Insights into the susceptibility of raspberries to Drosophila suzukii oviposition

The spotted wing drosophila (SWD) causes massive losses in red raspberry (Rubus idaeus L.) cultivation by direct oviposition leading to damages of the soft skin fruits. Knowledge of the fly's host preference could help farmers in managing the pest. We used a laboratory-based oviposition assay for screening the germplasm of Rubus to ascertain whether the spotted wing drosophila prefers certain cultivars to others for oviposition and if preference is based on citric acid and Brix content or firmness. Correlation analyses of evaluated characters with no-choice tests results in 3 years obtained no correlation between citric acid, Brix content and oviposition. Primocane raspberries were lower affected by SWD than floricane raspberries. The Rubus hybrid cultivar “Dorman Red” and the primocane cultivar “Pokusa” showed the lowest oviposition rate compared to the other 58 evaluated genotypes. We found that oviposition correlates to firmness of the investigated cultivars, which strongly indicates that host preference is partly connected to that character in raspberries. The results are discussed regarding the use of Rubus genetic resources in breeding and integrated pest management to control spotted wing drosophila in the field.     

Influences of blackberry margins on population dynamics of Drosophila suzukii and grape infestation in adjacent vineyards

Understanding the dynamics of pest insect populations in relation to the presence of non‐crop habitats and infestation levels of adjacent crops is essential to develop sustainable pest management strategies. The invasive pest species Drosophila suzukii (Diptera: Drosophilidae) is able to utilize a broad range of host plants. In viticulture, scientific risk assessment for D. suzukii has only recently started and studies assessing the effects of field margins containing wild host plants on D. suzukii population dynamics and on infestation risks in adjacent vineyards are lacking. Thus, in a one‐year field study, the role of different field margins on fly abundance and crop infestation in adjacent vineyards of Vitis vinifera, variety “Pinot Noir,” were investigated. Different monitoring methods were conducted to assess fly distribution, sex ratio and grape infestation in 14 vineyards adjacent to field margins containing either blackberry (BB) Rubus spp. or non‐host (NH) plants. Our results show that blackberries strongly enhanced D. suzukii abundance within field margin vegetation all year long, whereas fly abundance in vineyards adjacent to BB margins was just enhanced in some seasonal periods. Moreover, the influence of BB margins was limited by distance. However, high fly numbers in BB field margins did result in zero egg infestation of “Pinot Noir” berries. These results may have important implications for winegrowers to make efficient management decisions: regardless of high abundance of adult D. suzukii, only grape berry monitoring can assess the actual infestation risk and the potential need to take management action.     

Diet quality mitigates intraspecific larval competition in Drosophila suzukii

The invasive frugivore Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) utilizes a wide range of host plants and damages important fruit crops, including blueberries, cherries, blackberries, raspberries, and strawberries. Field infestations of D. suzukii often exceed one larva per berry, suggesting that intraspecific competition may frequently occur. Because dietary resources are also likely to vary across the host range of D. suzukii, we designed a laboratory assay to measure larval performance across diets of varying quality: a standard artificial diet, a fruit‐based medium, a low‐protein, and a low‐carbohydrate diet. We manipulated egg density across these diets to provide increasing levels of competition and measured larval performance by observing survival to pupation and adulthood, and development times for both life stages. Although increasing density generally negatively impacted D. suzukii performance across diets, the magnitude of these impacts varied by diet type. Drosophila suzukii performance was generally similar in fruit and standard diets, although larval development was more rapid in fruit diets at lower densities. Even at low densities (5 or 10 eggs per arena), survival was reduced and development time increased in low‐protein diets relative to standard and fruit diets. At the two highest larval densities (20 or 40 eggs per arena), survivorship was reduced in low‐carbohydrate diets as compared to standard and fruit diets. There is evidence that larvae compensated in both low‐quality diets by extending development time, which could have consequences for population dynamics. Population models for use in D. suzukii management may need to account for both host nutritional quality and relative competition to accurately predict turnover and geographic expansion.     

