3.8 L) to serve as a surfactant. In each plot, three host plants were selected and on each plant a trap was hung at a height of 1–1.5 m above the ground and on the north side of the canopy. Traps were checked weekly. The liquid contents and collected insects were collected in separate containers and the traps were refilled with fresh apple cider vinegar. The samples were taken to the laboratory where the numbers of D. suzukii, all other drosophilids, and drosophilid parasitoids were recorded. All D. suzukii females were preserved in 70% ethanol for additional processing. From this material, a sub-sample of D. suzukii females from each trap at the Brentwood, Courtland and Parlier locations were dissected to determine mature egg load of female flies. Additionally, adult D. suzukii from Parlier and Courtland were examined at 30× magnification for the presence of a black capsule inside the fly’s abdomen to determine possible attack from parasitoids. Many larvae of Drosophila species are able to defend themselves from parasitoid eggs placed inside their body by surrounding the egg with blood cells that eventually melanize and form a black capsule surrounding the egg and resulting in the immature parasitoid’s death by asphyxiation (Chabert et al. 2012). This additional information was therefore collected to determine if resident parasitoids were attempting to attack D. suzukii, but might have been unable to develop from it due to the host’s immune response (Chabert et al. 2012, Kacsoh and Schlenke 2012). 21 21 Weather Data and Degree-Days Climatic data were obtained from the California Irrigation Management Information System stations located nearest to each sampled site: Station 140 (Courtland), Station 70 (Stockton), Station 47 (Brentwood), and Station 39 (Parlier). The accumulated degree-days over the adult fly’s activity range (10–30 °C), rather than daily maximum, minimum or average temperatures, were used to determine the effect of temperature on trap captures. The degree-days for each trap monitoring interval in each location were calculated with the double Sine Wave Method based on the low and high threshold of 10 and 30 °C, respectively, using the online program at the University of California Integrated Pest Management website (http://www.ipm.ucdavis.edu).
Data Analysis Data are presented as mean numbers of weekly trap catches of adult D. suzukii, other frugivorous drosophilid species or drosophila parasitoids for each sampled location and host plant. The weekly trap captures were analyzed to examine possible effects of the accumulated degree-days during the trapping period, presence of susceptible (mature) fruit in the field, and the interaction of both variables, using Generalized Linear Models with Poisson distribution and a log link function. Fruit ripening period and availability was determined based on our field observation during the trapping periods and the fruiting chart by Dave Wilson Nursery (http://www.davewilson.com) (Table A2). Simple correlation analysis was used to further examine correlation in the trap captures between any two adjacent or close sites in each location. Data were logit transformation before the analysis to stabilize the variation. Here we used the slope of the statistical association between trap captures in cherry orchards and other orchards surrounding the cherry orchards to infer source or sink effects, where a significant 22 22 negative association indicates a sink effect and a positive association indicates a source effect. Data of mean number of mature egg load per female and percentage of females without mature eggs per trap were pooled for each of three seasons from April to July, August to October, and December to March. The host crop species/trap site, trapping season and a possible interaction between these two variables were considered as factors for the analyses of their effects on the number of mature egg load per captured female and the percentage of females without mature eggs per trap, using Generalized Linear Models with Poisson distribution and a log link function for the numbers of mature egg load, and with a Binomial distribution and a logit link function for the percentage of female without eggs. All analyses were conducted using JMP software (JMP, V. 11, SAS 2013, Cary, NC).
Results
Adult Trap Captures From April 2013 to May 2014, there were a total of 4,412 traps deployed and successfully recovered from the field, from which 49,095 adult D. suzukii (range: 0–2,256 per trap per collection) and 1,232,038 other adult frugivorous fruit flies (range: 0–8,630 per trap per collection) were captured Adjusted for the number of days each trap was deployed, the season-long capture rate was 10.17 ± 1.15 adult D. suzukii trap per week (4.34 ± 0.44 females and 5.82 ± 0.72 males) and 275.38 ± 8.89 other frugivorous fruit flies per trap per week. Mean number of weekly captures varied among geographic locations, host plant ecosystems monitored and seasonal sampling periods. Across all host plants monitored, most fruit flies were captured at sites near Brentwood, with 96.93 ± 13.52 adult D. suzukii per trap per week and 331.08 ± 28.87 other adult frugivorous fruit flies per 23 23 trap per week (Table A3). In comparison, at the other three locations (Courtland, Parlier and Stockton) there were fewer adult D. suzukii (less than 10.3 per trap per week) and relatively similar numbers of other species of frugivorous fruit flies captured (Table A3). Different crops or site locations monitored cannot be statistically compared because of the varying management practices used. Still, there were clearly different adult D. suzukii trap captures at the organic sites located near Brentwood compared with the conventional sites located near Courtland, Parlier and Stockton (Table A4). For example, the year-long average trap capture at the organic cherry site near Brentwood was about 3 higher than the three cherry sites monitored near Courtland, 5 higher than the cherry site monitored near Parlier, 2.5 higher than the six cherry sites monitored near Stockton (Table A4). Also of interest, year-long counts of D. suzukii at the organic Knoll farm near Brentwood (labeled as ‘Fig’) averaged 342.9 ± 102.1 D. suzukii per trap per week. The traps were placed in a 12-tree block of figs, but the 5 ha organic farm had a rich mixture of susceptible host plants such as figs, apricots, plums, nectarines, cherries, citrus and a variety of vegetables. The second largest counts were at the three riparian sites near Brentwood that averaged 74.9 ± 11.5 D. suzukii per trap per week (Table A4). The primary host plants in these riparian sites were blackberry. Season-long captures of adult D. suzukii per trap per week at sites near Parlier were as low, with only 0.4 ± 0.1 in blueberry orchards and as high as 6.2 ± 1.0 in cherry and 3.4 ± 0.5 in citrus (Table A4). There were clear seasonal patterns in trap captures, which were influenced by host plant species monitored. Nevertheless, whereas there were differences among locations and host plant/site monitored, the seasonal capture patterns were similar in different crops or sites and across different regions, with two capture 24 24 peaks in the spring and fall and numbers of captured flies dramatically dropped during the hot summer or cold winter months (Figs. A1-A3, see Appendix A for Study 1 figures). In early season fruit such as cherry, the spring peak was higher than the fall peak. However, during the non-cherry season and in the middle or later season fruits such as apricot, plum, fig, apple, citrus or in the multiple fruit crop garden, the fall capture peak was much higher than the spring peak in all sites (Figs. A1-A3). Similarly high numbers of D. suzukii were captured in riparian sites during the spring and fall peak in Brentwood (Fig. A2). Generalized Linear Model analyses showed that the weekly mean number of adult captures in most sites and locations were affected by the accumulated degree-days within the adult fly’s active temperature range (10˗30 °C), and the presence of susceptible (mature or overripe) fruit, as well as the interactions of these two factors (Table A5). Correlation analyses showed that numbers of captured D. suzukii were positively correlated between two close sites (< 2 km) (Table A6). For example, in Courtland the numbers of captured D. suzukii between the cherry and the adjacent pear orchard were highly correlated. However, the correlation relationship shift from positive to negative with increasing gap between two fruit ripening seasons in Parlier (Fig. A4). Of captured D. suzukii from Courtland and Parlier (flies from Brentwood and Stockton were not examined), 10 adult (5 from each location) were found to contain a black capsule in their abdomen, i.e. field evidence of the immune capacity of D. suzukii against some resident larval parasitoids. The cider vinegar traps also captured drosophilids other than D. suzukii (Fig. A5), and some common drosophilid parasitoids (mainly the two common larval parasitoids, Leptopilina boulardi and L. heterotoma) (Fig. A6). The seasonal capture patterns of other drosophilids and parasitoids were similar to D. suzukii, with two capture 25 25 peaks in the spring and fall. More other drosophilids were captured in Brentwood or Stockton during the fall peak than in Courtland and Parlier (Fig. A5). More parasitoids were captured in Courtland than Parlier during the two peaks of catches (Fig. A6).
Mature Egg Load Dissections were made on 1,992, 1,331 and 2,185 adult female D. suzukii from Brentwood, Courtland, and Parlier, respectively. The mature egg load number was affected by the trapping season, but not the monitoring sites or interaction between these two factors in Brentwood or Courtland (Table A5). At these locations, the mature egg load was highest early in the season (April to July), decreased during late summer and fall (August to October) and was lowest during the colder weather from late fall to early spring (November to March), regardless the host plant monitored (Fig. A7). In Parlier, there was a large variation in mature egg load among D. suzukii captured in the different host plants monitored (Fig. A7) and, as a result, mature egg load per female was affected seasonally and by the monitoring site (host plants) as well as the interaction between these two factors. In all three locations, the percentage of females without mature eggs (y) was affected by the trapping season, but not the monitoring sites or interaction between these two factors (Table A5), and was negatively related to minimum daily temperature (x) (Courtland: y = 93.2 ˗5.6 x, r2 = 0.822, p < 0.001; Brentwood: y = 94.1˗5.4 x, r2 = 0.639, p < 0.001; Parlier: y = 80.2˗3.7 x, r2 = 0.710, p < 0.001) (Fig. A8). Of interest is that from these dissected females, 61, 50 and 51 females had at least one hatched, live larva in the uterus, suggesting facultative ovoviviparity, 26 26 although it is unknown if a live larva could be successfully deposited into host plant material.
Discussion The temporal and spatial distribution of the pest population must be understood to implement sustainable pest management strategies. The current study reports the seasonal occurrence of adult D. suzukii in major fruit crops and adjacent non-crop habitats in some regions of California’s San Joaquin Valley. Adult trap captures showed a population peak in the spring and another in the fall. High trap captures early in the season were most common in favorable host crops, such as cherries. The adult trap captures indicate the fly population utilized other crop systems (e.g., apricot, fig and pear) in mid-summer and early fall periods before moving to citrus and non–crop habitats during colder months in late fall through early spring. Therefore, the source (e.g., D. suzukii pest population increasing in or coming from) or sink (e.g., D. suzukii pest population decreasing in) potential of particular habitats could vary during the seasons due to changes in host suitability or fruit preference. For example, cherries would be the preferred host from color change through harvest, but a harvested orchard will not hold the pest population. Previous studies of D. suzukii in other North American locations showed adult abundance was also associated with temperatures in certain crops (Dalton et al. 2011, Harris et al. 2014, Wiman et al. 2014). Temperature is known to affect the population abundance of D. suzukii (Wiman et al. 2014). The estimated temperature range for immature development is from 10–30 °C (Tochen et al. 2014); adult D. suzukii are most active in temperatures ranging from 20–25 °C, and their activity is reduced when temperatures exceed 30 °C (Kinjo et al 2014, 27 27
Tochen et al. 2014). No reproductive behavior was observed during laboratory experiments where D. suzukii was kept for the entire life cycle at temperatures below 10 °C, and low levels of reproduction or no reproduction were found at temperatures above 30 °C (Mitsui et al. 2010, Tochen et al. 2014). In the California Central Valley of California, daily maximum temperature during the hottest months of the year (June – August) can exceed 40 °C, while the daily minimum temperature during the coldest months (December – January) can drop to 0 °C. However, the daily minimum temperature in the winter or the daily maximum temperature in the summer is typically conjunct with a few hours of favorable temperatures when adult D. suzukii may be able to move around and survive longer periods in the field when adequate food sources are available (Kacar et al. unpublished data). Although many other factors (e.g., harvesting, use of pesticides in the focal orchards and nearby orchards) could affect the adult population dynamics, we show that both temperatures and availability of host plants significantly affected the number of captured adult D. suzukii. These two factors can interact to influence the number of captured flies. Trends in catches in fruit trees with similar fruiting seasonality were similar in different regions and did reflect the effect of these two factors. However, the relative importance of climate and density-dependent factors could depend on local condition. The phenomenon of why D. suzukii adults appeared in many of those monitored crops during the seasons when fruits were obviously absent is not fully understood. For example, flies were found in fall in cherry orchards and in deciduous fruit orchards in winter. We propose several possible explanations for this phenomenon including adult food attraction, present of breeding hosts, and shelter for overwintering. First, adult D. suzukii may be attracted by volatiles from green plants (Cha 2012, Landolt et al. 2012). Plant leaves such as those of cherries 28 28 produce extrafloral nectaries that may provide a resource for adult D. suzukii at times of the year when suitable host fruit are not present for them to feed or host in. Yee and Chapman (2008) showed that the cherry fruit fly Rhagoletis indifferens Curran survived longer with cherry branches with leaves present rather than absent in field tests. Blossoms may also provide food for adult and larval D. suzukii as adults have reared from Japanese Snowbell (Styrax japonicas) blossoms collected from field in Japan (Mitsui et al. 2010). Second, damaged or overripe fruits may be still present in the field after harvest and D. suzukii can complete development in most of these common fruits (e.g., Bellamy et al. 2013, Stewart et al. 2014). A recent survey in the Central Valley of California found that D. suzukii emerged from field-collected damaged fruits of apricot, apple, fig, loquat, peach, pear, persimmon, plum, pomegranate, and prickly cactus, and laboratory tests further confirm that the fly can develop on these alternative crop hosts as well as some non-crop hosts (cherry plum, wild plum, prickly cactus) as they did in favorable hosts such as cherries (Wang et al. in preparation). These crops could also be used by the flies as adult food as a recent study showed that various fruit juices (e.g., pomegranate, grapes and oranges) could support adult D. suzukii longevity (Kacar et al. unpublished data). Captures of abundant other drosophilids in those orchards provided an indirect evidence of presence of breeding hosts. Third, in the case of winter aggregation patterns in citrus, i.e. higher trap catches of adult D. suzukii from citrus than other deciduous crop fields during the winter, the results suggest that citrus orchards are likely overwintering sites for D. suzukii in the Central Valley of California. It may be difficult to directly measure the movement of adult D. suzukii among different crops or between crops and non-crop habitats over the season as little is known