Genetic characterisation of the Rhodanian twaite shad, Alosa fallax rhodanensis

Abstract  Data on the genetic characteristics of the rhodanian shad, Alosa fallax rhodanensis Roule, using three Mediterranean populations (rivers Rhône, Aude and Hérault) and seven protein markers are presented. Allelic frequency distribution, and absolute and relative genetic diversity show a genetically homogeneous structure in the Mediterranean populations. Results when compared with allis ( Alosa alosa Linné) and twaite ( Alosa fallax fallax Lacépède) shad Atlantic populations confirm that the Rhodanian shad belongs to the Alosa fallax Roule species. Furthermore, genetic characteristics of the twaite shad Mediterranean group were quite different from Atlantic populations but remained close to the most southern Atlantic population in the Oued Sebou (Morocco). These findings suggest that Rhodanian shad should be considered a well identified homogeneous group within Alosa fallax rather than a subspecies because of the low genetic distance and some relatively different biological characteristics between the Atlantic and Mediterranean twaite shad groups.     

Novel insights into the marine phase and river fidelity of anadromous twaite shad Alosa fallax in the UK and Ireland

1. Most research on anadromous fishes has been invested in their freshwater life‐phases, resulting in a relatively sparse understanding of their spatial ecology during marine life‐phases. However, understanding the marine dispersal of anadromous fishes is essential to identify threats and to implement conservation measures that fully encompass their lifecycle.
2. The twaite shad Alosa fallax is an anadromous fish increasingly imperilled across its range due to pollution, harvesting, and impediments to freshwater migration, but little is known about its distribution and movements during its marine life‐phase. Here, the application of acoustic telemetry provided novel insights into the coastal dispersal of twaite shad in the UK and Ireland during 2018–2019, and the freshwater entry of individuals during the 2019 spawning season.
3. Of 73 twaite shad acoustic‐tagged during their upstream migration in the River Severn in May 2018, 58 emigrated from the river. Twelve were subsequently detected 200 km to the south‐west at the Taw–Torridge Estuary between July 2018 and April 2019, where estuarine movements up to 5.8 km inland occurred in summer, winter, and spring. One was subsequently detected in the Munster Blackwater Estuary (Ireland) and then in the River Severn, indicating a minimum movement distance of 950 km. Thirty‐four (59%) of the emigrating individuals from 2018 re‐entered fresh water in the rivers Severn (n = 33) and Wye (n = 2) in April and May 2019.
4. These results suggest year‐round use of estuarine and nearshore habitats by at least a subset of the twaite shad population during their marine phase, providing evidence of potential range overlap between populations that spawn in different areas in the UK and Ireland, which may be facilitated by substantial dispersal. The results also highlight the potential of telemetry for estimating freshwater and marine mortality, and the benefits of sharing detection data across networks.

     

Estuarine phase during seaward migration for allis shad Alosa alosa and twaite shad Alosa fallax future spawners

Abstract –  The timing and duration of the estuarine phase during seaward migration were addressed for allis shad and twaite shad, closely related sympatric species in the Gironde system. Otolith Sr:Ca and daily growth increments were used to retrospectively estimate the age at entrance in the estuary, the time spent in estuary and the age at seaward exit from returning adults. The two species exhibited different seaward migration strategies: allis shad entered the estuary at an older age than twaite shad (88 days vs. 59 days), stayed shorter in the estuary (11 days vs. 17 days) and exited to the sea at an older age (99 days vs. 77 days). In the Gironde system, the estuarine phase is very short and occurs within a particular time 'window' for the juveniles that will become future spawners. In this context, it might be suggested that the estuarine phase is a bottleneck for these sympatric shad populations.     

Temporal and spatial variability in growth and condition of dab (Limanda limanda) and sprat (Sprattus sprattus) larvae in the Irish Sea

