First records of the fishhook waterflea Cercopagis pengoi Ostroumov, 1891 in the Mississippi River Basin, Illinois Waterway, USA

This paper reports the first records of Cercopagis pengoi in the Mississippi River Basin, Illinois, USA. In 2017, a total of 11 C. pengoi were collected from two different locations during zooplankton sampling in the Illinois Waterway. One location was in the Chicago Sanitary and Ship Canal and the second location was in the Calumet-Sag Channel, approximately eight and 35 river kilometers from Lake Michigan, respectively. In 2021, eight additional individuals were encountered in ichthyoplankton samples collected in the Chicago Sanitary and Ship Canal, three river kilometers upstream of the 2017 capture location. The extent and density of C. pengoi within the Illinois Waterway is currently unknown; therefore, additional sampling is warranted to better understand their distribution and abundance to facilitate an assessment of the risks of spread to other connected waterways.


Introduction
Modern commercial and recreational shipping traffic has contributed to the dispersal of non-native and invasive species worldwide (Cohen and Carlton 1998).Highly modified large river systems are utilized as commercial and recreational corridors between larger water bodies and thus have a higher susceptibility to introductions of non-native species (Strayer 2010).The Illinois Waterway (IWW; Illinois, United States of America -USA) is a large river system that connects the Laurentian Great Lakes and Mississippi River basins.This river system has been heavily modified from its natural state (e.g., Lake Michigan flow diversion and creation of levee and drainage districts and lock and dams) to control flooding and facilitate vessel navigation (Lian et al. 2012) and remains a heavily trafficked corridor used to transport millions of tons of goods (Grippo et al. 2014) through the center of the USA.  1.
The main stem of the Illinois Waterway consists of nine navigational lock and dams and is comprised of the Chicago Area Waterway System (CAWS), portions of the Des Plaines River, and the entire Illinois River (Figure 1).Historically, a variety of non-native and potentially invasive species have entered the IWW from both an upstream pathway via the Great Lakes (e.g., round goby Neogobius melanostomus, Irons et al. 2006;dreissenid mussels, Benson 2013) and a downstream pathway by way of the Mississippi River (e.g., silver carp Hypophthalmichthys molitrix, Sass et al. 2010; scud Apocorophium lacustre, Keller et al. 2017).
The probability of detecting invasive species present at low densities can be challenging because it requires extensive sampling effort due to their low density and potentially fragmented spatial distribution within a system.The behavior (e.g., aggregation), noticeability (e.g., size, Harvey et al. 2009), and catchability of some taxa can further hinder detection.For example, smallbodied species such as zooplankton, phytoplankton, and macroinvertebrates are often difficult to detect because of their small size and specialized sampling techniques.Furthermore, sample processing and identification of small taxa is tedious and labor intensive relative to larger organisms such as fish.Consequently, new observations of these species from their invaded range are often not discovered until higher densities are present in a system (e.g., MacIsaac et al. 2002;Harvey et al. 2009).
Commonly known as the fishhook waterflea, Cercopagis pengoi (Cladocera: Cercopagidae; Figure 2) is a large (6-13 mm), predaceous zooplankton native to the Ponto-Caspian region that was first reported from North America in Lake Ontario in 1998 (MacIsaac et al. 1999) and then quickly spread to Lake Michigan (Charlebois et al. 2001), Lake Erie (Vanderploeg et al. 2002), and Lake Huron (USEPA 2008) by 2002.Cercopagis pengoi is a prolific invader that has the ability to reproduce through parthenogenesis (Uitto et al. 1999;Gutkowska and Paturej 2010) and can tolerate a wide range of temperature, salinity (Gorokhova et al. 2000), and water depth (Gorokhova et al. 2000;Laxson et al. 2003).Invasive C. pengoi negatively impacts native zooplankton communities directly through predation and indirectly through changes in native zooplankton behavior and size structure (Laxson et al. 2003;Vanderploeg et al. 2002).Their invasion in North American lakes has been associated with declines in native zooplankton density (e.g., herbivorous Cladocera, cyclopoid copepods, and rotifers, Makarewicz et al. 2001;BenoÍt et al. 2002;Laxson et al. 2003;Brown and Balk 2008) and biomass (e.g., calanoid and cyclopoid copepods, Bowen and Johannsson 2011) and disruption of native zooplankton diel vertical migrations within the water column (BenoÍt et al. 2002).The direct and indirect effects of C. pengoi on native fish communities have been challenging to quantify (Ojaveer et al. 2001;Vanderploeg et al. 2002) but range from direct resource competition with native planktivorous fishes to C. pengoi serving as a food resource.For example, adult alewife (Alosa pseudoharengus) in the Great Lakes incorporate C. pengoi into their diets (Charlebois et al. 2001), but juvenile alewife do not consume C. pengoi -presumably because the long caudal process of this non-native Cladoceran makes ingestion difficult (Bushnoe et al. 2003;Warner et al. 2006).Cercopagis pengoi also impacts the Great Lakes commercial and recreational fishing industry by fouling fish nets and lines when they attach in dense clusters to fishing gear with their hook-shaped caudal process (Gutkowska and Paturej 2010).
These observed environmental changes in the Great Lakes plankton and fish communities indicate C. pengoi establishment in newly invaded areas could have widespread environmental, economic, and social impacts (Grippo et al. 2014).In 2014, the transfer of C. pengoi to the Mississippi River Basin from the Great Lakes through the IWW was identified as high-risk due primarily to natural downstream drift, with the likelihood of establishment ranging from low-high in 0-50 years (Grippo et al. 2014).Here we report the first occurrences of C. pengoi in the Mississippi River Basin from the IWW, representing the southernmost record in Illinois, and among the southernmost known locations in North America (Benson et al. 2023).