Susceptibility of cold hardy grapes to Drosophila suzukii (Diptera: Drosophilidae)

Drosophila suzukii (Diptera: Drosophilidae), known commonly as spotted wing drosophila, is a vinegar fly originating from South‐East Asia and a major pest to many soft‐skinned fruits. Due to the species recent arrival in North America in 2008, many fruit varieties are yet untested for susceptibility to infestation. While previous work has focused on Vitis vinifera, this study aimed to determine grape susceptibility of cold hardy varieties based on hybrids of V. labrusca, V. riparia and V. vinifera. Field sampling was conducted in Southern Wisconsin (USA) vineyards to establish adult and larval abundance and determine whether the number of adults caught in traps correlates with fruit infestation. Host susceptibility was further assessed through no‐choice bioassays of both intact and damaged fruits. The field study found D. suzukii adults present in all varieties, low larval abundance and no correlation between adult abundance and larval presence. Peak adult abundance occurred mid‐season between veraison and harvest, while larval infestation rates were highest near harvest. In laboratory no‐choice tests, significantly more eggs, larvae and adults occurred in damaged than undamaged grapes. In damaged grapes, larvae and adult abundance was comparable between varieties and to the highly susceptible control of undamaged raspberry; however, D. suzukii developed significantly faster in raspberry than grapes. Fruit characteristics (°Brix, titratable acidity, pH) in grapes were uncorrelated with D. suzukii performance. Together, these findings suggest that cold hardy grapes are overall resistant to D. suzukii if intact and highly susceptible if damaged.     

Seasonal morphotypes of Drosophila suzukii differ in key life‐history traits during and after a prolonged period of cold exposure

Seasonal polyphenism in Drosophila suzukii manifests itself in two discrete adult morphotypes, the “winter morph” (WM) and the “summer morph” (SM). These morphotypes are known to differ in thermal stress tolerance, and they co‐occur during parts of the year. In this study, we aimed to estimate morph‐specific survival and fecundity in laboratory settings simulating field conditions. We specifically analyzed how WM and SM D. suzukii differed in mortality and reproduction during and after a period of cold exposure resembling winter and spring conditions in temperate climates. The median lifespan of D. suzukii varied around 5 months for the WM flies and around 7 months for the SM flies. WM flies showed higher survival during the cold‐exposure period compared with SM flies, and especially SM males suffered high mortality under these conditions. In contrast, SM flies had lower mortality rates than WM flies under spring‐like conditions. Intriguingly, reproductive status (virgin or mated) did not impact the fly survival, either during the cold exposure or during spring‐like conditions. Even though the reproductive potential of WM flies was greatly reduced compared with SM flies, both WM and SM females that had mated before the cold exposure were able to continuously produce viable offspring for 5 months under spring‐like conditions. Finally, the fertility of the overwintered WM males was almost zero, while the surviving SM males did not suffer reduced fertility. Combined with other studies on D. suzukii monitoring and overwintering behavior, these results suggest that overwintered flies of both morphotypes could live long enough to infest the first commercial crops of the season. The high mortality of SM males and the low fertility of WM males after prolonged cold exposure also highlight the necessity for females to store sperm over winter to be able to start reproducing early in the following spring.     

Metagenomic analysis reveals changes of the Drosophila suzukii microbiota in the newly colonized regions

The spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a highly polyphagous pest of a wide variety of wild or cultivated berry and stone fruit. Originating from Southeast Asia, it has recently invaded a wide range of regions in Europe and North America. It is well known that insect microbiotas may significantly influence several aspects of the host biology and play an important role in invasive species introduction into new areas. However, in spite of the great economic importance of D. suzukii, a limited attention has been given so far to its microbiota. In this study, we present the first in‐depth characterization of gut bacterial diversity from field (native and invasive range) and lab‐reared populations of this insect. The gut bacterial communities of field insects were dominated, regardless of their origin, by 2 families of the phylum Proteobacteria: Acetobacteraceae and Enterobacteriaceae, while Firmicutes, mainly represented by the family Staphylococcaceae, prevailed in lab‐reared population. Locality was the most significant factor in shaping the microbiota of wild flies. Moreover, a negative correlation between diversity and abundance of Enterobacteriaceae and the time elapsed since the establishment of D. suzukii in a new region was observed. Altogether our results indicate that habitat, food resources as well as the colonization phase of a new region contribute to shape the bacterial communities of the invasive species which, in turn, by evolving more quickly, could influence host adaptation in a new environment.     