Variability in the high mortality rate during early life stages is considered to be one of the principal determinants of year‐class variability in fish stocks. The influence of water column stability on the spatial distribution of fish larvae and their prey is widely acknowledged. Water column stability may also impact growth through the early life history of fishes, and consequently alter the probability of survival to maturity by limiting susceptibility to predation and starvation. As a test of this concept, the variability in condition and growth of dab (Limanda limanda) and sprat (Sprattus sprattus) larvae was investigated in relation to seasonal stratification of the water column in the north‐western Irish Sea. RNA/DNA ratios and otolith microincrement analysis were used to estimate nutritional status and recent growth rates of larvae captured on four cruises in May and June of 1998 and 1999. Dab and sprat larvae were less abundant in 1999 and were in poorer condition with lower growth rates than in 1998. Dab larvae of <13 mm also exhibited spatial variability with higher RNA/DNA ratios at the seasonal tidal‐mixing front compared with stratified and mixed water masses. However, the growth and nutritional status of sprat larvae was uncorrelated to water column stability, meaning the more favourable feeding conditions generally associated with the stratified pool and tidal‐mixing front in the Irish Sea were not reflected in the growth and condition of these larvae. This suggests that the link between stability, production and larval growth is more complicated than inferred by some previous studies. The existence of spatio‐temporal heterogeneity in the growth and condition of these larvae has implications for larval survival and the recruitment success of these species in the Irish Sea.     

Modeling environmental,temporal and spatial effects on twaite shad (Alosa fallax) by‐catches in the central Mediterranean Sea

Modeling the relationships between environmental factors and the distribution at sea of species of conservation interest can be useful in predicting their occurrence from a local to a regional scale. This information is essential for planning management and conservation initiatives. In this study, generalized additive models (GAMs) were applied to investigate the influence of environmental, temporal and spatial variables on the catch rates of the twaite shad Alosa fallax (Lacepède) by the pelagic trawl fishery in the north‐central Adriatic Sea. Presence/absence and abundance [catch per unit effort (CPUE)] data between 2006 and 2012 were separately modeled, and the two models were then validated using a test data set. The most important factor influencing the presence and abundance of adult twaite shads was the spatial predictor (latitude × longitude). Two areas of major shads aggregations were observed, the most important of which being located near the estuaries of three main river systems of northern Italy. The twaite shad presence was also significantly affected by season, the largest and lowest occurrences being observed in autumn and spring, respectively. Among the environmental variables tested, only sea surface temperature was included in both models. Alosa fallax showed a wide thermal tolerance (6–27°C) with preference for temperature around 23°C. The model developed from the abundance data showed a moderate predictive power, whereas the accuracy of the presence/absence model was rather low. Some conclusions on the ecological requirements of A. fallax at sea arising from this study are useful to orient future monitoring and research programs and to develop effective conservation actions.     

Feeding larvae of winter flounder Pseudopleuronectes americanus (Walbaum) with live prey or microencapsulated diet: linear growth and protein, RNA and DNA content

A commercial microencapsulated diet was used as a total or partial replacement of live prey for feeding larvae of winter flounder Pseudopleuronectes americanus (Walbaum), a potential alternative finfish species for coldwater marine aquaculture. Growth performance (morphometric measurements and biochemical composition) and nutritional condition (RNA/DNA ratios) of larvae fed live prey (Brachionus plicatilis Müller), a microencapsulated diet or a mixed diet of live prey and microcapsules were compared. Newly hatched larvae were unable to digest microencapsulated diet; live prey at initial feeding was required for their survival and growth. Larvae offered a mixed diet showed slower growth than larvae fed exclusively with live prey. However, at the onset of stomach differentiation, RNA/DNA ratios (indicators of protein synthesis potential) of the larvae fed both diets became similar. We suggest that, at that stage (size 5.5–6.3 mm), enzymatic activity had developed enough to allow digestion of inert food. As the RNA/DNA ratio is a good indicator of nutritional condition, it appears to be an interesting tool for the assessment of diet adequacy in marine larval feeding technology.     

Diel Variation in RNA/DNA Ratios of Japanese Flounder Paralichthys olivaceus

Laboratory-reared Japanese flounder Paralichthys olivaceus larvae (11.2 ± 0.2 mm total length; age 26 d) were sampled over a 48-h period to determine any diel patterns in RNA/DNA ratios. RNA/DNA ratios were highest during daytime periods (0800, 1100, 1400, 1700, 2000 h) and significantly reduced at night (2300, 0200, 0500 h). Findings from this study indicate a diel variation in biochemical condition and suggest that special care should be taken in sampling design and data analysis to account for this source of variability.     