Materials and methods
Cercopagis pengoi was collected in the IWW as part of a long-term zooplankton monitoring project by the Illinois Natural History Survey -Illinois River Biological Station and Kaskaskia Biological Station (see Chará-Serna and Casper 2021).The long-term zooplankton monitoring project collected depthintegrated microzooplankton (≤ 20 µm) and macrozooplankton (> 55 µm) samples with a diaphragmatic pump connected to a weighted 7.6 cm diameter hose moved vertically through the entire water column.At each site, a total of 90 L of river water was filtered through a 55 µm mesh filter for macrozooplankton samples and a single 10 L sample for microzooplankton was filtered through a 20 µm mesh filter (Chará-Serna and Casper 2021).All samples were preserved using either Lugol's solution or a 12% sugar-buffered formalin solution and stained with Rose Bengal for laboratory identification.Zooplankton sampling using this methodology was conducted intermittently at 11 locations in the CAWS from 2010-2021 (Table 1; Figure 1).
Table 1.List of sampling sites from 2010-2021 and their latitude, longitude, reach/waterbody within the Illinois Waterway, year(s) sampled, number of 55 µm and 20 µm zooplankton samples collected, number of C. pengoi collected, and date detected.At each site, 90 L of river water was filtered through a 55 µm mesh filter for macrozooplankton samples and 10 L was filtered through a 20 µm mesh filter for microzooplankton.Note that the Arnold's Barge Slip location was sampled by the Shedd Aquarium using 500 µm mesh ichthyoplankton nets rather than zooplankton nets sampling an estimated 22 m 3 via rotary flow meter (22000 L).Abbreviations for names of sample sites are used in Figure 1.Macro-and microzooplankton samples were sub-sampled prior to identification.All macrozooplankton samples were enumerated and identified from a minimum 10% volumetric subsample from a known volume of all individuals in a Ward Whipple counting wheel or a gridded petri dish under a dissecting scope at 10x-60x.Subsamples were processed until at least 100 individuals across all taxa were counted and categorized as either the phylum Rotifera, order Cladocera, or subclass Copepoda (juvenile copepods classified as nauplii; Thorp and Covich 2001).Microzooplankton samples were enumerated and identified from a minimum 10% volumetric subsample from a known concentrated volume in a Sedgewick-Rafter counting cell under a compound microscope.Subsamples of microzooplankton were processed until at least 400 individuals across all taxa were counted and categorized as either the phylum Rotifera, copepod nauplii, or veligers.Most taxa were identified to genus except for Copepoda nauplii.When C. pengoi was detected in a subsample, the entire sample was scrutinized for C. pengoi to avoid missing large numbers of individuals because they can form clusters by attaching to each other with their hook-shaped caudal process (Grippo et al. 2014).