Oviposition and development potential of the spotted‐wing drosophila,Drosophila suzukii (Diptera: Drosophilidae), on uninjured Campbell Early grape

The spotted‐wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), originally distributed across a few Asian countries including South Korea, has invaded North America and Europe but is absent from Australia. In order to export the South Korean grape cultivar Campbell Early to Australia, its potential to serve as an oviposition and development medium for SWD must first be determined. In this study, we determined the oviposition and development potential of SWD on Campbell Early, after elucidating the SWD life cycle and establishing an artificial diet‐based mass‐culturing system. An investigation of the life cycle under five temperature regimes (16, 19, 22, 25 and 28°C) showed that the durations of the egg, larval and adult stages were shortened when temperature was increased from 16, 19, 22, 25 and 28°C, but pupal duration was shortest at 25°C and extended again at 28°C. A test of oviposition and development potential of SWD on Campbell Early grape clusters showed oviposition of 30.8 ± 6.8 eggs per cluster of injured grapes and 157.7 ± 16.2 eggs on a culture dish of artificial diet. However, in a similar experiment using uninjured grape clusters, only a single egg was deposited on the grape skin, which soon dried. In light of these results, newly harvested grapes left at vineyards during daily harvests are unlikely to serve as an oviposition and development medium for SWD, as long as the grapes remain uninjured.     

The effect of border sprays and between‐row soil tillage on Drosophila suzukii in organic blackberry production

Drosophila suzukii (Matsumura) is a major pest of soft‐skinned fruit. Females have an enlarged serrated ovipositor that is used to cut into ripening fruit and lay their eggs. Larvae develop inside infested fruit, rendering fruit unmarketable. Previous research has indicated that D. suzukii can move from adjacent woodlands into cultivated fields. Furthermore, multiple generations can occur in a single season as a result of fallen, infested fruit in the fields. Our hypothesis was that border sprays and soil tillage of field aisles can reduce D. suzukii presence in commercial blackberry fields (Rubus spp.). To test our hypothesis, we conducted split‐plot field trials in organic blackberry fields for 3 and 4 weeks in 2014 and 2015, respectively. Treatments were border sprays (whole plot, pyrethrins + azadirachtin) and tillage (subplot, ~15 cm). We evaluated adult D. suzukii in both years and berry infestation and natural enemies in 2015 only. We found that plots with border treatments had fewer D. suzukii (larvae and adults) than plots without border sprays. Tilling the soil between rows of blackberry bushes did not have a significant effect on adult captures or larval infestation of fruit. Natural enemies were unaffected by the border spray and tillage treatments. Our results confirmed our hypothesis that border sprays can be utilized to reduce populations of D. suzukii in organic blackberry fields, while maintaining populations of natural enemies. However, the effect of soil tillage is unclear and requires further investigation. Additional research should investigate the timing of border sprays and their effect on high infestations of D. suzukii as well as quantify fruit fall and depth of burial to reduce D. suzukii emergence using soil tillage.     

Effects of fluctuating thermal regimes on cold survival and life history traits of the spotted wing Drosophila(Drosophila suzukii)

Drosophila suzukii is an invasive pest causing severe damages to a large panel of cultivated crops.To facili tate its biocontrol with stratcgies such as sterile or incompatible insect techniques,D.suzukid must be mass-produced and then stored and transported under low temperature.Prolonged cold exposure induces chill injuries that can be mitigated if the cold period is interrupted with short warming intervals,referred to as fluctuating thermal regimes(FTR).In this study,we tested how to optimally use FTR to extend the shelf life of D.suzukii under cold storage.Several FTR parameters were asessed:temperature(15,20,25℃),duration(0.5,1,2,3 h),and frequency(every 12,24,36,48 h)of warming intervals,in two wild-type lines and in two developmental stages(pupac and adults).Generally,FTR improved cold storage tolerance with respect to constant low temperatures(CLT).Cold mortality was lower when recovery temperature was 20℃ or higher,when duration was 2 h per day or longer,and when warming interruptions occurred frequently(every 12 or 24 h).Applying an optimized FTR protocol to adults greatly reduced cold mortality over long-term storage(up to 130 d).Consequences of FTR on fitness-related traits were also investigated.For adults,poststorage survival was unaffected by FTR,as was the case for female fecundity and male mating capacity.On the other hand,when cold storage occurred at pupal stage,postorage survival and male mating capacity were altered under CLT,but not under FTR.After storage of pupae,female fecundity was lower under FTR compared to CLT,suggesting an energy trade-off between repair of chill damages and C22 production.This study provides detailed information on the application and optimization of an FTR-based protocol for cold storage of D.suzuki that could be useful for the biocontrol of this pest.     