of the fly’s dispersal range. Trap captures may not necessarily 29 29 reflect population density if the traps are not fairly evenly distributed over the studied areas (Byers 2012), or distributed in relatively small orchard blocks, as volatile compounds from neighboring blocks could compete with and/or mask the effect of the individual fruit species, while trap efficiency may also influence trap capture (Harries et al. 2014). Other environmental factors (i.e., climate and geography) and landscape traits (i.e., crop arrangement, connectivity, quality of habitat patches and propagule density) could all essentially affect insect dispersal (Mazzi and Dorn 2008, Zappalà et al. 2012). Strong correlation of catches between subsequently fruiting adjacent orchards and between orchard and adjacent non- crop habitats may reflect local population movement. Our results from the Brentwood site indicated a shift in location of D. suzukii out of crop fields and toward adjacent non-agricultural habitats. On a micro-geographic scale, for example in Parlier, positive correlations among trap catches were clearly related to proximity of fruiting as it was significant for crops fruiting subsequently, whereas this relationship was quite weak between the early fruiting (cherry) and late fruiting (Citrus) crops. Other approaches such as the use of distinct protein markers (Klick et al. 2014) or genetic markers with the recent availability of D. suzukii genome (Chiu et al. 2013) may aid to the understanding of local dispersal or long distance movement patterns of the fly in the future. Also, adult catches do not necessarily indicate the population dynamics, though they do indicate the temporal distribution of adult flies or aggregation patterns over the seasons. We found that captured D. suzukii females during the winter season had low mature egg loads and a high proportion of females did not contain mature eggs at all. This supports an early hypothesis that adult female D. suzukii may enter reproductive diapause when host fruit is not available during the later fall and winter seasons (Mitsui et al. 2010). In Japan, Mitsui et al. (2010) found that D. 30 30 suzukii populations migrated to higher altitudes for better host sources during the summer and were mostly composed of reproductively mature individuals, while those that returned to lower altitudes for overwriting were mainly composed of sexually immature individuals, suggesting that they entered a winter reproductive diapause. Most females captured during the fruiting period in most crops had mature egg loads. An alternative explanation may be that D. suzukii migrates into the orchards from other host crops. This also suggests that many fruit crops, especially cherries, can be very vulnerable as most D. suzukii females contained mature eggs during the fruiting seasons. Observed differences in egg load among different crops and locations over time could be due first to different depletion rate in different crops as flies would form mature eggs in advance of host availability, and then begin to lay eggs in suitable hosts, and the degree of depletion largely depend on the host availability. It is also possible that captured female D. suzukii may have lower egg loads due to differential attraction between traps and fruit, with reproductively mature females more likely to orient toward fruit in which to lay eggs than to traps emitting fermentation volatiles, assumed to be food attractants for insects. Most growers and researchers still use traps baited with apple cider vinegar or a combination of sugar-water and yeast to monitor adult D. suzukii (Cha et al. 2012, Landolt et al. 2012, Lee et al. 2012). The cider vinegar traps seemed to be non-specific, as we showed in this study that a lot of parasitoids and other drosophilids were captured. We also noticed captures of tephritids, beetles and other insects. Leptopilina boulardi and L. heterotoma are the two most common larval drosophila parasitoids that readily attack other drosophilids such as D. melanogaster (Hertlein 1986). These resident parasitoids could attack D. suzukii but are unable to develop from D. suzukii due to the host’s immune response 31 31
(Chabert et al. 2012, Kacsoh and Schlenke 2012). Although presence of a black capsule in their abdomen of captured D. suzukii was rare, it provides field-drawn evidence of the immune capacity of D. suzukii against resident larval parasitoids. The high number of captures of parasitoids suggests that the apple cider vinegar traps could cause non-target catches of beneficial parasitoids. Ovoviviparity occurs when fertilized eggs remain within the mother until they hatch or are about to hatch and is common in Diptera including drosophilids (Meier et al. 1999, Markow et al. 2008). The majority of dissected female D. suzukii found to be oviparous (i.e., contained only eggs), but some dissected female D. suzukii exhibited retention of one larva (always only one larva) and eggs, clearly showing that facultative ovoviviparity can occur in D. suzukii. The adaptive significance of ovoviviparity is still not well understood. It may benefit the population by shortening the developmental time or larval life thereby reducing exposure of the vulnerable immature stage to natural enemies, or facilitating accessibility to breeding substrates. Most drosophila researchers have observed that in many species, including D. melanogaster, females kept away from appropriate oviposition sites will retain fertilized eggs, hence exhibiting some degree of facultative ovoviviparity. In other Diptera, ovoviviparity is associated with a reduction in ovariole number and an increase egg size (Meier et al. 1999). The current observations do demonstrate facultative ovoviviparity in D. suzukii but the ecological mechanisms and in the degree to which it occurs is unclear and deserve further studies. In conclusion, the more favorable California climate and availability of fallen fruits such as citrus and fruiting ornamentals could enable D. suzukii to remain active and reproducing through the winter (Yu et al. 2013, Hamby et al. 2014, Harris et al. 2014, Stewart et al. 2014, Wiman et al. 2014, Wang et al. 32 32 unpublished date). Non-crop habitats may contribute to increasing or decreasing pest density in cultivated crops. An understanding of D. suzukii seasonal use of host plants and potential dispersal patterns would be of aid in the timing of and need for pest treatments, and may allow for implementation of alternative management strategies such as crop ‘border-sprays’, mass trapping or bait sprays, or possible targeting for future release of biological control agents to reduce the source populations in the environment. Management strategies to reduce D. suzukii populations in cherries may include lessening the source potential of certain crops and uncultivated habitats, or manipulating the spatial arrangements of source and sink habitats.
STUDY 2: FACTORS LIMITING PEACH FRUIT (PRUNUS PERSICA) AS A POTENTIAL HOST FOR DROSOPHILA SUZUKII 1
Abstract The spotted wing drosophila, Drosophila suzukii Matsumura, has widely established in North America and become an economic concern for a variety of fruit crops. To better understand fruit susceptibility, we evaluated peach surface characteristics on the pest’s oviposition success. The number of D. suzukii eggs laid into the fruit flesh was tested on (1) peaches with or without indumenta (commonly referred to as peach fuzz), (2) peaches physically damaged by harvest operations, (3) peaches damaged by the peach twig borer Anarsia lineatella Zeller or the fork tailed bush katydid, Scudderia furcata Brunner von Wattenwyl, and (4) peaches with punctures that simulated stink bug damage. Female D. suzukii did not lay eggs in intact fuzzy sections of the fruit or into small punctures (0.3 or 0.5 mm), but readily laid eggs in sections without fuzz, with insect damage, and with large punctures (1 mm). The number of eggs per treatment was positively related to the area of the damaged section; the overall fruit firmness and sugar content was not related to the number of eggs laid in treated or damaged spots. Direct observations of D. suzukii oviposition confirmed that peach fuzz appeared to be an obstacle for the fly’s oviposition success, and female flies ceased ovipositional attempts on fuzzy peach sections after a short period of time. Successful oviposition times were associated with substrate firmness, with shorter oviposition time in damaged spots than in cherry fruit or shaved spots of the peach. The results
1 This study has been published as Stewart, T. J., Wang, X.-G., Molinar, A., and Daane, K. M. 2014. Factors limiting peach as a potential host for Drosophila suzukii (Diptera: Drosophilidae). Journal of Economic Entomology 107(5): 1771-1779. DOI: http://dx.doi.org/10.1603/EC14197 34 34 indicate that intact, prior to harvest peach fruit are unlikely to be infested by the fly, but any surface damage could render the fruit susceptible to the fly.
Introduction Whereas several studies conducted on D. suzukii confirm that the drosophilid can infest numerous fruit crops, most biological studies on D. suzukii have focused on cherry and berry crops (Mitsui et al. 2006, Lee et al. 2011b, Burrack et al. 2013, Kinjo et al. 2013, Tochen et al. 2014). Previous studies suggest that many external (e.g., firmness) or internal (e.g., sugar content, pH values) fruit characteristics can affect the susceptibility of the fruit to D. suzukii (Mitsui et al. 2006, Lee et al. 2011b, Burrack et al. 2013). For example, D. suzukii laid more eggs in blueberry (Vaccinium corymbosum L. and Vaccinium virgatum Aiton) cultivars with softer rather than firmer fruits (Kinjo et al. 2013). Female attraction to susceptible fruits is likely queued by olfactory stimulus. Hamby et al. (2012) indicate that an association exists between D. suzukii and certain yeast species and that these yeasts may attract D. suzukii females for oviposition and feeding. Apart from chemical stimulus and requirements, physical characteristics of suitable host fruits might also facilitate oviposition, as was shown by Steffan et al (2014) with D. suzukii oviposition into wounded or unwounded cranberries. Many fruit species may be physiologically suitable hosts for D. suzukii growth and development, but are less likely infested by the fly simply because the adult fly is unable to successfully lay eggs in the fruit flesh (hereafter referred to oviposition success) due to the fruit surface characteristics such as a relatively hard (firm) or thick surface skin. In this sense, behavioral adaptation must precede the physiological adaptation in the evolution of host specificity of insects (Futuyma and Moreno 1988). Other than fruit firmness, the effects of other fruit surface 35 35 characteristics (e.g., trichomes) on the ovipositional success by adult D. suzukii, are largely unknown. Bellamy et al. (2013) evaluated D. suzukii to develop a relative ‘host potential index’ to numerous fruit hosts based on combined results from studies on larval performance on a fruit and agar diet, flight bioassay (adult fly attraction to host volatiles), population oviposition, and individual oviposition. They report peach (Prunus persica L.) ranked 5 of 7 fruit in total host potential, ahead of grape (Vitis vinifera L.) and blueberry (Vaccinium spp.), and below cherry (Prunus spp.), blackberry (Rubus spp.), strawberry (Fragaria x ananassa Duchesne), and raspberry (Prunus spp.). In their individual studies, a peach-agar mixture was a suitable diet for larval D. suzukii (1 of 7 fruits); however, in a multiple-choice test where the different fruits were simultaneously exposed to female D. suzukii they report that no adult D. suzukii emerged from exposed peach (7 of 7 fruits). The potential of a plant serving as a host might best be described using a hierarchal structure of an insect’s host location, oviposition behavior, and larval development rather than an accumulation of these criteria (Futuyma and Moreno 1988). A non-choice test and direct evaluation of oviposition behavior (determining the initial number of eggs laid rather than emerged adults) might be needed to determine if D. suzukii can oviposit on fresh peach, and the mechanisms that determine oviposition success or failure. Peach skin is covered by dense, short trichomes called indumenta (commonly referred to as peach fuzz) that could influence a fly’s oviposition success in fresh intact peaches. Other peach skin characteristics that influence D. suzukii oviposition might include feeding damage by other insects or animals that break the fruit surface and hand-picking during harvest operations that result in minor damage to the stem-end area of the fruit. Any surface damage could result in potential exposure of bare peach flesh to the 36 36 fly, and many of these wounds can be small and difficult to detect in post-harvest cull operations. We sought to determine various factors that could impact oviposition by adult D. suzukii on peach. Specifically, we examined the effects of peach fuzz and insect feeding or harvest damage on the fly’s oviposition success. We selected feeding damage by two common insect pests in stone fruit: the peach twig borer, Anarsia lineatella Zeller (Lepidoptera: Gelechiidae), and the fork-tailed katydid Scudderia furcata Brunner (Orthoptera: Tettigoniidae). Both pests can cause ‘grazing’ damage to the peach surface and the peach twig borer will also bore into the fruit; damage from these insects likely occurs from fruit color break to harvest (Bentley and Steffan 2001). We also simulated damage from some true bugs that can probe into ripening fruit using their needle-like mouthparts, typically causing little visible damage. Finally, we directly observed the fly’s ovipositional behavior on peaches with different types of surface characteristics, as well as on cherry (one of the fly’s preferred hosts) and on artificial diet substrate.
Materials and Methods
Insects and Fruit Tested Insect colonies were maintained at the University of California’s Kearney Agricultural Research and Extension Center (Kearney) in Parlier (Fresno County), CA. The D. suzukii colony was established from field collections of infested cherries at Kearney in May 2013. Thereafter, fly larvae were maintained on a cornmeal-based artificial diet, using similar methods as described by Dalton et al. (2011) and adult flies were held in Bug Dorm2 cages (BioQuip Products Inc., Rancho Dominguez, CA) supplied with a 10% honey-water solution as food. Both larvae and adults were held inside incubators at 23 ± 1 °C, 12L: 12D, 60-75% RH. 37 37
All adult female flies tested were between one and 2 weeks old and had been kept with males since emergence (and therefore assumed to be mated). The A. lineatella colony was established with larvae recovered from commercial peach orchards near Parlier, CA. The moth larvae were maintained on a bean-based artificial diet, as described by Krugner (2005) for the oblique banded leaf roller, Choristoneura rosaceana (Harris). Adults were held in BugDorm2 cages with pieces of felt cloth provided as an oviposition substrate for the adult moths. First instar peach twig borer were collected from the felt and placed into the bean-based diet. The fork-tailed katydid colony was established with katydids collected in commercial peach orchards near Visalia, CA (Tulare County) and thereafter maintained either on peach, citrus, almond, or grape, depending on their seasonal availability, in a greenhouse where temperatures fluctuated from a 30 °C maximum in summer to a 5 °C minimum in winter. The colony was exposed to 20–50 potted plants at any given time to maintain the colony density between 100- 400 adult katydids. The yellow-flesh peach fruit cultivar ‘Earlirich’ was used for most tests; Earlirich is one of the most common varieties grown in California’s San Joaquin Valley. When harvested, the fruit is relatively firm and features an acidic flavor. Two other cultivars ‘Spring Snow’ and ‘Babcock’, both white-flesh peaches, were also used. Relative to Earlirich, Spring Snow has longer and heavier hairs on the fruit surface while Babcock has less fuzz. Fruit tested were collected fresh from orchards at Kearney that had not received any insecticides during the fruiting season, but that had received a dormant insecticide sprays for peach twig borer (the insect growth regulator diflubenzuron) and mites (the pyrethroid esfenvalerate). 38 38
The tested fruit were hand-picked using a pruning shear to cut them off just above the stem-end; unless otherwise stated, fruit collections were made during commercial harvest periods in order to match fruit ripeness in our study with that in a commercial operation.