Predator response to releases of American shad larvae in the Susquehanna River basin

Abstract— Predation on American shad ( Alosa sapidissima ) larvae within the first two hours of release was examined from 1989 to 1992 on 31 occasions at stocking sites in the Susquehanna River basin. Twenty-two fish species consumed shad larvae; the dominant predators were spotfin shiner ( Cyprinella spiloptera ), mimic shiner ( Notropis volucellus ) and juvenile smallmouth bass ( Micropterus dolomieu ). The number of shad larvae found in predator stomachs ranged from 0 to 900. Mortality of shad larvae at the stocking site was usually less than 2%. The greatest mortality (9.6%) occurred at the highest stocking level (1.5 million larvae). Highly variable predation rates and release levels of shad insufficient to achieve predator satiation hindered the ability to determine a specific type of functional response of predators. Predator numbers increased with stocking density, indicating short-term aggregation at the release site. Because of practical problems associated with releasing the large numbers of larvae that would be required to satiate predators, routine stocking at these levels is probably unreasonable. Releases of 400,000 to 700,000 larvae may reduce predation by offsetting depensatory mechanisms that operate on small releases and the effects of increased predation due to predator aggregation on large releases. Night stocking may reduce predation on larval shad at the release site.     

Effect of weaning time on the performance of Trachinotus ovatus (Linnaeus 1758) larvae

Effect of weaning time on the larval performance of golden pompano Trachinotus ovatus was studied in the experimental condition. The same weaning regime started on four different days of posthatch (DPH): 13 DPH (W13), 16 DPH (W16), 19 DPH (W19) and 22 DPH (W22), respectively. Growth, survival, RNA/DNA ratios and mid‐gut cell height of fish were assessed in each weaning regime over time. Growth and survival rates of fish larvae weaned in the W19 and W22 regimes were significantly higher than those in the W13 and W16 regimes. No significant differences were found either between W19 and W22, or between W13 and W16 treatments. Fish showed a decrease in the RNA/DNA ratio in W13, but a trend of increase in W16, W19 and W22. Weaning time significantly affected the mid‐gut cell height in larval golden pompano. On 24 DPH, the mid‐gut cell height of fish in the W13 and W16 treatments was significantly thinner than those in the W19 and W22 treatments. Based on fish growth, survival, RNA/DNA ratio and mid‐gut cell height, weaning can be started on 13 DPH in golden pompano larvae, but we recommend that the best weaning window be 16–22 DPH. These results are supported by histological and nutritional evidence and would guide the weaning process for golden pompano larvae, and offer a useful approach to explore the weaning protocol in larval fish rearing.     

Salinity tolerance in young Allis shad larvae (Alosa alosa L.)

Abstract –  Estuarine migration in Allis shad ( Alosa alosa ) usually does not take place before mid-summer, but exposure to salinity in brackish water may occur earlier as many spawning areas are of necessity located in upper estuarine zones due to the placement of dams. Therefore, Allis shad conservation programmes need to consider the risk of mortality due to the ability of young Allis shad to tolerate salinity. To evaluate larval mortality due to the inability of larvae to withstand salinity during early ontogeny, we exposed larvae of different ages (from few days old to 27 days old) to salinities ranging from 0 to 30 g·l⁻¹. Results indicate that direct seawater exposure induces high mortality in young larvae whatever the ontogenetic stage. However, young larvae can easily deal with upper estuary salinity conditions.     

From salmon to shad: Shifting sources of marine‐derived nutrients in the Columbia River Basin

Like Pacific salmon (Oncorhynchus spp.), nonnative American shad (Alosa sapidissima) have the potential to convey large quantities of nutrients between the Pacific Ocean and freshwater spawning areas in the Columbia River Basin (CRB). American shad are now the most numerous anadromous fish in the CRB, yet the magnitude of the resulting nutrient flux owing to the shift from salmon to shad is unknown. Nutrient flux models revealed that American shad conveyed over 15,000 kg of nitrogen (N) and 3,000 kg of phosphorus (P) annually to John Day Reservoir, the largest mainstem reservoir in the lower Columbia River. Shad were net importers of N, with juveniles and postspawners exporting just 31% of the N imported by adults. Shad were usually net importers of P, with juveniles and postspawners exporting 46% of the P imported by adults on average. American shad contributed <0.2% of the total annual P load into John Day Reservoir, but during June when most adult shad are migrating into John Day Reservoir, they contributed as much as 2.0% of the P load. Nutrient inputs by American shad were similar to current but far less than historical inputs of Pacific salmon owing to their smaller size. Given the relatively high background P levels and low retention times in lower Columbia River reservoirs, it is unlikely that shad marine‐derived nutrients affect nutrient balances or food web productivity through autotrophic pathways. However, a better understanding of shad spawning aggregations in the CRB is needed.     