Distribution and activity of Drosophila suzukii in cultivated raspberry and surrounding vegetation

Spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), may utilize wild ‘Himalaya’ blackberry (HB) Rubus armeniacus Focke or other non‐crop plants as refugia and possibly exploit adjacent field margins before colonizing cultivated fruiting crops. Studies were conducted to determine the role of field margins containing HB and their effect on D. suzukii activity, density and distribution in an adjacent commercial red raspberry crop. One‐ha plots adjacent to field margins containing HB or known non‐host (NH) grass crops were established in 2011 and 2012 and replicated three times. Each plot contained two transects with monitoring traps for D. suzukii in the field margin (0 m) and spaced approximately 10 (crop boundary), 40, 70 and 100 m into the adjacent crop (n = 10 traps/plot). Field margin vegetation was treated with a 10% chicken egg white mark solution weekly from pre‐harvest until the end of harvest using a cannon sprayer. Adult D. suzukii were collected from traps weekly and analysed for the presence of the egg white mark using an egg white‐specific enzyme‐linked immunosorbent assay (ELISA). During both years, marked flies and total flies were captured in higher numbers in HB field margins, whereas virtually no flies were captured in field margins containing no known alternative host. Similarly, more flies were captured in the crop near HB than near NH. Spatial Analysis by Distance IndicEs (SADIE) and mean D. suzukii trap captures additionally displayed significantly higher fly densities in the raspberry field near HB than near NH. These results suggest that HB may contribute to elevated D. suzukii populations and pest pressure in comparison with field margins containing no known alternate host vegetation for D. suzukii. Having closely adjacent non‐crop alternate host landscapes may result in increased D. suzukii pest pressure.     

Effects of sweet alyssum flowers and their volatile compounds on Drosophila suzukii (Matsumura) in the laboratory

Drosophila suzukii (Matsumura) is a major global pest of soft fruit crops. Based on field observations, we tested in the laboratory whether sweet alyssum (Lobularia maritima (L.)) flower volatiles and their major constituent compounds, acetophenone and benzaldehyde, repelled D. suzukii flies. Volatiles from cut flowers and acetophenone reduced numbers of D. suzukii attracted to raspberries, and acetophenone reduced numbers of larvae in the raspberries. Testing of sweet alyssum plantings or dispensing acetophenone to repel D. suzukii in fields and lower fruit infestation should be conducted.     

Surveying native egg parasitoids and predators of the invasive Halyomorpha halys in Northern Italy

The invasive Halyomorpha halys (Heteroptera: Pentatomidae) is a key pest of fruits in the Emilia‐Romagna region of Italy. For the development of a sustainable management programme, knowledge of its native natural enemy community and its efficacy is essential. A three‐year field survey was conducted exposing H. halys egg masses in different types of habitats to investigate the efficacy of native natural enemies in reducing the H. halys populations in the Emilia‐Romagna region, where the stinkbug was first detected in 2012. Over the first year of the study, sentinel eggs from laboratory cultures were stapled to the underside of leaves in various host trees, whereas in following years H. halys adults were directly caged on branches in sleeve cages to allow natural oviposition. Over the examined years, low rates of parasitism (1%–3%) and predation (2%–5%) were observed. Parasitism was caused exclusively by the generalist parasitoid Anastatus bifasciatus.     

Going feral: Time and propagule pressure determine range expansion of Asian house geckos into natural environments

Upon establishment in a new area, invasive species may undergo a prolonged period of relatively slow population growth and spread, known as a lag period. Lag periods are, apparently, common in invasions, but studies of the factors that facilitate subsequent expansions are lacking in natural systems. We used 10 semi‐independent invasions of the Asian house gecko (Hemidactylus frenatus) to investigate which factors facilitate expansion of this human‐associated species across the urban–woodland interface. We conducted 590 surveys over 12 months on 10 transects running from the urban edge to 2 km into adjacent natural woodland. We recorded H. frenatus out to 2 km from the urban edge on nine of 10 transects, and at high abundance at many woodland sites. Body size, body condition, sex ratio and proportion of gravid females did not vary with distance from the urban edge, suggesting viable, self‐sustaining populations in natural habitats. The extent of expansion was, however, strongly dependent on propagule pressure (the abundance of H. frenatus at the urban edge), and time (time since H. frenatus established in the urban area). The size of the urban area and the structure of the surrounding environment did not impact invasion. Our results show that an invasive species that is deemed ‘human‐associated’ over most of its range is invading natural habitats, and propagule pressure strongly controls the lag time in this system, a finding that echoes results for establishment probability at larger scales.     