Fruit Surface Damage and Test Procedures All hand-picked fruit were carefully examined and sorted into ‘intact fruit’ (no visible signs of damage) or harvest-damaged fruit (a small amount of the peach skin, near the petiole, was removed during the harvest operations). Randomly selected intact fruit were also manipulated to create the following six types of fruit surface treatments: (1) Intact fruit: Hand-picked fruit without any visible damage. A total of 45 Earlirich and 21 Babcock fruit were tested, respectively. (2) Shaved fruit: Hand-picked and intact fruit were selected and a small section of peach fuzz on each fruit was then removed using a razor and wet tissue paper. The length and width of the shaved area was 3.0-3.5 cm. To reflect the relative size of shaved vs. unshaved surface area, the length (L) and width (W) of each tested fruit were also measured using a caliper (to nearest 0.1 mm), and the surface area (SA) of each fruit (prolate spheroid) was calculated as SA = 2×p×W2 + 2×p×L×W×arcsin (E)/E, where E = [(1-W2/L2)1/2] (Weisstein 2014). A total of 44 Spring Snow fruit were tested. For this test, we compared the intact and shaved sections only within the Spring Snow fruit. (3) Harvest damage: Hand-picked fruit with a small amount of the peach skin, near the petiole, that had been removed during the harvest operations. A total of 26 Earlirich and 15 Spring Snow fruit were tested, respectively. 39 39
(4) Peach twig borer damage: Intact fruit that were picked using a pruning shear, thereby keeping the petiole intact to better mimic fruit on the tree, were individually exposed to peach twig borer larvae (2nd to 4th instar) for 1-2 d. The use of differently sized larvae and exposure periods was intentional to produce fruit with variable sizes of damaged area. The shape of feeding damage by peach twig borer was irregular; therefore, a relative measure of damage size was taken by measuring the maximum length and width of each damaged section with an ocular micrometer. A total of 33 Earlirich fruit were tested, with a combined total of 68 feeding wounds (each fruit had 1-3 separate feeding wound sites). (5) Katydid damage: Intact fruit were picked using a pruning shear, as described previously, and then exposed to 30 katydid nymphs and adults in a large (30 ´ 30 cm) screened cage for a 24 h period. The maximum length and width of each damaged area were measured, as described previously. A total of 35 Earlirich fruit were tested, with a combined total of 78 feeding wounds (individual fruit had 1-8 wounds). (6) Puncture damage: To simulate hemipteran feeding damage, intact fruit were punctured using 0.3, 0.5, and 1.0 mm diameter-sized needles. The three puncture sizes were lined up within a 1 cm area near the shoulder of each fruit, with the order of each puncture size random. A total of 21 Earlirich fruit were tested (each with the three different puncture sizes). Additionally, 10 randomly selected and newly laid fly eggs from the colony were measured for their length and width with an ocular micrometer to be compared with the size of the punctures. Because the intent of the experiment was to determine only if these treatments facilitate oviposition, and not which type of damage is preferred by female D. suzukii, no choice oviposition tests were conducted for each fruit 40 40 treatment using similar procedures. For each trial, immediately after the treatment preparation, fruit were exposed to female flies (2–4 flies per fruit) in a BugDorm2 cage for a 24 h oviposition period; cages were provisioned with a 10% honey- water solution as food for the adult flies. Each exposed fruit was then checked thoroughly under a microscope to record the location and number of eggs laid. Here, we define a successful oviposition when an egg was vertically inserted into the fruit flesh with the egg filaments visible, but an aborted egg if the entire egg was horizontally placed over the fruit surface as previous observations showed that larvae from these eggs rarely survived. Three different locations (damaged or treated spots, stem-end region, and the rest of fruit) of the eggs on the fruit surface were recorded. All tests were conducted under controlled conditions (23 ± 1°C, 16L: 8D, 40–60% RH). For each fruit tested, brix (sugar content) and fruit firmness (surface penetration force) were measured. A portable refractometer (ATC-1E Brix 0–32%, ATAGO USA Inc., Bellevue, WA, USA) was used to measure the sugar content. A piece of fruit from the cheek area (middle section or the widest part of the fruit) was removed and juiced to measure brix. Surface penetration force was measured using a penetrometer with 1 mm test tip (L-500 g, 5 g/Div., AMETEK Inc., Berwyn, PA, USA). A value of 100 g mm-1 indicates that 100 g of force is needed to penetrate the 1 mm diameter section of fruit surface. Preliminary tests of 32 ripe Earlirich fruit used five readings at the stem-end (an area about 2 cm around the petiole), top shoulder, cheek, bottom shoulder, and bottom (or tip) of each fruit. The results found that the stem-end area was softer (174.4 ± 8.7 g) than other surface areas of the fruit, which were not significantly different from each other and that combined required an average 293.0 ± 11.9 g penetration force (t-test, t = 8.1, df = 31, P < 0.001). Therefore, one reading was taken from the middle of 41 41 stem-end area to represent the firmness of the stem-end area, and three readings were taken from the area between the shoulder and tip, which were averaged to represent average fruit firmness.
Fruit Brix and Firmness Additionally, for shaved fruit and harvest-damaged fruit tests, fruit with a range of different sugar content and firmness levels were used (additional fruit were collected after the commercial harvest dates). For the shaved fruit and treated fruit sections, the fruit firmness on the shaved or damaged spot was also measured after the count of eggs. Therefore, fruit brix levels and firmness based on three readings taken from the intact areas as mentioned previously were used to determine whether these two characteristics were correlated with D. suzukii oviposition.
Ovipositional Behavior Direct observations on the ovipositional behavior of adult female D. suzukii were made and the success or failure of each oviposition attempt was recorded when the adults were presented treatments of artificial diet media (provided in a 2 ´ 8 cm Petri dish), cherry fruit (Rainier cv.), intact peach fruit, harvest-damaged peach fruit, and shaved peach fruit (all peach fruit were Earlirich cv.), respectively. For each observation, artificial diet media or tested fruit were first placed inside a plastic cage (30 ´ 30 cm) with 20-30 adult female flies. After the fly landed on the tested substrate, oviposition behaviors were viewed using a stereomicroscope for closer observation of the behaviors. The entire process was timed for each successful oviposition, from probing to the withdrawal of the ovipositor. For each treatment, 15–30 observations were completed. After each observation period, the brix and firmness of the tested substrate were measured, as described previously. 42 42 Data Analysis Data are presented as treatment means (± SE) for the number of eggs laid, brix, and fruit firmness; each fruit or damage patch is considered a replicate. Because the type of damage was different for the different treatments, numbers of eggs per treated or damaged area were compared with the intact fruit, as well as the intact area of the damaged fruit, using Analysis of Variance (ANOVA). For peach twig borer and fork-tailed katydid treatments, the relationships between numbers of eggs and damaged area (based on maximum length and width of areas damaged) were analyzed using linear regression. Ovipositional times on different fruits or artificial substrates were compared using survival analysis (log-rank test). If a female fly did not successfully complete oviposition, the observation was considered as censored data. If the overall log-rank test for the survival analysis was significant between group comparisons, the significance of each paired comparison was adjusted to a table-wide level of 5% using the sequential Bonferroni adjustment. Unsuccessful ovipositional attempts (i.e., giving-up time) were also compared using the survival analysis. All analyses were performed using JMP V10 (SAS 2010, Cary, NC, USA).
Results
Fruit Tests Drosophila suzukii eggs were not found on intact fruit or any intact- sections (e.g., fuzzy and undamaged) of tested Earlirich, Babcock, or Spring Snow cultivars in any of the treatments (shaved, harvest damaged, insect damaged, or puncture damaged fruit) (Fig. B1, see Appendix B for Study 2 figures), with the exception of the less fuzzy stem-end region. Female D. suzukii readily laid eggs 43 43 into the shaved area of every tested fruit (Fig. B1), and no egg was found on the rest of the fruit surface (i.e., unshaved area), even though the average shaved area (10.8 ± 0.6 cm2, n = 44) was less than 10% of the total surface area of the fruit (116.9 ± 2.9 cm2, n = 44). Female flies also laid eggs into stem-end sections damaged by harvest operations, peach twig borer and fork-tailed katydid feeding damage, and large (1.0 mm) puncture wounds (Fig. B1). No eggs were found in smaller (0.3 and 0.5 mm) punctures (Fig. B1). The number of D. suzukii eggs showed a positive relationship with maximum width of the damaged section by peach twig borer or fork-tailed katydid feeding (Fig. B2). There was a similar relationship to maximum length of the damaged area (peach twig borer: y = 0.039 + 0.903x, r2 = 0.298, P < 0.001 and fork-tailed katydid feeding: y = - 0.032 + 0.713x, r2 = 0.204, P < 0.001). Eggs were found in sections with > 0.5 and > 0.9 mm wide damage by peach twig borer and fork-tailed katydid, respectively. Note that the mean length and width of each newly laid D. suzukii egg were 0.594 ± 0.004 mm and 0.212 ± 0.004 mm, respectively (n =10).
Test fruit varied in sugar content (F7, 317 = 20.4, P < 0.001) and firmness
(F7, 317 = 53.8, P < 0.001) across different treatments or within each treatment, especially within the shaved or harvest-damaged treatments because of the variable harvest dates for these trials (Table B1, see Appendix B for Study 2 tables). Within each cultivar, sugar content generally increased with decreased firmness in Earlirich (brix = 10.9 - 0.007 firmness (g) (n = 150, r2 = 0.136, P < 0.001), but the relationship was not significant for Babcock (n = 21, r2 = 0.027, P = 0.515) or Spring Snow (n = 59, r2 = 0.008, P = 0.485). The mean firmness between the shaved (90.1 ± 7.4 g) and unshaved (84.7 ± 6.3 g) areas were not significantly different (F1,86 = 0.290, P = 0.592). Considering the overall peach 44 44 condition (i.e., outside of the damaged area), there was no relationship between the number of eggs laid in the treatment area and fruit brix or firmness (Table B2), with the exception of the fork-tailed katydid treatment and fruit firmness where the linear regression was significant but basically a flat slope (y = - 0.009 + 0.012x) indicating no positive or negative relationship. Drosophila suzukii eggs were found in intact stem-end region; however, this region near the petiole often has little or no trichomes (observed rather than measured) and these oviposition events were not common even though the experimental design created extreme pest pressure. Across all treatments there were 133 eggs found on 22 out of 230 tested fruit. The stem-end area was also about half as firm as the other fruit sections tested in all three tested varieties (Fig. B3); however, fuzz may have been a determining factor as all 22 fruit with eggs inside the intact stem-end area varied widely in firmness (157.2 ± 22.4 g, ranged 5.0-367.5 g). Aborted D. suzukii eggs were found over the fruit surface but rarely on damaged, shaved or punctured spots. These aborted or misplaced eggs were not common. Across all treatments, 218 aborted eggs were found on 50 fruit; the majority of them were found on intact (66 eggs on 20 fruit) or harvest-damaged (154 eggs on 24 fruit) fruit, and only were a few found on shaved (4 eggs on 4 fruit) and insect damaged fruit (3 eggs on 2 fruit).
Ovipositional Behavior After landing on cherry fruit, a female D. suzukii first walks around the fruit surface, possibly searching for a suitable place for egg deposition. She then extends her ovipositor, curls her abdomen inward, and probes the fruit surface for 5-10 s. The complete oviposition process generally consisted of two phases: 45 45 scratching (i.e., breaking the fruit skin) and insertion (depositing the egg into the fruit flesh). Once the female fly settles on an oviposition point, her ovipositor is angled so that only the end or point of each blade remains in contact with the fruit skin. She quickly alternates the movement of the left and then right blade of her serrated ovipositor in a lateral direction, at a rate of about two pumps per second, until the fruit exocarp breaks. Afterwards, she begins to spread and contract her ovipositor to insert one egg into the fruit mesocarp (flesh). The serrated section of the ovipositor is used only to break the skin and create a puncture deep enough for about one quarter of the egg, which is then pushed completely into mesocarp with only the oxygen filament remaining outside of the fruit exocarp. After the egg is inserted, the female slowly retracts her ovipositor and straightens her abdomen releasing the remaining portion of oxygen filament. The oviposition behavior observed on cherry is similar to that on a shaved or damaged peach section or artificial diet. On the artificial diet and damaged peach sections, there was less obvious surface-scratching behavior and the female inserted her ovipositor almost immediately after probing. Also, the female inserted her ovipositor sheath much further into the peach flesh than the cherry flesh, possibly to create more space for the egg. This is accomplished by alternately spreading and sawing the peach skin with her ovipositor, a more intensive behavior that is likely necessary due to the increased density and firmness of peach compared with cherry flesh. All observed females successfully completed oviposition on artificial diet media or damaged sections (Table B3). Some females ceased an ovipositional attempt on cherry (3 out of 21) or shaved peach (13 out of 30), but no female successfully completed oviposition on the fuzzy (intact) sections of a peach.