Temperature,plankton and conspecific density influence dynamics of age‐0 gizzard shad: implications for a gape‐limited piscivore

Age‐0 gizzard shad Dorosoma cepedianum are the main prey fish for white crappies Pomoxis annularis in many US reservoirs. However, these prey fish commonly outgrow their vulnerability to white crappie predation in some, but not all, northern Missouri reservoirs. Potential variables that could influence abundance, growth and mortality of age‐0 gizzard shad were examined in three reservoirs that differed with respect to age‐0 gizzard shad growth rates. Because of thermal effluent from a power plant, gizzard shad spawned earlier in Thomas Hill Lake and initial densities of larvae were greater than in the other reservoirs. Larval and juvenile gizzard shad grew slowest in Thomas Hill Lake, followed by Mark Twain Lake and Long Branch Lake. Growth rate of larvae increased with increasing water temperature and food abundance, but decreased with increasing conspecific density. Similar relationships were found for juvenile growth, except that growth declined with increasing temperature. The slower growth of larvae and juveniles in Thomas Hill Lake was probably a consequence of their greater densities relative to their food abundance and higher water temperatures during the juvenile stage. Conversely, both larvae and juvenile gizzard shad grew more rapidly and juveniles attained large sizes in Long Branch Lake owing to their lower densities relative to their available food. Mortality of larvae and juveniles was mostly similar among the reservoirs. Because of their greater abundance and slower growth, gizzard shad were available as prey for white crappies for a longer period in Thomas Hill Lake than in the other reservoirs.     

Differences in food availability for Japanese sardine larvae between the frontal region and the waters on the offshore side of Kuroshio

The availability of food for larvae of the Japanese sardine, Sardinops melanostictus , was investigated in the Kuroshio frontal region and the waters on the offshore side of the Kuroshio, the Pacific coast of central Japan, in March 1990 and 1991, respectively. Food availability was assessed by changes in biomass and production of nauplii and small copepods, and RNA/DNA ratios of the larvae during about 2.5 days (the frontal region) or 3 days (the offshore waters) of tracking a drifter released in a pitch of the larvae. The biomas of the nauplii tended to increase with time in the frontal region and to decrease in the waters on the offshore side of the Kuroshio during the drifter tracking periods. The production of small copepods including nauplii in the waters on the offshore de of the kuroshio was 14% of that in the frontal region. The sum of the mean food requirements of the carnivorous macrozooplankters and sardine larvae was 11% of the production of small copepods including nauplii in the frontal region, compared with 136% in the waters offshore of the Kuroshio. The RNA/DNA ratios of postlarvae smaller than 8 mm in the frontal reqion were significantly higher than those in the waters on the offshore side of the Kuroshio ( P < 0.001) It is considered that the food availability for sardine larvae was relatively high in the frontal region and low in the waters on the offshore side of the Kuroshio. The food availability for the larvae probably deteriorated with the offshore shift of the main spawning ground from the frontal region to the waters on the offshore side of the Kuroshio in the latter half of the 1980s.     

First report of a cystic malformation on the upper jaw of hatchery‐reared allis shad Alosa alosa

The anadromous allis shad Alosa alosa has suffered dramatic population declines throughout Europe and is currently considered as endangered throughout its entire distribution range. In order to reestablish allis shad in the River Rhine, which formerly housed one of the largest and most important populations, an EU‐LIFE Project ‘The re‐introduction of allis shad in the Rhine system’ was started in 2007. In course of the LIFE+ Projects, allis shad larvae bred from genitor fish of the Gironde–Garonne–Dordogne population in France were reared in a pilot ex situ stock plant pilot facility in Aßlar, Germany. At an age of 1–2 months, about 100% of these fish developed approximately 0.5‐ to 0.8‐cm large, fluid‐filled, transparent cysts in conjunction with the upper jaw. The performed microbiological, virological, parasitological and histological examinations did not detect any infectious agents. Possible causative agents are discussed with regard to environmental factors and the nutrition of larvae. In conclusion, the observed malformations are considered a sign for a severe health problem and therefore a risk for the successful breeding of allis shad in aquaculture.     