Oviposition preference and larval development of the invasive moth Cydalima perspectalis on five European box‐tree varieties

The box‐tree pyralid Cydalima perspectalis (Walker 1859) (Lepidoptera: Pyralidae), native to Eastern Asia, is a newly introduced species causing severe damage to box‐trees (Buxus sp.) in private and public gardens as well as in semi‐natural box‐tree forests in Central Europe. It is so far not known whether different box‐tree subspecies (or varieties) are similarly affected by this invasive moth. In a choice experiment offering branches of five different box‐tree varieties as oviposition sites, we found a preference of female moths for laying their egg clusters on the variety ‘Rotundifolia’, while other varieties were less frequently considered. The preference for ‘Rotundifolia’, the variety with the largest leaves in the tests, remained when intervariety differences in foliar area (mean leaf size × number of leaves) were taken into account. Feeding larvae on leaves of either of the five box‐tree varieties revealed a significant effect of the seasonal generation of C. perspectalis on the growth rate of individuals but no influence of the box‐tree variety. Larvae from the spring generation show the highest growth rate, those from the summer generation a moderate and those from the autumn generation the lowest growth rate. The moths used in the experiments may belong to the 10th to 12th generation present in Europe. The time elapsed since their introduction may be too short for an optimal adaptation to the partly novel diet encountered by the invasive moth.     

Drosophila suzukii in Michigan vineyards,and the first report of Zaprionus indianus from this region

Drosophila suzukii is a new invasive pest that in recent years has become established in the Great Lakes region of the United States. Understanding the level of infestation in potentially susceptible crops is an important first step for planning appropriate management responses. This study was conducted in 2010–2012 to determine the infestation potential of this pest in native Vitis labrusca, French hybrid and V. vinifera grape cultivars grown in Michigan vineyards. Drosophila suzukii adults were reared out of collected grape samples in all 3 years, comprising a low proportion of all emerged drosophilids in each of the years. This trend was also found in vacuum sampling, conducted in 2011, with the majority of flies collected being non‐D. suzukii drosophilids. Another recently introduced invasive fly species, Zaprionus indianus, was also reared out of grape samples collected in 2012. While the results of this study indicate no immediate threats to commercial grape production from D. suzukii, further research is needed to elucidate possible secondary effects that this species may have on vineyards, such as the introduction of diseases to the fruit.     

Asian Long‐horned Beetle dispersal potential estimated in computer‐linked flight mills

The Asian Long‐horned Beetle (ALB) is a highly polyphagous species invasive in North America and Europe. This species has been reported to have low dispersing potential, but long‐distance dispersal could occasionally happen. We conducted a preliminary study on laboratory‐reared adults from invasive populations to measure the flying potential of beetles using computer‐linked flight mills. Under standardized conditions, ALB was capable of flying over longer distances than previously described. The highest distance recorded over an adult lifespan outreached 14 km. Flight mill method is therefore useful to estimate the maximum physiological flight abilities of the species that should be taken into account to improve management of invasive populations.     

Effects of temperature and relative humidity on mating and survival of sterile Drosophila suzukii

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a widely distributed pest species of soft-skinned fruits. Recent studies suggest the use of sterile insect technique (SIT) as a control method for this species; however, many factors can impact effectiveness of a SIT programme, including the environmental conditions. Environmental condition is critical at the time of the release and in the days afterwards, since it may impact sterile insects’ survival and ability to mate. Thus, we verified the influence of temperature and relative humidity on mating and survival of fertile and sterile D. suzukii, when insects were food provided or deprived. Highest mating rates occurred when sterile or fertile flies provided with food were exposed to 25ºC or 81%–100% relative humidity, while temperatures of 10 and 35ºC and humidity below 60% impaired mating. Overall, mating rate among food-deprived flies was low in all temperatures and humidity levels tested, but fertile insects were more prone to mate when compared to sterile flies. Survival was negatively influenced by high temperatures, low relative humidity and food deprivation. The information present in this study is useful to be considered for release of sterile D. suzukii.     