Cherry not only had higher sugar content (F3, 91 = 471.8, P < 0.001) but also softer surface (F2, 64 = 122.4, P < 0.001) than peach, whereas the pressure needed to 46 46 penetrate the surface of artificial diet media or damaged peach was so slight as to be unmeasurable with the penetrometer used (Table B3). The difficulty of ovipositing on peach skin naturally covered with indumenta was apparent by how rapidly flies ceased oviposition attempts on intact peach sections (85.6 ± 28.2 s, n =15) compared with shaved sections (221.1 ± 46.1 s, n =15) (Fig. B4(A)). In those trials with successful oviposition, the individual ovipositional time varied among the tested substrates, which correlated with substrate firmness. Female flies took a longer time to complete oviposition on the shaved peach than on cherry, likely because the flesh of the peach is more fibrous and dense than a cherry (Table B3, Fig. B4(B)). The ovipositional time was shorter in artificial diet media or in a damaged peach section than on cherry or shaved section of the peach.
Discussion Our results suggest that harvestable peach fruit are unlikely to be attacked by D. suzukii largely because of their fuzzy surface that discourages or prevents successful oviposition. This was further evidenced by the fly’s rapid cessation of ovipositional attempts and deposition of poorly placed eggs on the intact peach surface. Under our experimental conditions with numerous flies enclosed with a single fruit, which created extreme fruit fly pressure for oviposition, adult D. suzukii occasionally laid eggs in the stem-end region near the petiole where there was less peach indumenta. We also showed that peach surface damage by insects feeding or harvest operations can facilitate D. suzukii oviposition. The combined results support the findings of early work by Kanzawa (1939) that lists peach as a D. suzukii host, but also supports the recent work by Bellamy et al. (2013) that found no D. suzukii emerging from exposed peach fruit. The peach surface might be the most important defense from D. suzukii oviposition, with intact fruit being a 47 47 poor host but wounded fruit being an acceptable host. Similarly, Steffan et al. (2013) showed that cranberries with a 5 mm deep surface wound were an acceptable host but unwounded cranberries did not support D. suzukii regardless of the fruit ripeness. Although in this cranberry study the authors suggest that a surface wound and decay were both necessary to support D. suzukii development, whereas in our study with fresh peach only the surface wound is needed. In fact, in cases of extreme pest pressure there can occasionally be successful oviposition in peach near the petiole where there is less of the obstructive fuzzy surface. We created scenarios whereby susceptible peach fruit might be exposed to D. suzukii just prior to harvest, using treatments that included intact, shaved, puncture-damaged, harvest-damaged, and insect-damaged fruit. Although the brix or firmness levels varied widely among cultivars as well as among treatments within each cultivar tested, we did not find significant relationships between the numbers of eggs laid in treatment sections and the overall peach condition (i.e., sugar content and firmness outside of the treated spots). Measurements of brix and firmness levels have provided mixed results in other studies. Burrack et al. (2013) modified the water to agar medium concentration of artificial diet to create a range of surface penetration forces and brix levels across test plates. Their research showed that no D. suzukii egg was deposited in the medium with the highest penetration force and brix level, but the relationship was not consistent across the four treatment levels measured. Lee et al. (2011b) reported that the number of D. suzukii eggs increased as brix levels increased within tested berry cultivars and Kinjo et al. (2014) reported that D. suzukii laid more eggs in blueberry cultivars that had softer rather than firmer fruit. Laboratory studies also showed that D. suzukii prefers ripening cherries to ripe ones and this may increase the chance of the larvae to fully develop and reach maturity before the fruit totally decays 48 48
(Poyet et al. 2014). In our studies, we suggest that the presence (intact) or absence (damaged) of peach fuzz has a greater influence on oviposition than the brix or firmness of the peach fruit at a harvest-time ripening stage. We present two explanations on why the numbers of eggs laid in treated spots were not related to the overall peach sugar content and firmness. First, peach fuzz may be the determining factor for the fly’s ovipositional site selection and oviposition success. In the shaved study, there was no difference in the firmness between shaved and un-shaved sections and the firmness of tested fruit within the treatment varied widely, but no eggs were found in the unshaved treatment. Second, the flies may be overwhelmingly attracted to fruit odor from the damaged spots, as shown in other studies (Yu et al. 2013). In trials with insect-damaged (peach twig borer and fork-tailed katydid) and harvest-damaged fruit, fruit firmness in the damaged sections was so soft that it could not be accurately measured with the penetrometer and brix level of these matured fruit may be too similar to be an oviposition determinant. In general, the size of the damaged section also played a role in D. suzukii oviposition, and the numbers of D. suzukii eggs positively increased with the damage wound size. Our direct observations confirmed that female D. suzukii use their serrated ovipositor only to break the fruit skin and then insert a single egg vertically into the fruit flesh. We would expect that the wound width must be larger than the egg width (0.212 ± 0.004 mm). The size of puncture wounds by hemipteran feeding would depend on the insect species, but the proboscis diameter of most stink bugs is < 0.3 mm. For example, the leaf footed bug Leptoglossus clypealis Heidemann is a common hemipteran pest in California orchards, and adult L. clypealis have a proboscis diameter of 2.0 ± 0.03 mm (n = 10, Wang et al. unpubl. data). Drosophila suzukii eggs were not found in small punctures wounds 49 49
(0.3 and 0.5 mm) that were still larger than the width of D. suzukii egg. It is possible that the large puncture (1 mm) may be more attractive to the fly than the small punctures because of the volatiles produced. Whereas there is no literature, that we are aware of, describing the importance of the size of the damaged area for D. suzukii oviposition, we note again that peach is a non-preferred host because of the surface skin and fuzz and that in preferred hosts (i.e., cherries) fruit damage is not needed to create an oviposition site. Furthermore, many other uncontrolled factors (e.g., the nature or degree of different damage) could have affected the number of eggs laid in the tested peaches. Poorly deposited (or aborted) D. suzukii eggs were found on intact peach fruit. As far as we are aware, other studies of D. suzukii oviposition do not report misplaced eggs occurring on fruit of the more susceptible host crops (Lee et al. 2011b, Burrack et al. 2013, Kinjo et al. 2013, Poyet et al. 2014, Tochen et al. 2014). We suggest that these misplaced eggs are more common in less preferred hosts such as peach, and this placement may reflect largely an unsuccessful attempt of the female flies to lay eggs into the fuzzy surface. The current study has practical applications for D. suzukii management. The presence of D. suzukii in peach is unlikely, as both Bellamy et al. (2013) and this study suggests. To further reduce the possibility of infested peach fruit, additional control programs for other insect pests should be applied – and this is already in place for most commercial operations. Harvest damage to the fruit’s stem-end area could also be a potential oviposition site, and these fruit should also be culled, but the time period between harvest and cold storage is so short that the window of opportunity for oviposition is quite narrow. Rapidly moving harvested fruit in bins from the field to storage will further reduce any opportunity of oviposition. Finally, peach trees are often harvested two or three times, typically 4–7 d apart to allow for fruit to ripen in 50 50 different parts of the tree. Care should be taken during harvest operations to reduce damage to the remaining fruit, even the abrasive removal of peach fuzz. Current peach Integrated Pest Management Programs do indeed utilize these insect control programs, as well as packing procedures to cull damaged fruit, and such efforts will decrease any risk of D. suzukii oviposition in peaches. Finally, the host potential index (HPI) proposed by Bellamy et al. (2013) used a combination of results from several independent studies, with the HPI index placing harvestable peach as more susceptible than blueberry and most similar to cherry. We focused on a hierarchal system whereby the drosophilid must first successfully oviposit into the fruit before larval development becomes important. We suggest that the most important role played by peach orchards in D. suzukii control programs may be the post-harvest utilization of damaged, unharvested peaches left in the orchard that serve as a population resource for D. suzukii populations before moving into more susceptible host fruits.
STUDY 3: ORANGE (CITRUS SINENSIS) AS A POTENTIAL OVERWINTERING HOST: ADULT SURVIVORSHIP AND FECUNDITY IN A CITRUS ORCHARD
Abstract The overwintering survival and development of Drosophila suzukii Matsumura were investigated in field-cages in California’s interior fruit growing region. Experimental treatments consisted of exposure of D. suzukii eggs, larvae, pupae, and adults to winter conditions in cages as well as the burial of pupae in the soil. Results found eggs exposed from late November to January did not survive but a low percentage of larvae or pupae (< 3%) survived and developed into adults. Survival of pupae was higher in the soil (buried) than on the tree (caged). From late January to March, the majority of the eggs, larvae and pupae survived and successfully developed into adults. Adult D. suzukii survived less than one week without food or with water only. Adult female D. suzukii that emerged during the field tests were able to survive and produce eggs when provided honey- water and sliced oranges as food. Adult survival varied among the food provision treatments and exposure periods, and was lowest in the 27 December water only trial (3.4 ± 0.9 d) and highest in the 27 December honey-water trial (44.1 ± 3.0 d). Adult female longevity was reduced when trials were launched in March, coinciding with increased ambient temperatures. D. suzukii readily oviposited into damaged fresh or rotting orange fruit and was able to completely develop on both. We discuss factors potentially influencing overwintering survival of D. suzukii in fruit-growing regions in California’s interior valleys
Introduction Despite its wide geographic distribution in native and invaded ranges, the field biology of spotted wing drosophila, Drosophila suzukii Matsumura, has only 52 52 recently been studied, and its overwintering survival is still relatively poorly understood. In central Japan, Kanzawa (1939) suggested that D. suzukii might overwinter as adults. Mitsui et al. (2010) found that the fly migrated from low altitudes to high altitudes during the summer seasons likely seeking out better host sources, but returned to the low altitudes during the winter seasons, possibly for more favorable environmental conditions to overwinter. These same researchers report that adult D. suzukii populations at higher elevations were composed primarily of reproductively mature individuals, whereas those at lower altitudes were composed primarily of sexually immature individuals possibly entering a winter reproductive diapause. Temperature appears to be key factor in adult D. suzukii flight, overwintering and reproductive behaviors. Adult D. suzukii are most active between 20 to 25 °C (Kinjo et al 2014, Tochen et al. 2014). In cold regions such as on the northern island of Hokkaido, Japan, where average temperature in winter is below 0 °C, it is unclear whether individuals of this species may overwinter in human-protected warm places, or migrate from the warmer southern regions (Kimura 2004). In Oregon, Dalton et al. (2011) reported that acclimated adult D. suzukii were unable to survive more than 17 d at 1 °C, and the fly could only survive 7 d after exposure to -2 °C. Adult longevity was positively correlated with temperatures from 1˗10 °C, and flies are estimated to survive for up to 103–105 d at 10 °C. Long-term survival of D. suzukii seems to be unlikely at constant temperatures below 10 °C. The estimated suitable temperature range for immature development is within 10–30 °C based on laboratory studies under constant temperature conditions (Tochen et al. 2014). Egg, larval, or pupal D. suzukii were found to not survive temperatures below freezing (CFIA 2011). However, D. suzukii is cold and heat tolerant relative to other drosophilids, and the low and 53 53 high lethal temperatures are -0.9 and 32 and °C, respectively (Kimura, 2004). D. suzukii may be able to rapidly evolve tolerance to local temperatures (Kimura 2004). No reproductive behavior was observed during laboratory experiments where D. suzukii was kept below 10 °C, and little to no reproduction occurs above 30 °C (Mitsui et al. 2010, Tochen et al. 2014). Mated females lay infertile eggs when exposed to temperatures above 31 °C (Kinjo et al 2014). It is therefore, essential to understand the overwintering survival of D. suzukii under the local ecological conditions. Overwintering survival is one of the major climatic obstacles to many invasive species successful establishment in new geographic ranges (e.g. Wang et al. 2013). It is unclear if immature D. suzukii can slowly develop in the winter conditions in the California’s Interior Valleys, or if adult D. suzukii can survive long periods when hosts or food sources are not available. Sugar sources are often critical for the survival of adult insects, including fruit flies (e.g. Siekmann et al. 2001, Wang et al. 2011). High captures of adult D. suzukii in citrus orchards suggest that citrus may provide food and an overwintering site for adult D. suzukii (Harris et al. 2014, Haviland et al. 2015), yet it is still unclear if D. suzukii can successfully overwinter in San Joaquin valley citrus, or if the fly can successfully reproduce in citrus fruit – which is not considered to be a host that is economically damaged by D. suzukii. The major aim of this study was therefore to investigate the overwintering survival of D. suzukii life stages in California’s San Joaquin Valley, and to understand the potential role of citrus in D. suzukii’s overwintering survival and reproduction. This work provides information critically needed to better understand the fly’s spring population densities and reproductive and dispersal potentials in susceptible cash crops. 54 54 Materials and Methods
Insects A laboratory colony of D. suzukii was maintained under controlled room conditions (23 ± 1°C, 12L: 12D, 4075% RH) at the University of California’s Kearney Agricultural Research and Extension Center (Kearney) near Parlier (Fresno County), CA. The D. suzukii colony was established from field collections of infested cherries at Kearney in May 2013. Thereafter, fly larvae were maintained on a cornmeal-based artificial diet, using similar methods as described by Dalton et al. (2011); adult flies were held in Bug Dorm2 cages (BioQuip Products Inc., Rancho Dominguez, CA) supplied with a 10% honey-water solution as food. Laboratory tests were conducted under these same conditions. Field- collected D. suzukii from Kearney were introduced into the colony periodically to maintain colony vigor. All insects tested were derived from this colony.