Results and discussion
Numerous zooplankton collections in the IWW from 2010 -2016 (Table 1) did not detect C. pengoi.Cercopagis pengoi was first collected in the IWW in 2017 near Western Avenue in the Lockport Pool of the Chicago Sanitary and Ship Canal and near Worth Avenue in the Calumet-Sag Channel (Table 1; Figure 1).One individual was collected at the Western Avenue location and 10 individuals were captured at the Worth Avenue location (Table 1).These are the first records of C. pengoi in the IWW-and to the best of our knowledge-are the first records of the species in the Mississippi River Basin, USA.In 2021, eight additional C. pengoi were collected in Arnold's Barge Slip located three river kilometers upstream of the Western Avenue location (Table 1; Figure 1).These eight specimens were encountered in samples collected using 500 µm mesh ichthyoplankton nets (50 cm diameter opening, towed 110 m sampling an estimated 22 m 3 via rotary flow meter [22000 L; General Oceanics]) by the Daniel P. Haerther Center for Conservation and Research, John G. Shedd Aquarium (hereafter "Shedd Aquarium"; Austin Happel, personal communication).Additional long-term zooplankton monitoring sites along the length of the IWW near Lake Michigan to its confluence with the Mississippi River were also routinely sampled from 2010-2021; no C. pengoi were collected, suggesting that its distribution is currently limited to within the CAWS.
Cercopagis pengoi was found sporadically-and in low numbers-in the IWW, indicating this population does not appear to have undergone a rapid, post-introduction-expansion like that experienced in the Great Lakes.It is likely that Lake Michigan is the source population of C. pengoi in the IWW given it shares several direct connection with Lake Michigan's southwestern coastline (Figure 1), but we have no genetic data to confirm this assumption.It remains uncertain whether these new records represent a new expansion or a low-density "sleeper" population that has existed undetected prior to our first records in 2017 (Spear et al. 2021).
In Eurasia, invasive populations of C. pengoi can be found in lotic systems (e.g., Kama and Volga rivers, Panov et al. 2007 and references therein; Lazareva 2019), but much of the IWW, and some connected lotic systems, may not be ideal habitats for C. pengoi (Muirhead et al. 2011).For example, Grippo et al. (2014) suggested that C. pengoi had a low probability of establishment in the CAWS due to potentially unsuitable habitat such as shallow water depth and high turbidity.Nonetheless, C. pengoi can invade lakes with a wide range of turbidity (4.4-105.2NTU; Muirhead et al. 2011) and have been found within the uppermost 20 m of the water column (Ojaveer et al. 2001).Water depth within the CAWS ranges from 2.4-8.2m (Sinha et al. 2013).Water turbidity in the IWW is highly variable and largely coincident with agricultural land use, with turbidity (average NTU values from June-November 2011-2015) increasing downstream from 20.2 NTU in the upper river to 42.4 NTU in the middle river to 47.7 NTU in the lower river (Parker et al. 2016;Chará-Serna and Casper 2021).During 2021, Shedd Aquarium measurements of turbidity (0.5 L water sample, tested via desktop turbidimeter within 24 hours of collection via 3 subsamples) at three mainstem locations within the CAWS, one location upstream of the 2021 detection site (Lawrence Fish and Shrimp's dock) and two locations downstream (Park 571 and Richard Daley Park docks), found a range of 4.0 to 10.0 NTU (mean = 5.7 NTU) across locations (Table 1; Figure 1).In 2022, an off-channel location (Bubbly Creek) near Arnold's Barge Slip where C. pengoi were found in 2021, had an average turbidity of 11.8 NTU (range 8.0-18.0NTU), whereas the same main stem sites averaged 6.3 NTU and generally ranged between 4.0 and 8.0 NTU.These average turbidity values in the IWW are within the range of preferred turbidity reported for C. pengoi found in lentic systems.As there are likely to be several factors influencing C. pengoi expansion into the IWW from Lake Michigan and the likelihood of further spread throughout the IWW and connected waterways, future work should seek a more comprehensive understanding of their distribution in the IWW and assess how their distribution relates to abiotic conditions, such as water chemistry.For now, it appears that C. pengoi is limited to areas in the extreme upper portion of the IWW (within ~ 8 river kilometers downstream of Lake Michigan near Chicago, IL and ~ 35 river kilometers downstream of Lake Michigan in the Calumet-Sag Channel) where water chemistry is influenced by a mixture of urban runoff, stormwater, wastewater discharge, and Lake Michigan water (Figure 1; Wang et al. 2021).
In summary, these are the first records of C. pengoi in the IWW and the Mississippi River Basin and are currently among the southernmost locations in North America (Benson et al. 2023).It remains unknown whether C. pengoi will thrive in the IWW (as it has in some European rivers within its invaded range) and have undesired ecosystem impacts, but the possibility of expanding throughout the IWW and into the Mississippi River should be considered.Vigilance during all types of aquatic sampling and diligent reporting of new species occurrences are needed to document the ongoing spread of C. pengoi and other non-native species in the IWW and worldwide.

Figure 1 .
Figure 1.Map of the Illinois Waterway from Lake Michigan to the confluence with the Mississippi River and zooplankton sample sites from 2010-2021.Site abbreviations are in Table1.

Figure 2 .
Figure 2. Photograph of a preserved Cercopagis pengoi collected from the Illinois Waterway in 2017.Photo taken by the Illinois River Biological Station.