Reproductive interference by male Drosophila subobscura on female D. persimilis: A laboratory experiment

While females often reject courtship attempts by heterospecific males, reproductive interference by harassment from such males can nonetheless occur, potentially reducing female fitness. Such effects may be profound following a range expansion, when males from a new species may suddenly encounter (and perhaps even become abundant relative to) females of related native species. Drosophila subobscura recently invaded North America and may impact native species through reproductive interference and other processes. We test for the potential for reproductive interference by D. subobscura males on D. persimilis females in the laboratory. D. subobscura males aggressively copulated with D. persimilis females, including many females that exhibit rejection behaviors. Despite females attempting to dismount the males, the heterospecific copulations are on average longer than conspecific copulations, and females exhibit some reluctance to remate with conspecific males following this harassment. Females confined with both conspecific and heterospecific males produce fewer adult progeny than those with either conspecific males only or with conspecific males and distantly related D. simulans males that do not engage in female harassment. Overall, our results illustrate how reproductive interference by an invasive species can have negative effects on resident natural populations.     

Single and combined effects of Drosophila suzukii and Drosophila melanogaster on sour rot development in viticulture

Sour rot is a disease complex that causes serious damage in viticulture. The common vinegar fly Drosophila melanogaster (Diptera: Drosophilidae) is associated with sour rot in overripe or otherwise damaged grapes. Drosophila suzukii (Diptera: Drosophilidae) is an invasive species, which is suspected to induce sour rot in previously undamaged grapes due to the flies' ability to infest healthy, undamaged soft fruits with its serrated ovipositor. As a consequence, infection of healthy grapes by D. suzukii may facilitate the colonization by D. melanogaster. We investigated the single and combined effects of D. suzukii and D. melanogaster on sour rot development by measuring volatile acidity under near-natural conditions in the vineyard, along with laboratory experiments under controlled climate. In 2017, the combined field and laboratory experiments suggested that the presence of D. suzukii and D. melanogaster increased the volatile acidity levels at a similar rate. In 2018, the field experiments showed an only marginal increase in sour rot development in treatments with both Drosophila species. Under more favourable laboratory conditions, the presence of D. suzukii, but not D. melanogaster triggered sour rot emergence. A facilitating effect of D. suzukii infestation for D. melanogaster was not detectable. These findings suggest that D. suzukii does in fact have the potential to trigger sour rot, but will probably rarely do so under field conditions in the vineyard, at least in the studied region. Instead, our study showed that D. melanogaster can have a similar impact on sour rot development as D. suzukii, emphasizing the need of comparative studies.     

Invasion genetics from eDNA and thousands of larvae: A targeted metabarcoding assay that distinguishes species and population variation of zebra and quagga mussels

Identifying species and population genetic compositions of biological invasions at early life stages and/or from environmental (e)DNA using targeted high‐throughput sequencing (HTS) metabarcode assays offers powerful and cost‐effective means for early detection, analysis of spread patterns, and evaluating population changes. The present study develops, tests, and applies this method with a targeted sequence assay designed to simultaneously identify and distinguish between the closely related invasive Eurasian zebra and quagga mussels (Dreissena polymorpha and D. rostriformis) and their relatives and discern their respective population genetic patterns. Invasions of these dreissenid mussel species have markedly changed freshwater ecosystems throughout North America and Europe, exerting severe ecological and economic damage. Their planktonic early life stages (eggs and larvae) are morphologically indistinguishable, yet each species exerts differential ecological effects, with the quagga often outcompeting the zebra mussel as adults. Our targeted assay analyzes genetic variation from a diagnostic sequence region of the mitochondrial (mt)DNA cytochrome oxidase I (COI) gene, to assess temporal and spatial inter‐ and intra‐specific genetic variability. The assay facilitates analysis of environmental (e)DNA from water, early life stages from thousands of individuals, and simultaneous analysis of 50–100 tagged field‐collected samples. Experiments evaluated its accuracy and performance using: (a) mock laboratory communities containing known DNA quantities per taxon, (b) aquaria with mixed‐species/haplotype compositions of adults, and (c) field‐collected water and plankton versus traditional sampling of adult communities. Results delineated species compositions, relative abundances, and population‐level diversity differences among ecosystems, habitats, time series, and life stages from two allopatric concurrent invasions in the Great Lakes (Lake Erie) and the Hudson River, which had separate founding histories. Findings demonstrate application of this targeted assay and our approach to accurately and simultaneously discern species‐ and population‐level differences across spatial and temporal scales, facilitating early detection and ecological understanding of biological invasions.