Overwintering Survival Field-cage experiments were conducted in a Kearney citrus orchard to determine, under San Joaquin Valley ambient late fall and winter temperatures, immature D. suzukii development; adult survival, longevity and reproductive potential, and pupal survival in the soil versus more exposed locations on the citrus tree. The treatments consisted of exposure of D. suzukii eggs, larvae, pupae, and adults in cages made of clear plastic storage containers (81119 cm). Each cage had a 70 cm² hole cut in both sides of the cage and was covered with organdy cloth to allow ample air flow, so that the inner temperature of the containers was as close to ambient as possible. For the pupal survival trial, pupae were also buried in the soil at a depth consistent with natural populations. 55 55
Immature development. The same procedures were initiated eight different times to test a range of winter temperatures and conditions. Every 2 weeks from 22 November 2013 to 28 March 2014 a new trial was launched in the field (eight trials). Treatments were different immature stages: eggs, larvae or pupae, Trials were the eight different start times. Each trial had 10 replicates per treatment and each replicate consisted of 10 individuals placed in a narrow drosophila vial (25 ×95 mm polystyrene, VWR International, Radnor, PA) provisioned with artificial diet and capped with a foam plug. For each replicate, each treatment (three separate vials) was placed into an organdy cage, with each of the 10 cages hung on a different tree in the east side of the citrus canopy (1.5 m high) with a Pherocon trap lid (Trece Inc., Adair, OK) fitted over the cage to protect it from direct rain and sun. Coinciding with each trial, soil-buried pupae were also tested. For each replicate, 10 young (< 24 h old) pupae were transferred to a drosophila vial with artificial diet, and the vials (caped with a foam plug) were buried 5˗10 cm deep in the soil underneath the canopy of each tree with a cage.
Adult survival, longevity, and fecundity. Treatments were different food provisions: (1) no food or water, (2) water only, (3) 10% honey-water only, and (4) 10% honey-water and a halved orange. For each of three replicates per treatment, 25 female and 10 male flies (< 24 h) were released into an organdy cage hung on individual citrus trees similar to the immature stage tests. In the cages, the water or honey-water was kept in reservoirs with cotton wicks, which were refilled as needed. The oranges were picked from the citrus trees on the same day each trail was initiated and placed in the cage after the rind was removed and the fruit halved orange was fitted into a small plastic cup to expose flies to a possible food 56 56 or/and ovipositional medium. The first trial was initiated on 19 November 2013 and was repeated every month until 28 March 2014 (a total of five trials). Both immature and adult trials were launched at 1100 hours to allow the flies to acclimate to the ambient temperatures. Both immature and adult treatments were checked Monday, Wednesday, and Friday for the duration of each trial. For the immature tests, the numbers of individuals that successfully developed to the next stage were recorded on each sample date. For the adult tests, dead flies were recorded and removed on each sample date. Orange pieces were replaced every Monday and the numbers of eggs laid or larvae that developed on the exposed pieces were counted under a dissecting microscope in the laboratory. All adult flies that developed from the immature stage tests were collected and released into cages in the laboratory, provisioned with honey-water and a sliced orange, to determine if these flies could successfully reproduce after their overwintering period. The study ended in late April when there was no longer fly emergence or adult survival.
Host Suitability of Citrus Fruit To determine if D. suzukii would oviposit and develop from damaged or rotting navel orange fruit (cv. Thomson Improved), individual adult female D. suzukii were exposed to treatments of (1) one whole fresh fruit, (2) one halved fresh fruit, (3) one rotted whole fruit or (4) one halved rotted fruit for 24 h. The test arenas were small (1111 cm) plastic containers that were covered with fine organdie screened lids. To simulate the natural decaying process of fallen oranges, fresh oranges were placed individually over wet sandy soil in the containers until the fruit began to rot (approximately 1 month under the laboratory conditions). The halved fruit were treated similarly but were then sliced in half just prior to 57 57 each test. Following the exposure period, the numbers of eggs laid on the fruits were counted and the fruit was kept in the container until the emergence of adult flies. There were 25 replicates per treatment, although a few replicates were discarded because of contamination from other drosophilid species. A sub-sample of 10 fruit was measured to determine the brix levels of the fresh or rotting fruit.
Data Analysis Data are presented as treatment means (± SE) for the percentage of immature stages that developed into adults and the number of offspring produced per female or brix level of tested fruit. Treatment effects were compared using one-way or two-way ANOVA. Prior to analyses, percentage data were arcsine square-root transformed as needed to normalize the variance. All longevity data were subject to survival analyses and data were pooled from different replicates for each treatment. If the overall log-rank test was significant, a paired test of any two groups was made, with the significance of paired comparisons adjusted to a treatment-wide level of alpha = 0.05 using the sequential Bonferroni adjustment. All analyses were performed using JMP V11 (SAS 2011, Cary, NC, USA). Climatic data were from the nearest California Irrigation Management Information System (CIMIS Station 39, Parlier, CA).
Results
Overwintering Survival Percentage of immature D. suzukii successfully developed into adults in the field cages was affected by the trial period, the fly’s developmental stage, and the interaction between these two factors (trial period: F 7,288 = 163.7, P < 0.001; immature stage: F 3,288 = 27.8, P < 0.001; interaction: F 21,288 = 7.1, P < 0.001). 58 58
When the trials began in November and December, no eggs developed and a low percentage (< 3%) of larvae and pupae developed to adults (Fig. C1(A), see Appendix C for Study 3 figures). In general, the percentage of developed pupae was higher when buried in the soil burial than on the citrus foliage, with 31% and 24% of the soil-buried pupae developing into adults in trials beginning on 20 December 2013 and 8 January 2014, respectively (Fig. C1(A)). The majority of eggs, larvae, and pupae successfully developed into adults when trials began in late January and thereafter (Fig. C1(A)). The seasonal periods of D. suzukii survival or development are effected by regional climate. During this trial, temperatures showed the expected seasonal warming trend in December, January, February and March, with daily mean minimum air temperatures of -0.6, 1.8, 6.9 and 8.2 °C, respectively, and maximum air temperatures of 15.7, 19.5, 19.1, and 23.1 °C. Soil temperatures were relatively stable, with mean daily soil temperatures for December, January, February and March of 8.5, 9.8, 12.5 and 15.9 °C, which was slightly higher than the corresponding mean daily air temperatures for the same periods (Fig. C1(B)). The only period of relatively cold winter temperatures was a 2 wk period from 4–18 December 2013 (Fig. C1(B)) and no D. suzukii immature stages survived during this period. In the field cage food provisioning trial, adult D. suzukii that were not provided food or water died within one week (Table C1, see Appendix C for Study 3 tables); for this reason, this treatment was discontinued after the first two trials and the data were not included in survival analyses comparisons with the other treatments. When only water was provided, adult flies survived for about one week (Table C1), which was shorter than treatments with honey-water and honey- water with an orange slice (Fig. C2). Adult longevity was similar between adult 59 59 male and female flies and between the honey-water and honey-water with an orange slice for each trial, except for two trials launched on 19 November 2013 and 24 February 2014 when the females provided honey-water with an orange slice survived longer than those provided honey-water only (Fig. C2, Table C1). When honey-water with an orange slice was provided, longevity was similar among all trial periods with the exception of the last trial period (beginning 28 March 2014) when longevity was shorter (Table C1, Fig. C3); this period also corresponded with increasing ambient temperatures (Fig. C1(B)). For example, female flies survived an average of 32.1 to 44.1 d in trials conducted from November through February but only 14.2 d in the trial that began on 28 March (Table C1). The female D. suzukii that emerged from soil-buried pupae and were tested for reproductive potential on sliced oranges produced offspring. A total of 98 females and 98 males were tested, from 6 January to 4 April 2014. On average, female and male survival was 21.8 ± 1.7 and 25.3 ± 2.1 d, respectively. Lifetime egg production, on orange slices, from these overwintered females was 4.7 ± 1.1 per female.
Host Suitability of Citrus Fruit The status of the tested orange fruit affected the number of eggs laid by D. suzukii (F3,96 = 24.6, P < 0.001) (Fig. C4). No eggs were laid into intact fruit; three eggs were found on the surface of two intact fruit but these were unsuccessful oviposition attempts because the egg was not inserted vertically into the fruit flesh, but placed horizontally over the fruit surface. The surface penetration pressure of the tested intact oranges was 321.1 16.9 g mm-1 (n =10), which was 2–3 times higher than that of cherry fruits tested as a preferred host (Table C2). The 60 60 treatment using intact but rotting orange fruit found a few eggs into the gelatinous rind of the rotting fruit, whereas eggs were readily laid into sliced fresh and sliced rotting fruit (Fig. C4). The percentage of eggs that successfully developed into adults from the sliced fresh fruit (39.7 8.3%) and sliced rotting fruit (67.9
9.8%) as not significantly different (F1,36 = 2.9, P = 0.099). At the time of oviposition, brix levels of the fresh (10.7 0.2) and rotting fruit (10.5 0.2) were similar (F1,18 = 0.3, P = 0.567).
Discussion Understanding environmental conditions that affect the geographic distribution and abundance of insect pests is fundamental to their effective management, particularly for invasive species. Temperature plays a vital role in the survival and reproductive success of insects and has been identified as a factor influencing the geographical abundance of D. suzukii populations (Dalton et al. 2011, Tochen et al. 2014, Emiljanowicz et al. 2014, Kinjo et al 2014, Wiman et al. 2014). In California’s interior fruit growing regions, daily minimum temperatures can drop below freezing (0 ºC) during the winter, but the daily temperatures will typically be high enough to support D. suzukii survival and even development. For example in Parlier, CA, where this study was conducted, from 1984 to 2014, the maximum, minimum and mean air temperatures for the coldest 2 months (December and January) were 13.3, 2.5 and 7.3 ºC, while mean soil temperature was 9.3 ºC. Under such variable winter conditions, this current study shows that adult D. suzukii were able to survive and female flies were able to oviposit when adequate food and ovipositional medium (in this case an orange slice) were avaiable. 61 61
Adult D. suzukii require food nutrients to meet the energetic and metabolic requirements for survival and reproduction, and this would be similar even during the winter period. Many adult fruit flies are capable of surviving on an energy source alone, such as yeast or sucrose (Bertrand et al. 1999, Min and Tatar 2006, Wang et al. 2011). This study investigated the possible food sources available in citrus, which appears to be one of the primary overwintering sites for D. suzukii in some San Joaquin Valley regions (Haviland et al. 2015). In this region, many commercial crops and ornamental plants are potential D. suzukii hosts, including apple, cherry, fig, grape, kiwifruit, loquat, nectarine, peach, pear, persimmon, plum, pomegranate and prune. The importance of citrus as an overwintering host in the southern and central San Joaquin Valley is due, in part, to the large acreage planted to this crop, the abundance of fruits that are not harvested during the winter and left on the ground, and the anti-frost techniques used to protect citrus (e.g., wind machines and micro sprinkler irrigation) that elevate temperatures in the orchard and may improve D. suzukii overwintering survival. Juices from split, damaged or overripe fruits could seep from wounds and provide food for adult D. suzukii, which are known to feed on other food sources such as flowers and yeasts (Mitsui et al. 2010, Hamby et al. 2012). These trials showed that low temperature exposure can be lethal to immature D. suzukii, especially eggs and larvae, which had the lowest survival percentage during the colder months (Fig. C1). Immature survival and development increased as temperatures increased in February, with minimum low temperatures above 4 ºC. There was also higher survival of soil-buried pupae in each trial period, which was also likely a result higher and more stable soil temperatures relative to the ambient air temperatures. Age-related variation in thermotolerance has been documented in various insects and theoretical studies 62 62 and suggests that thermotolerance should decline with advanced life stages because the more mobile insect stages can compensate behaviorally by increased mobility (Bowler and Terblanche 2008). In this study, we found the less mobile stages (egg and larva) of D. suzukii appeared to be less cold-resistant than the adults. A few individual eggs or larvae (< 3%) survived but took >2 months to develop into adults during the earlier trials. Whether this was due to stage-related variation in lower thermal tolerance or the cumulative effect of cold stress on various immature stages cannot be determined, and requires further investigation into separate exposures for each developmental stage. Various abiotic and biotic factors, such as temperature, humidity, photoperiod, and host stimuli are known to influence ovarian development in insects (Papaj 2000). Earlier observations in Japan found that adult D. suzukii females which had migrated from high to low latitudes did not contain mature eggs in winter seasons (Mitsui et al. 2010). The authors suggested that reproductive dormancy occurred in D. suzukii, presumably due to the lack of host fruit. Recent field studies found that field-captured adult D. suzukii females in cider vinegar traps also contained low mature eggs during the later fall and winter seasons than in other seasons (Stewart et al. 2014). Previous studies also reported that adult D. suzukii flies ceased most reproductive activities when ambient temperatures were lower than 10 ºC (Dalton et al. 2011, Tochen et al. 2014). Adverse environmental conditions and lack of host availability appear to induce reproductive dormancy in D. suzukii. However, in this study, adult female D. suzukii were able to mature and lay eggs when host fruit were absent, and adult D. suzukii were observed to be active in all trial periods, typically during the warmest period day, providing windows for oviposition even during the winter. Apparently, adult experience with host fruit (field emerged flies) or pre-imaginal conditioning 63 63
(laboratory-cultured flies) could stimulate egg maturation of D. suzukii, although it remains unclear whether the stimulatory effect of host fruit results directly from chemical or ovipositional stimuli or indirectly from feeding on fruit containing protein cues. Here, factors affecting egg maturation were not addressed and require further research. Adult D. suzukii have been caught in apple cider vinegar traps in California interior fruit growing regions throughout the year, although the numbers were low during the winter seasons (Harris et al. 2014, Haviland et al. 2015). This current study suggests that the cold tolerance of adult D. suzukii, combined with high dispersal ability, may allow winter survival and development in the San Joaquin Valley. Although the resulting offspring may not complete development when temperatures are low in December and January, eggs deposited in late January were able to develop into adults, although this temperature-dependent activity will vary among years depending on the winter conditions and the 2013-2014 winter was relatively mild and dry. Proper hosts and food are critical for the survival of adult flies and unharvested fruit may play an important role as reservoirs in sustaining the fly populations that may migrate into surrounding crops in the spring. Therefore, future management strategies may consider field fruit sanitation to reduce the sources of D. suzukii populations.