Effects of starvation and recovery on the survival, growth and RNA/DNA ratio in loliginid squid paralarvae

The ability of Loligo opalescens paralarvae to resist and recover from starvation was examined by measuring their survival, growth rate and RNA/DNA ratios during starvation and refeeding. Paralarvae were fed Artemia sp. nauplii, zooplankton and mysid shrimp. Fourteen days after hatching they were separated into five feeding treatments: a control treatment (food was always available) and treatments starved for 2, 3, 4 and 5 days, and then refed. Each day, 5–7 paralarvae from each treatment were anesthetized to measure mantle length and wet weight (WW), and then RNA and DNA were extracted using an ethidium bromide fluorometric technique. Paralarvae did not survive 4 and 5 days of starvation, showing that at 15 days of age and at 16 °C the limit to recovery was 3 days of starvation. Paralarvae starved for 2 and 3 days showed compensatory growth that mitigated the effects of starvation, in that at the end of the experiment (10 days), they attained mean final body weights similar to the control treatment. Differences in the RNA/DNA ratios between control and starved paralarvae were detected within 2 days of food deprivation. For paralarvae starved 2 and 3 days, it took 1 day after refeeding to attain RNA/DNA ratios not significantly different from the control treatment. Additionally, RNA/DNA ratios were highest during the day (0800, 1200, 1600 h) and lowest at night (0000, 0400 h), suggesting daytime feeding activity. Growth rates ranged from − 14% to 21% WW day^− 1 and the resulting equation between RNA/DNA ratio and growth rate (GR) of paralarvae was GR = 1.74 RNA/DNA − 11.79 (R² = 0.70). After starvation, there was a reduction in growth variability in all starved treatments, while growth variability remained high in the control treatment. Findings from the present study indicate that nucleic acids are a valid indicator of nutritional condition and growth in squid paralarvae.     

Developmental changes in digestive enzyme activity in American shad, <Emphasis Type="Italic">Alosa sapidissima</Emphasis>, during early ontogeny

In order to assess the digestive physiological capacity of the American shad Alosa sapidissima and to establish feeding protocols that match larval nutritional requirements, we investigated the ontogenesis of digestive enzymes (trypsin, amylase, lipase, pepsin, alkaline phosphatase, and leucine aminopeptidase) in larvae, from hatching to 45 days after hatching (DAH). We found that all of the target enzymes were present at hatching, except pepsin, which indicated an initial ability to digest nutrients and precocious digestive system development. Trypsin rapidly increased to a maximum at 14 DAH. Amylase sharply increased until 10 DAH and exhibited a second increase at 33 DAH, which coincided with the introduction of microdiet at 30 DAH, thereby suggesting that the increase was associated with the microdiet carbohydrate content. Lipase increased until 14 DAH, decreased until 27 DAH, and then increased until 45 DAH. Pepsin was first detected at 27 DAH and then sharply increased until 45 DAH, which suggested the formation of a functional stomach. Both alkaline phosphatase and leucine aminopeptidase markedly increased until 18 DAH, which indicated intestinal maturation. According to our results, we conclude that American shad larvae possess the functional digestive system before mouth opening, and the significant increases in lipase, amylase, pepsin, and intestinal enzyme activities between 27 and 33 DAH suggest that larvae can be successfully weaned onto microdiets around this age.     

RNA/DNA ratio is an early responding,accurate performance parameter in growth experiments of noble crayfish Astacus astacus (L.)

Recent intensification efforts of astacid culture considerably depend on the appropriate assessment of the animal's physiological condition both for research and application. We conducted a 4 weeks feeding experiment to assess temporal resolution and accuracy of different response parameters (RNA/DNA ratio, RNA per wet weight, carapace length, wet weight, specific growth rate). Juvenile noble crayfish were exposed to five feeding regimes that differed in feeding frequency and food availability. Continuous growth was detected in all feeding regimes with individual increase up to 90% (wet weight) and 17% (carapace length), respectively. Morphometric parameters allowed separation of three weight‐groups or two length‐groups. During the experimental period RNA/DNA ratios showed both decrease (?17%) and increase (+35%), with superior accuracy than morphometric parameters, separating four groups. Based on RNA/DNA ratios, different feeding regimes were detected earlier, with two groups separated already after 3 weeks. RNA/DNA ratio was clearly superior to RNA per unit wet weight, as the latter failed to detect any differences between groups. In conclusion, RNA/DNA ratio is a valuable tool in nutritional studies with freshwater crayfish if overall growth is the key variable of interest.     