GENERAL CONCLUSIONS
Understanding the localized life history and population dynamics of an economically significant pest such as D. suzukii can aid in the development of effective integrated pest management practices. In my first study, I elucidated the possible dispersion patterns of D. suzukii populations in some regions of California’s San Joaquin Valley of California. The data shows aggregations of the fly in susceptible fruit orchards during ripening and harvest phases followed by aggregations in alternative hosts such as citrus during the winter months. This suggests that adequate control of D. suzukii infestations likely requires consideration of local landscape dynamics and alternative hosts as the pest is able to host in numerous fruit crops throughout different times of the year. For example, managing elusive adults in the cash crop alone may be far less effective than reducing source populations in nearby overwintering or over summering hosts. I also monitored the sexual maturity of fly populations throughout the year via female abdomen dissection and provided evidence that D. suzukii may overwinter as adults in a partial reproductive diapause. In the second study, I examined the host suitability of peach fruit, a local stone fruit seemingly exempt from infestation by D. suzukii. I showed that under certain circumstances, peach fruit becomes a highly suitable host for the pest. The removal or destruction of the indument by any means, including harvest methods, yielded the fruit susceptible to oviposition and therefore development of offspring from the host. This suggests that the limiting factor of suitability of peaches as a D. suzukii host is simply the fruit fuzz. Unpublished and preliminary data have shown high numbers of D. suzukii in Citrus orchards in winter months, thus, in the final study I explored the 65 65 suitability of citrus orchards and the fruit itself as an overwintering host in the Central Valley of California. First, I showed the ability of the adult flies to survive a mean of 32.2 to 44.1 d, with a maximum survival time of 118 d. This study also showed that immature stages of the pest had a low rate of survival during winter months in the Central Valley, but showed that individuals in the pupal stage that were within the soil had a relatively higher rate of survival. Second, I showed that D. suzukii can successfully develop within fresh or rotting citrus fruit. However, females were only able to oviposit in severely damaged or rotted fruit. This study provides further evidence that D. suzukii can host in citrus orchards during the winter months when few other hosts are available.
LITERATURE CITED
LITERATURE CITED
Atallah, J., L. Teixeira, R. Salazar, G. Zaragoza, and A. Kopp. 2014. The making of a pest: the evolution of a fruit-penetrating ovipositor in Drosophila suzukii and related species. Proc. R. Soc. B 281: 1781.YYY
Beers, E. H., R. A. Van Steenwyk, P. W. Shearer, W. W. Coates, and J. A. Grant. 2011. Developing Drosophila suzukii management programs for sweet cherry in the western United States. Pest Manage. Sci. 67: 1386–1395.
Bellamy, D. E., M. S. Sisterson, and S. S. Walse. 2013. Quantifying host potentials: Indexing postharvest fresh fruits for spotted wing drosophila, Drosophila suzukii. PLoS One 8: e61227.
Bentley, W. J., and S. A. Steffan. 2001. Notes on biology of katydids in San Joaquin Valley stone fruit orchards. Univ. Calif. Plant Prot. Quart. 11: 1–4.
Berry, J. A. 2012. Pest risk assessment: Drosophila suzukii: spotted wing drosophila (Diptera: Drosophilidae) on fresh fruit from the USA. Ministry for Primary Industries, Wellington, New Zealand, pp 42.
Bertrand, H. A., J. H. Herlihy, Y. Ikeno, and B. P. Yu. 1999. Dietary restriction. In: Yu, B.P. (Ed.), Methods in Aging Research. CRC Press, Boca Raton, pp. 271–300.
Bolda, M. P., R. E. Goodhue, and F. G. Zalom. 2010. Spotted wing drosophila: potential economic impact of a newly established pest. Giannini Foundation of Agricultural Economics, University of California.
Bowler, K., and J. S. Terblanche. 2008. Insect thermal tolerance: what is the role of ontogeny, aging and senescence? Biol. Rev. 83: 339335.
Brown, P. H., P. W. Shearer, J. C., Miller, and H. M. A. Thistlewood, 2011. The discovery and rearing of a parasitoid (Hymenoptera: Pteromalidae) associated with spotted wing drosophila, Drosophila suzukii, pp. 13-16. In: Oregon and British Columbia. Entomol. Soc. Am. 59th Annual Meeting.
Bruck, D. J., M. Bolda, L. Tanigoshi, J. Klick, J. Kleiber, J. DeFrancesco, B. Gerdeman, and H. Spitler. 2011. Laboratory and field comparisons of insecticides to reduce infestation of Drosophila suzukii in berry crops. Pest Manage. Sci. 67: 1375–1385. 68 68 Burrack, H. J., G. E. Fernandez, T. Spivey, and D. A. Kraus. 2013. Variation in selection and utilization of host crops in the field and laboratory by Drosophila suzukii Matsumara (Diptera: Drosophilidae), an invasive frugivore. Pest Manage. Sci. 69: 11731180.
Calabria, G., J. Maca, G. Bachli, L. Serra, and M. Pascual. 2012. First records of the potential pest species Drosophila suzukii (Diptera: Drosophilidae) in Europe. J. Appl. Entomol. 136: 139147.
California Agricultural Production Statistics. Rep. California Department of Food and Agriculture, 2014. Web. 10 Aug. 2014.
California Crop Profiles and Other Data Sources, California Pest Management Center. (2014, September 27). Retrieved September 27, 2014.
Carrière, Y., P. B. Goodell, C. Ellers-Kirk, G. Larocque, P. Dutilleul, S. Naranjo, and P. C. Ellsworth. 2012. Effects of local and landscape factors on population dynamics of a cotton pest. PLoS One 7: e39862.
Cha, D. H., T. Adams, H. Rogg, and P. J. Landolt. 2012. Identification and field evaluation of fermentation volatiles from wine and vinegar that mediate attraction of spotted wing drosophila, Drosophila suzukii. J. Chem. Ecol. 38: 1419–1431.
Chabert, S., R. Allemand, M. Poyet, P. Eslin, and P. Gibert, P. 2012. Ability of European parasitoids (Hymenoptera) to control a new invasive Asiatic pest, Drosophila suzukii. Biol. Control 63: 40–47.
Cini, A., C. Ioriatti, and G. Anfora. 2012. A review of the invasion of Drosophila suzukii in Europe and a draft research agenda for integrated pest management. Bull. Insectology 65: 149–160.
Cini, A., G. Anfora G., L. A. Escudero-Colomar, A. Grassi, U. Santosuosso, G. Seljak, and A. Papini. 2014. Tracking the invasion of the alien fruit pest Drosophila suzukii in Europe. J. Pest Sci. 87: 559–566.
Dalton, D. T., V. M. Walton, P. W. Shearer, D. B. Walsh, J. Caprile, and R. Isaacs 2011. Laboratory survival of Drosophila suzukii under simulated winter conditions of the Pacific Northwest and seasonal field trapping in five primary regions of small and stone fruit production in the United States. Pest Manag. Sci. 67: 1368–-1374. 69 69 Deprá, M., J. L. Poppe, H. J. Schmitz, D. C. De Toni, and V. L. S. Valente. 2014. The first records of the invasive pest Drosophila suzukii in the South American continent. J. Pest. Sci. 87: 379–383.
Dreves, Amy J., Vaughn Martin Walton, and Glenn C. Fisher. 2009. A new pest attacking healthy ripening fruit in Oregon: spotted wing Drosophila: Drosophila suzukii (Matsumura). Corvallis, Oregon: Extension Service, Oregon State University, 2009.
Emiljanowicz, L. M., G. D. Ryan, A. Langille, and J. Newman. 2014. Development, reproductive output and population growth of the fruit fly pest Drosophila suzukii (Diptera: Drosophilidae) on artificial diet. J. Econ. Entomol. 107: 1392-1398.
Futuyma, D. J., G. Moreno. 1988. The evolution of ecological specialization. Annu. Rev. Ecol. Syst. 19: 207–234.
Goodhue, R. E., M. Bolda, D. Farnsworth, J. C. Williams, and F. G. Zalom. 2011. Spotted wing drosophila infestation of California strawberries and raspberries: economic analysis of potential revenue losses and control costs. Pest Manage. Sci. 67: 1396–1402.
Hamby, K. A., A. Hernández, K. Boundy-Mills, and F. G. Zalom. 2012. Associations of yeasts with spotted-wing Drosophila (Drosophila suzukii: Diptera: Drosophilidae) in cherries and raspberries. Appl. Environ. Microbiol. 78: 48694873.
Hamby, K. A., M. P. Bolda, M. E. Sheehan, and F. G. Zalom. 2014. Seasonal Monitoring for Drosophila suzukii (Diptera: Drosophilidae) in California Commercial Raspberries. Environ. Entomol. 43: 10081018.
Harris, D. W., K. A. Hamby, H. E. Wilson, and F. G. Zalom. 2014. Seasonal monitoring of Drosophila suzukii (Diptera: Drosophilidae) in a mixed fruit production system J. Asia-Pacific Entomol. 17: 857–864.
Hauser, M. 2011. A historic account of the invasion of Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) in the continental United States, with remarks on their identification. Pest Manage. Sci. 67: 13521357.
Hertlein, M. B. 1986. Seasonal development of Leptopilina boulardi (Hymenoptera: Eucoilidae) and its hosts, Drosophila melanogaster and D. simulans (Diptera: Drosophilidae), in California. Environ. Entomol. 15: 859– 866. 70 70 Ideo, S., M. Watada, H. Mitsui, and M. T. Kimura. 2008. Host range of Asobara japonica (Hym.: Braconidae), a larval parasitoid of drosophilid flies. Entomol. Sci. 11: 1–6.
Kacsoh, B. Z., and T. A. Schlenke. 2012. High hemocyte load is associated with increased resistance against parasitoids in Drosophila suzukii, a relative of D. melanogaster. PLoS One 7: e34721.
Kanzawa, T. 1939. Studies on Drosophila suzukii Mats. Kofu, Yamanashi Agricultural Experiment Station 49 pp. (abstr.). Rev. Appl. Entomol. 29: 622.
Kasuya, N., H. Mitsui, S. Ideo, M. Watada, and M. T. Kimura. 2013. Ecological, morphological and molecular studies on Ganaspis individuals (Hymenoptera: Figitidae) attacking Drosophila suzukii (Diptera: Drosophilidae). Appl. Entomol. Zool. 48: 87–92.
Kimura, M. T. 2004. Cold and heat tolerance of drosophilid flies with reference to their latitudinal distributions. Oecologia 140: 442–449.
Kinjo, H., Y. Kunimi Y, T. Ban, and M. Nakai. 2013. Oviposition efficacy of Drosophila suzukii (Diptera: Drosophilidae) on different cultivars of blueberry. J. Econ. Entomol. 106: 17671771.
Kinjo, H., Y. Kunimi, and M. Nakai. 2014. Effects of temperature on the reproduction and development of Drosophila suzukii (Diptera: Drosophilidae). Appl. Entomol. Zool. 49: 297–304.
Krugner, R., K. M. Daane, A. B. Lawson, and G. Y. Yokota. 2005. Biology of Macrocentrus iridescens (Hymenoptera: Braconidae): A parasitoid of the obliquebanded leafroller (Lepidoptera: Tortricidae). Environ. Entomol. 34: 336343.
Landolt, P. J., T. Adams, and H. Rogg. 2012. Trapping spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), with combinations of vinegar and wine, and acetic acid and ethanol. J. Appl. Entomol. 136: 148–54.
Lee, J. C., D. J. Bruck, A. J. Dreves, C. Ioriatti, H. Vogt, and P. Baufeld. 2011a. Spotted wing drosophila, Drosophila suzukii, across perspectives. Pest Manage. Sci. 67: 1349–-1351. 71 71 Lee, J. C., D. J. Bruck, H. Curry, D. Edwards, D. R. Haviland, R. A. Van Steenwyk, B. M. Yorgey. 2011b. The susceptibility of small fruits and cherries to the spotted wing drosophila, Drosophila suzukii. Pest Manag. Sci. 67: 1358–-1367.
Lee, J. C., H. J. Burrack, L. D. Barrantes, E. H. Beers, A. J. Dreves, K. Hamby, D. R. Haviland, R. Isaacs, T. Richardson, P. Shearer, C.A. Stanley, D. B. Walsh, V. M. Walton, F. G. Zalom, and D. J. Bruck. 2012. Evaluation of monitoring traps for Drosophila suzukii (Diptera: Drosophilidae) in North America. J. Econ. Entomol. 105: 1350–1357.
Lee, J. C., D. J. Bruck, H. Curry, D. L. Edwards, D. R. Haviland, R. Van Steenwyk, and B. Yorgey. 2011. The susceptibility of small fruits and cherries to the spotted wing drosophila, Drosophila suzukii. Pest Manage. Sci. 67: 1358–6137.
Min, K. J., and Tatar M. 2006. Drosophila diet restriction in practice: Do flies consume fewer nutrients? Mech. Aging Devel. 127: 93–96
Mitsui, H., and M. T. Kimura. 2010. Distribution, abundance and host association of two parasitoid species attacking frugivorous drosophilid larvae in central Japan. Eur. J. Entomol. 107: 535–540.
Mitsui, H., K. H. Takahashi, and M. T. Kimura. 2006. Spatial distributions and clutch sizes of Drosophila spp. ovipositing on cherry fruits of different stages. Pop. Ecol. 48: 233–237.
Mitsui H., K. Van Achterberg, G. Nordlander, and M. T. Kimura. 2007. Geographical distributions and host associations of larval parasitoids of frugivorous Drosophilidae in Japan. J. Natur. His. 41:1731-1738.
NAPIS (National Agricultural Pest Information System). 2014. Survey status of spotted wing Drosophila-Drosophila suzukii (2009 to 2013). http://pest.ceris.purdue.edu/map.php (accessed on 0/1/07/2015).
Oku T. 2003. Drosophila suzukii (Matsumura) in Japan. Agricultural Pest Encyclopedia. Zenkoku Noson Kyoiku Kyokai pp. 381
Papaj, D. R. 2000. Ovarian dynamics and host use. Annu. Rev. Entomol. 45: 423–448. 72 72 Poyet, M., P. Eslin, M. Héraude, V. Le Roux, G. Prévost, P. Gibert, and O. Chabrerie. 2014. Invasive host for invasive pest: when the Asiatic cherry fly (Drosophila suzukii) meets the American black cherry (Prunus serotina) in Europe. Agri. Forest Entomol. 16: 251–259.
Siekmann, G., B. Tenhumberg B., and M. A. Keller. 2001. Feeding and survival in parasitic wasps: Sugar concentration and timing matter. Oikos 95: 425–430.
Steffan, S. A., J. C. Lee, M. E. Singleton, A. Vilaire, D. B. Walsh, L. S. Lavine, and K. Patten. 2014. Susceptibility of cranberries to Drosophila suzukii (Diptera: Drosophilidae). J. Econ. Entomol. 106: 24242427.
Stewart, T. E, X. G. Wang, A. Molinar, and K. M. Daane. 2014. Factors limiting peach as a potential host for spotted wing drosophila. J. Econ. Entomol. 107:1771-1779.
Tochen, S., D. T. Dalton, N. G. Wiman, C. Hamm, P. W. Shearer, and V. M. Walton. 2014. Temperature-related development and population parameters for Drosophila suzukii (Diptera: Drosophilidae) on cherry and blueberry. Environ. Entomol. 43: 501510.
USDA. 2013. Non-citrus fruits and nuts 2012 summary. Publication Fruit and Nuts 1–3 (09), Washington, D.C.
Vlach, J. 2010. Identifying Drosophila suzukii. Version from June, 2, 2010.
Walse, S. S., R. Krugner, and J. S. Tebbets, J. S. 2012. Postharvest treatment of strawberries with methyl bromide to control spotted wing drosophila, Drosophila suzukii. J. Asia-Pacific Entomol.15: 451–456.
Walsh, D. B., M. P. Bolda, R. F. Goodhue, A. J. Dreves, L. Lee, D. J. Bruck, V. M. Walton, S. D. O’Neal, and F. G. Zalom 2011. Drosophila suzukii (Diptera: Drosophilidae): invasive pest of ripening soft fruit expanding its geographic range and damage potential. J. Integ. Pest Manage. 2(1): G1– G7(7)
Wang, X. G., K. Levy, H. Nadel, M. W. Johnson, A. Blanchet, Y. Argov, C. H. Pickett, and K. M. Daane. 2013. Overwintering survival of olive fruit fly and two introduced parasitoids in California. Environ. Entomol. 42: 467–476. 73 73 Wang, X. G., M. W. Johnson, S. B. Opp, R. Krugner, and K. M. Daane. 2011. Honeydew and insecticide bait as competing food resources for a fruit fly and common natural enemies in the olive agroecosystem. Entomol. Exp. Appl. 139: 128–137.
Weisstein, E. W. 2014. Prolate spheroid. From MathWorld-A Wolfram Web Resource. http://mathworld.wolfram.com/ProlateSpheroid.html.
Wu, S. R., H. K. Tai, Z. Y. Li, X. Wang, S. S. Yang, W. Sun, and C. Xiao. 2007. Field evaluation of different trapping methods of cherry fruit fly, Drosophila suzukii. J. Yunan Agricul. Univ. 44:776–-782.
Wilson, H. E., K. A. Hamby, and F. G. Zalom. 2013. Host susceptibility of “French Prune” Prunus domestica to Drosophila suzukii (Diptera: Drosophilidae). Acta Horticul. 985: 249–254.
Wiman, N. W., G. Anfora, H. J. Burrack, J. Chiu, K. M. Daane, D. T. Dalton, A. Grassi, C. Ioriatti, B. Miller, S. Tochen, X. G. Wang, and V. M. Walton. 2014. Integrating temperature-dependent life table data into a matrix projection model for Drosophila suzukii population estimation. PLoS One 9:e106909.
Yu, D., F. G. Zalom, and K. A. Hamby. 2013. Host status and fruit odor response of Drosophila suzukii (Diptera: Drosophilidae) to figs and mulberries. J. Econ. Entomol. 106: 19321937.
APPENDICES
APPENDIX A: STUDY 1 TABLES AND FIGURES 76 76
Table A1. Geographical locations and monitoring sites of D. suzukii adult in California Central Valley during 2013 and 2014 Location Site Latitude Longitude Orchard or habitats (N) (W) Brentwood 1 37°54'53" 121°42'21" Mixed fruit: cherry, grape, loquat peach, pears, persimmon)
2 37°54'50" 121°42'49" Plum 3 37°54'18" 121°39'27" Apricot 4 37°54'37" 121°39'28" Pear 5 37°54'43" 121°42'54" Riparian area (three spots) 6 37°54'07" 121°38'38" Mixed fruit garden Courtland 1 38°19'03" 121°34'58" Cherry 1 Kiwi (adjacent to Cherry 1) 2 38°17'60" 121°35'03" Cherry 2 3 38°17'40" 121°35'07" Cherry 3 4 Pear (adjacent to Cherry 3) Stockton 1 37°48'47" 121°06'33" Cherry 1 2 38°02'05" 121°06'15" Cherry 2 3 Peach (adjacent to Cherry 2) Parlier 1 1- 36°35'59" 119°30'56" Apricot, Apple, Blackberry, 14 Blueberry Cherry (1), Cherry (2), Citrus, Fig, Grape, Kiwi, Peach, Plum, Persimmon, Pomegranate
1 All orchards were located within a 330-acre research farm (University of California, KEARNEY station) and separately at least by one block of other crops except peach and persimmon which were adjacent to each another. 77 77
Table A2. Periods of ripening and fruit availability of monitored fruit crops in California Central Valley
Table A3. Sum and average (± S.E.) number of adult Drosophila suzukii and other species of frugivorous fruit flies captured in vinegar traps placed in four locations in some of California’s San Joaquin Valley fruit growing regions Total Frugivorous Traps Total D. D. suzukii per Location frugivorous flies per trap recovered suzukii trap per week flies per week Brentwood 351 34,021 96.20 ± 115,548 325.3 ± 32.3 13.97 Courtland 460 5,085 9.93 ± 0.95 179,494 357.0 ± 33.0 Parlier 3,229 5,984 1.85 ± 0.11 709,574 224.6 ± 8.6 Stockton 372 4,004 9.04 ± 0.99 227,422 570.6 ± 51.3
78 78
Table A4. Average (± S.E.) number of adult Drosophila suzukii captured in vinegar traps placed in different plant ecosystems at four locations in some of California’s San Joaquin Valley fruit growing regions from April 2013 to May 2014. The number of traps used is provided in parenthesis and varied according to the number of ecosystem sites monitored each location or the number of traps used at each site Plant Average (± S.E.) adult D. suzukii per trap per collection ecosystem Brentwood Courtland Parlier Stockton Apple – – 1.5 ± 0.2 (234) – Apricot 54.5 ± 16.7 (39) 1 – 3.2 ± 0.6 (234) – Blackberry – – 1.7 ± 0.3 (234) – Blueberry – – 0.4 ± 0.1 (234) – 12.2 ± 1.4 Cherry 33.2 ± 8.8 (40) 1 9.8 ± 1.2 (276) 6.2 ± 1.0 (234) (245) Citrus – – 3.4 ± 0.5 (234) – 342.9 ± 102.1 (39) Fig – 1.8 ± 0.5 (234) – 1,2 Grape – – 0.3 ± 0.1 (234) – Kiwi – 10.3 ±1.9 (138) 1.2 ± 0.2 (234) – Peach 9.4 ± 2.2 (39) 3 – – 2.8 ± 0.5 (126) Pear 8.4 ± 2.4 (38) 9.6 ± 1.6 (46) – – Persimmon – – 1.0 ± 0.1 (234) – Riparian 74.9 ± 11.5 (117) – – – 1 Organic production 2 The trap was placed within a group of 12 fig trees, but this highly diverse 5 ha organic farm had numerous fruit, nut, vegetable and herb crops intermixed. 3 Mixed stone fruit that contained cherry, peach, pears, persimmon, grape, and loquat. 79 79
Table A5. Generalized Linear Model analyzing the effects of presence of mature fruit (Fruit), accumulated degree-days (DD), as well as their interaction on weekly captures of adult D. suzukii in different geographical locations and host plants Location Host plant Variable1 Residual 2 P deviance Courtland Cherry Fruit 253.7 16.8 < 0.001 DD 25.6 < 0.001 Fruit DD 191.6 < 0.001 Kiwi Fruit 217.3 116.4 < 0.001 DD 27.1 < 0.001 Fruit DD 111.7 < 0.001 Pear Fruit 165.6 142.7 < 0.001 DD 0.1 0.760 Fruit DD 1.2 0.281 Stockton Cherry Fruit 316.7 82.3 < 0.001 DD 50.5 < 0.001 Fruit DD 4.0 0.045 Peach Fruit 32.9 5.5 0.039 DD 4.3 0.020 Fruit DD 1.2 0.282 Brentwood Cherry Fruit 998.4 788.6 < 0.001 DD 20.5 < 0.001 Fruit DD 17.7 < 0.001 Plum Fruit 61.7 35.4 < 0.001 DD 2.1 0.147 Fruit DD 9.8 < 0.001 Apricot Fruit 452.3 111.4 < 0.001 DD 189.4 < 0.001 Fruit DD 96.1 < 0.001 Pear Fruit 270.3 240.2 < 0.001 DD 37.8 < 0.001 Fruit DD 4.7 < 0.001 Riparian Fruit 1540 126.8 < 0.001 DD 490.7 < 0.001 Fruit DD 9.6 < 0.001 Fig/Garden Fruit 21291 6022 < 0.001 DD 4762 < 0.001 Fruit DD 54.4 < 0.001
80 80
Location Host plant Variable1 Residual 2 P deviance Parlier Apple Fruit 76.2 54.4 < 0.001 DD 2.6 0.107 Fruit DD 9.2 0.002 Apricot Fruit 26.8 10.2 0.001 DD 13.1 < 0.001 Fruit DD 10.7 0.001 Blackberry Fruit 48.6 14.7 < 0.001 DD 5.7 0.017 Fruit DD 0.01 0.772 Blueberry Fruit 9.5 6.5 0.011 DD 0.1 0.884 Fruit DD < 0.1 0.926 Cherry Fruit 156.5 15.3 < 0.001 DD 14.0 < 0.001 Fruit DD 26.3 < 0.001 Citrus Fruit 273.8 49.3 < 0.001 DD 5.6 0.024 Fruit DD 1.7 0.189 Fig Fruit 250.3 98.2 < 0.001 DD 14.0 < 0.001 Fruit DD 4.7 0.031 Grape Fruit 52.2 18.1 < 0.001 DD 0.4 0.541 Fruit DD 4.4 0.035 Kiwi Fruit 130.1 70.7 < 0.001 DD 1.1 0.285 Fruit DD 0.2 0.626 Peach Fruit 36.1 5.7 0.016 DD 2.0 0.153 Fruit DD 33.5 < 0.001 Persimmon Fruit 45.2 31.4 < 0.001 DD 2.8 0.092 Fruit DD 1.6 0.212 Plum Fruit 51.5 39.1 < 0.001 DD 0.1 0.796 Fruit DD 6.1 0.014 Pomegranate Fruit 36.1 28.9 < 0.001 DD 3.3 0.071 Fruit DD 3.3 0.071 1 Presence of fruit was treated as a category variable and coded as 1 for presence and 0 for absence.