胭脂鱼仔鱼的生长和消化酶活力变化

对胭脂鱼(Myxocyprinus asiaticus)孵化后1～37 d仔鱼的体长、体重、RNA/DNA比率、胰蛋白酶、脂肪酶和淀粉酶的酶活力变化进行了测定。结果显示:胭脂鱼仔鱼1～37 d体长稳定增长。体重增加呈阶段性,在1～21 d体重增长缓慢,21～29 d体重增加较快,29～37 d体重快速增加。仔鱼从7 d开始摄食,1～6 d,RNA/DNA比率变化很小,蛋白含量逐渐降低;7～21 d,RNA/DNA比率波动较大,蛋白含量增长缓慢;21～37 d,RNA/DNA比率和蛋白质均增长迅速。生长率(GR)与RNA/DNA比率的回归方程为:GR=1.01RNA/DNA+1.6077(R2=0.8516)。从2、5和7 d开始分别检测到明显的胰蛋白酶、脂肪酶和淀粉酶活力,之后,消化酶活力随着发育先快速地上升然后波动性地下降到一个稳定水平。结果表明:胭脂鱼仔鱼从内源性到外源性营养转化过程中,不同的消化酶能够检测到明显酶活力的时间不同,到仔鱼开始摄食时已经具备消化蛋白质、脂肪和淀粉的能力;RNA/DNA比率可以作为判定仔鱼生长率的可靠指标。     

Effect of starvation on biochemical composition and gametogenesis in the Pacific oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The effects of starvation on biochemical composition and gametogenesis were investigated in the Pacific oyster Crassostrea gigas. Histological analysis, combined with oocyte examination and measurements of protein, glycogen and lipid levels and RNA/DNA ratio from gonad, adductor muscle and mantle tissue of each sex were performed. In the starved groups, C. gigas showed gonad development, but the progress was delayed during the experiment. Glycogen was the first substrate used by C. gigas for dealing with lack of food. While glycogen was rapidly consumed, protein and lipid contents decreased gradually. A decrease in the RNA/DNA ratio in the starved groups in all the body components was found during starvation, illustrating that RNA/DNA ratio was a valid indicator of nutritional condition in C. gigas. A significant increase in water and ash contents and a corresponding decrease in condition index were observed in the starved groups, showing that the water and ash content and condition index were related to the usage of glycogen, lipid and protein reserves in body composition. During starvation, energy reserves were mobilized for survival and gonad development, but spawning was arrested. The information obtained in this study is useful for broodstock management in the Pacific oyster industry.     

Environmental DNA reveals the temporal and spatial extent of spawning migrations of European shad in a highly fragmented river basin

1. Artificial barriers on lowland rivers impede the spawning migrations of anadromous fishes, preventing access to historical spawning areas. In the cryptic European shads Alosa alosa and Alosa fallax (‘shad’ hereafter), this has resulted in population declines across their range. Conservation programmes aim to facilitate the passage of migrators over these barriers and so require baseline information on the spatial and temporal extent of current migrations.
2. Here, a shad-specific environmental DNA (eDNA) assay was used to quantify the spatial extent of shad spawning migrations in the River Severn basin, western England. This basin is characterized by the presence of multiple barriers in the lower catchment. In 2017, the eDNA assay was piloted in the River Teme, an important shad spawning tributary, and then applied in 2018 and 2019 across the lower Severn basin.
3. In all years, shad DNA was detected between mid-May and mid-June, with the maximum spatial extent of shad distribution being in early June when shad eDNA was detected upstream of weirs that were generally considered as impassable. In 2018, this included the detection of shad above the most upstream weir on the main River Severn that required individual fish to have passed six weirs.
4. Although barriers inhibit the spawning migrations of shad, this eDNA assay showed that some highly vagile individuals might be able to ascend these barriers and migrate considerable distances upstream. This suggests that efforts to increase the permeability of these barriers could result in relatively high numbers of migrating shad reaching upstream spawning areas. These results demonstrate that this eDNA assay could also be used across their range, to further quantify the spatial extent of their spawning, including in highly fragmented rivers and those where shad are believed to spawn only occasionally and are rarely observed.