Table A6. Correlation coefficients in the relationship between trap captures of adult D. suzukii in two closely located sites in different geographical locations in California’s Central Valley Location Site A Site B Distance (km) Slope r2 df F P Courtland Cherry 1 Kiwi Adjacent 0.997 0.802 1,44 120.5 < 0.001 Cherry 3 Pear Adjacent 0.553 0.382 1,44 27.2 < 0.001
Stockton Cherry 2 Peach 1.29 0.199 0.109 1,39 4.8 0.036
Brentwood Riparian Cherry 0.82 0.583 0.434 1,39 30.0 < 0.001 Plum 0.29 0.248 0.149 1,39 6.8 0.013 Garden Apricot 1.1 0.576 0.404 1,39 26.4 < 0.001 Pear 1.3 0.455 0.452 1,39 32.2 < 0.001
Parlier Cherry Apricot 0.42 0.444 0.503 1,63 63.8 < 0.001 Fig 1.1 0.287 0.180 1,63 13.8 < 0.001 Pomegranate 1.2 0.153 0.189 1,63 14.7 < 0.001 Apricot Fig 1.1 0.787 0.533 1,63 71.9 < 0.001 Pomegranate 1.2 0.384 0.457 1,63 52.9 < 0.001 Fig Citrus Adjacent 0.981 0.617 1,63 101.5 < 0.001 Pomegranate Citrus Adjacent 1.314 0.309 1,63 20.8 < 0.001
81
82
Table A7. Results of Generalized Linear Model testing the effects of trapping site (different orchard or non-orchard habitats) and seasons on the number of mature eggs of captured female D. suzukii or the percentage of captured females without mature egg in different geographical locations in California’s Central Valley Mature eggs per % Females without
Location Variable1 female mature egg df χ2 P df χ2 P Courtland Site 2 1.0 0.375 2 1.2 0.314 < Season 2 37.8 < 0.001 2 73.7 0.001 Site × Season 4 0.8 0.555 4 1.2 0.326
Brent Site 6 2.0 0.309 6 0.6 0.697 wood 123. < Season 2 36.8 < 0.001 2 3 0.001 Site × Season 12 0.2 0.998 12 1.7 0.056
Parlier Site 7 1569 < 0.001 7 1.6 0.121 < Season 2 55.3 < 0.001 2 22.6 0.001 Site × Season 14 369 < 0.001 14 0.7 0.743 1 Site refers to the orchard or non-orchard habitat where the trap was placed, while season was divided into April to July, August to October, and November to March, and data were pooled from each season.
83
Fig. A1. Weekly mean D. suzukii trap captures in cherry and other commercial fruit crop orchards in (A) Courtland (Sacramento County) and (B) Stockton (San Joaquin County), CA. Data for the three cherry orchards (within 2 km) in Courtland were pooled.
84
Fig. A2. Weekly mean D. suzukii trap captures in (A) cherry and other commercial fruit orchards, (B) a riparian habitat area adjacent to the commercial orchards, and (C) a mixed fruit garden in Brentwood (Contra Costa County), CA. Data from the three riparian sites were pooled.
85
Fig. A3. Weekly mean D. suzukii trap captures in different crop orchards within a 330-arca research farm at the University of California Kearney Agricultural Research and Extension Center in Parlier (Fresno County), CA. Data are grouped based on the fruiting seasons of monitored crops (A-D). 86
Fig. A4. Shifting correlations between the trap captures of D. suzukii in two different crops in Parlier over a range of gap between fruit ripening seasons. 87
Fig. A5. Weekly mean trap captures of other drosophilids (except D. suzukii) in (A) Courtland, (B) Stockton and Brentwood, and (C) Parlier, CA. Data were pooled from all traps in different fruit orchards or site for each location. 88
Fig. A6. Weekly mean trap captures of drosophilid parasitoids (Leptopilina spp.) in cider vinegar traps for D. suzukii in (A) Courtland and (B) Parlier, California. Data were pooled from all traps in different fruit orchards for each location. 89
Fig. A7. Number of mature eggs of D. suzukii females captured in different orchards orsites in (A) Courtland, (B) Brentwood, and (C) Parlier. Data were pooled for each of three seasons (November to March, April to July and August to October). Bars refer to mean ± SE. 90
Fig. A8. Percentage of captured D. suzukii females without mature eggs in different orchards or sites in (A) Courtland, (B) Brentwood, and (C) Parlier. Data were pooled from all traps for each month. Lines refer to mean monthly minimum daily temperature.
APPENDIX B: STUDY 2 TABLES AND FIGURES
Table B1. Brix and firmness of various types of tested peach fruit Surface penetration force No. of Brix 2 Fruit type Peach variety (g)2 fruit Mean SE Range Mean SE Range Intact ‘Earlirich’ 45 9.4 0.26 c 6.0 13.0 296 11.7 ab 100 425 ‘Babcock’ 21 12.8 0.28 a 10.0 15.0 219 16.7 c 63 385 Punctured ‘Earlirich’ 21 7.5 0.22 d 6.5 10.0 320 8.3 a 248 – 400 ‘Spring Shaved 44 10.6 0.33 b 7.0 17.5 85 6.3 d 5 208 Snow’ ‘Spring Damaged 15 10.0 0.35 b 8.0 14.5 250 10.4 bc 5 320 Snow’ (harvest) ‘Earlirich’ 26 10.4 0.51 b 6.0 15.0 113 8.6 d 30 175 Damaged (PTB) 1 ‘Earlirich’ 33 9.3 0.16 c 6.5 11.5 219 7.6 c 108 388 Damaged ‘Earlirich’ 25 8.6 0.81 c 7.0 11.5 217 9.5 c 113 391 (Katydid) 1 PTB = peach twig borer. 2 Different letters indicate significant difference (One-way ANOVA and Tukey’s HSD, P < 0.05).
92
Table B2. Relationship (linear regression) of fruit brix and fruit firmness with the number of D. suzukii eggs per intact or damaged area (shaved to remove peach fuzz, simulated harvest damage, insect damage from peach twig borer and fork-tailed katydid feeding, and simulated hemipteran damage with variable sized punctures) using Earlirich (Er), Babcock (Bc) or Spring Snow (Sp) cv. peaches Fruit brix 1 Fruit firmness 1 Treatment Slope Statistics Slope Statistics Intact (Er and Bc) ------
F 1,41 = 0.001, P = F 1,42 = 0.427, P = Shaved (Sp) y = 5.02 – 0.01x y = 4.45 + 0.02x 0.975 0.517
F 1,24 = 0.605, P = F 1,24 = 0.001, P = Harvest (Er) y = 25.20 – 0.83x y = 16.36 + 0.002x 0.444 0.980
F 1,12 = 0.252, P = y = - 31.17 + F 1,13 = 3.885, P = Harvest (Sp) y = 25.02 – 1.27x 0.625 0.18x 0.070
Peach twig borer F 1,66 = 0.281, P = F 1,66 = 0.021, P = y = 1.46 + 0.15x y = 2.72 + 0.001x (Er) 0.598 0.886
F 1,75 = 3.807, P = y = - 0.009 + F 1,75 = 5.541, P = Katydid (Er) y = 9.06 – 0.71x 0.055 0.012x 0.021
Puncture 1.0 mm F 1,18 = 1.421, P = F 1,18 = 0.978, P = y = -1.74 + 0.27x y = 4.92 – 0.01x (Er) 0.250 0.335 1 Readings on brix or firmness were taken from outside of treated areas and reflected the overall fruit conditions as firmness on damaged spots were unmeasurable. 93
94
Table B3. Ovipositional time by D. suzukii on different fruits or artificial diet media Successful Ovipositional Surface penetration Substrate n Brix 3 oviposition time (sec) 2 force (g) 3
Artificial 27 27 13.3 0.7 a N/A Unmeasurable diet media
Cherry 21 16 103.9 15.4 c 25.1 0.5 a 62 5.2 a
Damaged 28 28 23.0 2.9 b 11.3 0.1 b Unmeasurable peach 1
Shaved 30 17 310.2 31.9 d 12.1 0.3 b 159 4.2 b peach
Unshaved 15 0 N/A 12.1 0.4 b 161 6.1 b peach 1 The skin of the fruit was removed. 2 Values are mean SE and different letters within the column indicate significant difference (Survival analysis, log-rank test) and the significance of pair comparison was adjusted to a table-wide level of 5% using the sequential Bonferroni adjustment. 3 Readings on firmness were taken from treated area and values are mean SE and different letters within the column indicate significant difference (One-way ANOVA and Tukey’s HSD, P < 0.05). 95
Fig. B1. The mean (± SE) number of eggs per treated area for intact Earlirich (Er.) and Babcock (Bc) fruit (minus the stem-end area), shaved Spring Snow sections ( 3 cm2), stem-end harvest damaged Earlirich and Spring fruit, peach twig borer (PTB) and the fork-tailed katydid (katydid) damaged Earlirich fruit, and simulated hemipteran feeding damage with variable sized needle punctures (0.3, 0.5 and 1.0 mm). Different letter above each bar represent a significant difference between the intact treatments means (the entire fruit) and the manipulated shaved or damaged fruit sections, regardless of damage size (ANOVA, P < 0.05).
96
Fig. B2. Relationship between the number of eggs deposited by D. suzukii per maximum width of area damaged by (A) the peach twig borer feeding (y = 0.552 + 2.098x, r2 = 0.251, P < 0.001) and (B) the fork-tailed katydid feeding (y = -1.070 + 1.569x, r2 = 0.247, P < 0.001). 97
500 Earlirich Babcock
and and tip (g) 400 Spring Snow
300
200
100
0
Fruit firmness firmness on shoulder Fruit 0 50 100 150 200 250 300
Fruit firmness on stem-end (g)
Fig. B3. Relationship between peach firmness on the stem-end area and the average firmness on the other fruit sections (mean values from three readings between the shoulder and tip of the fruit) for Earlirich (y = 5.214 + 0.484x, n = 130, r2 = 0.715, P < 0.001), Spring Snow (y = –94.054 + 0.949x, n = 15, r2 = 0.681, P < 0.001) and Babcock (y = 26.969 + 0.478x, n = 21, r2 = 0.541, P < 0.001). 98
100 (A) 80 Shaved peach fruit Intact peach fruit 60
40
of oviposition attempt (%) attempt oviposition of 20
0
Cessation 0 100 200 300 400 500 600
100 (B) Shaved peach fruit 80 Intact cherry fruit Damaged peach fruit 60 Artificial diet
of oviposition (%) oviposition of 40
20
Completion 0 0 100 200 300 400 500 600 Time (in seconds)
Fig. B4. Effects of different treatments substrates for (A) unsuccessful oviposition attempts the cessation or giving-up time with intact or shaved peach sections before the female fly left the arena (log-rank test, 2 = 6.0, P = 0.014) and (B) successful ovipositional time with substrates of shaved peach, intact cherry, damaged peach, and artificial diet (log-rank test, 2 =166.2, P < 0.001).
APPENDIX C: STUDY 3 TABLES AND FIGURES
99
Table C1. Maximum and mean (± SE) survival days and number of eggs reproduced per female D. suzukii under the different food provision conditions in field cages in 2013 to 2014 winter Set-up No food Water Honey water Honey water + Orange date Max Mean Max Mean 1 Max Mean1 Max Mean1 Eggs per female2 19 Nov 8 4.4 ± 0.2 10 6.7 ± 0.3 68 22.8 ± 1.9 118 39.3 ± 3.4 4.3 ± 0.5a
27 Dec 7 3.4 ± 0.9 19 4.8 ± 0.4 82 43.4 ± 3.0 87 44.1 ± 3.0 9.5 ± 2.5ab
22 Jan - - 23 7.2 ± 0.8 82 25.4 ± 2.9 90 32.2 ± 2.3 13.4 ± 0.3b
24 Feb - - 11 7.1 ± 0.2 57 23.6 ± 1.4 60 34.1 ± 2.3 13.4 ± 1.6b
28 Mar - - 12 5.0 ± 0.3 25 16.6 ± 0.5 31 14.2 ± 0.8 9.9 ± 0.5ab
1 Longevity data are subject to the survival analyses (see Figs. 1-2). 2 Different letters indicate significant difference (ANOVA and Tukey’s HSD, P < 0.05).
100
101 101
Fig. C1. Survival and development of immature (egg, larva and pupa) D. suzukii in field-cage test in Parlier, CA. (A) Mean daily air and soil temperature during the winter of 2013 to 2014 near the field study site. (B) Mean (± SE) percentage of each immature stage successfully developed into adults when the tests were launched at the 8 different dates from 22 November 2013 to 28 March 2014. Climatic data were obtained from a nearest (300 m) weather station. 102 102
Fig. C2. Survival of adult D. suzukii under different food provision conditions in field cages launched on (A) 19 November, (B) 27 December, (C) 22 January, (D) 24 February, and (E) 28 March during the winter of 2013 to 2014. W = water, HW = honey water, HWO = honey water + orange, F = female, M = male. Different letters to the nearest survival curves indicate significant difference between different food treatments (Survival Analysis, log-rank test, with the significance of paired comparisons adjusted to a treatment-wide level of alpha = 0.05 using the sequential Bonferroni adjustment).
103 103
Fig. C3. Comparison of adult female D. suzukii longevity in the field cage tests during the winter of 2013 to 2014 when honey water and orange as a food and/or ovipositional medium were provided for the adults. Dates on the top of the curves indicate the launching date of the field tests while different letters to the right of the nearest survival curves indicate significant difference among monthly releases (Survival Analysis, log-rank test, with the significance of paired comparisons adjusted to a treatment-wide level of alpha = 0.05 using the sequential Bonferroni adjustment). 104 104
Fig. C4. Oviposition by D. suzukii on post-harvest orange fruit with different conditions. Bars refer to mean ± SE and different letters over the bars indicate significant difference (One-way ANOVA and Tukey’s HSD, P < 0.05)
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