Research Article |
Corresponding author: Eric R. Larson ( erlarson@illinois.edu ) Academic editor: Christoph Chucholl
© 2024 Elle K. Sawyer, Jordan H. Hartman, Daniel K. Szydlowski, Eric R. Larson.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Sawyer EK, Hartman JH, Szydlowski DK, Larson ER (2024) Investigating Calico Crayfish (Faxonius immunis Hagen, 1870) as a possible “sleeper” invasive species in northern Wisconsin, United States. Aquatic Invasions 19(2): 191-209. https://doi.org/10.3391/ai.2024.19.2.119829
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“Sleeper” invaders are non-native populations that experience time-lags post-establishment before subsequent spread or negative impacts, challenging managers to differentiate harmless non-native species from invasive species. In lakes of northern Wisconsin, United States, Rusty Crayfish (Faxonius rusticus Girard, 1852) has dominated as an invasive species for decades, but this species has recently experienced population declines. Following these F. rusticus declines, we rediscovered in 2020 a population of non-native Calico Crayfish (Faxonius immunis Hagen, 1870) that had not been documented since the 1970s. Declining F. rusticus populations may create opportunities for F. immunis spread to other lakes and impacts as a sleeper invader. We conducted additional sampling in summer 2021 that suggests F. immunis remains isolated in only one lake within this watershed. We used mitochondrial DNA barcoding to confirm these crayfish were F. immunis and had not been misidentified as a congener. Next, we investigated whether biotic interactions with F. rusticus may have prevented F. immunis spread over the past several decades. We measured agonistic behaviors using F. immunis and F. rusticus pairs in the laboratory, and then modeled differences in aggression between species while controlling for size and reproductive form. We found that F. rusticus were consistently dominant over F. immunis, suggesting that competition with an established hyper-abundant invasive species may have restricted past spread by F. immunis. Managers and policy makers should consider whether precautionary actions against F. immunis are warranted while the population of this species remains small and localized, especially in the context of F. rusticus declines.
Barcoding, behavior, over-invasion, Faxonius rusticus, Rusty Crayfish, serial invasion
Invasive species are non-native species that spread in a new region (
The Rusty Crayfish (Faxonius rusticus Girard, 1852) has been a prominent invasive species in lakes of northern Wisconsin for half a century (
During routine population monitoring for F. rusticus, we captured three Calico Crayfish (Faxonius immunis Hagen, 1870) across 24 overnight traps in Wild Rice Lake, Vilas County, Wisconsin on August 28th, 2020 (Figure
Calico Crayfish (Faxonius immunis) were collected in summer 2020 at two locations (stars) in Wild Rice Lake of the Manitowish River watershed, Vilas County, Wisconsin, United States (inset map). Population declines of Rusty Crayfish (Faxonius rusticus) in six regularly monitored lakes of the watershed (blue) from
We report here a series of studies on the rediscovered F. immunis population in Vilas County, Wisconsin. First, because the majority of sampling for crayfish in this region has occurred in lakes (
Lakes of Vilas County have been monitored for crayfish populations since
Vilas County crayfish monitoring has historically prioritized lentic ecosystems in this lake-rich region, but crayfish also occur in streams and rivers (
Results of summer 2021 baited trapping for crayfish in streams and rivers of the Manitowish River watershed, Vilas County, Wisconsin (Figure
Site | Date | Latitude, Longitude | Traps (#) | Result |
Allequash Creek | 8/19/2021 | 46.0238, -89.6528 | 3 | F. rusticus (4 F, 4 M) |
Mann Creek | 8/19/2021 | 46.0111, -89.6760 | 3 | F. rusticus (1 M) |
Trout River | 8/19/2021 | 46.0278, -89.7377 | 3 | No crayfish |
Gresham Creek | 8/19/2021 | 46.0603, -89.7423 | 3 | No crayfish |
Nixon Creek | 8/20/2021 | 46.0986, -89.5608 | 3 | No crayfish |
White Sand Creek | 8/20/2021 | 46.0964, -89.6112 | 3 | No crayfish |
Manitowish River | 8/20/2021 | 46.1113, -89.6911 | 3 | F. virilis (1 M) |
Rice Creek | 8/20/2021 | 46.1400, -89.7484 | 3 | F. rusticus (1 F), F. virilis (1 F) |
Trout River | 8/21/2021 | 46.8076, -89.8076 | 1 | No crayfish |
Trout River | 8/21/2021 | 46.0708, -89.8073 | 1 | No crayfish |
Trout River | 8/21/2021 | 46.0724, -89.8092 | 1 | No Crayfish |
Trout River | 8/21/2021 | 46.0737, -89.8102 | 1 | F. virilis (1 F) |
Trout River | 8/21/2021 | 46.0748, -89.8114 | 1 | No crayfish |
Trout River | 8/21/2021 | 46.0754, -89.8142 | 1 | No crayfish |
Trout River | 8/21/2021 | 46.0777, -89.8145 | 1 | No crayfish |
Trout River | 8/21/2021 | 46.0798, -89.8130 | 1 | F. virilis (1 F) |
Trout River | 8/21/2021 | 46.0798, -89.8130 | 1 | F. virilis (1 F) |
Identification errors are possible between F. immunis and similar congeners like F. virilis, particularly when only female or juvenile crayfish are collected because taxonomic keys for cambarid crayfishes rely on form I male gonopods. Accordingly, we used mtDNA barcoding to confirm identifications of F. immunis individuals used in behavioral trials with F. rusticus (below), including all individuals collected from Wild Rice Lake during the summer of 2021. We also sequenced two F. virilis individuals collected from the Manitowish River watershed in summer 2021 to confirm their identifications. We prioritized female or juvenile F. virilis individuals with life colors or morphology most similar to F. immunis for sequencing.
We froze crayfish used for molecular sequencing in a -20 °C freezer, and subsequently dissected muscle tissue from the frozen crayfish abdomens, which was then preserved in 99% ethanol. We extracted DNA from muscle tissue using a DNeasy Blood and Tissue Kit following the protocol for animal tissues (Qiagen, Hilden, Germany). We then used
We used Geneious Prime 2020.2.2 (
Because F. immunis were so rare in Wild Rice Lake in
At the University of Illinois at Urbana-Champaign, crayfish were kept in individual 3.79 L aquariums at ambient room temperature (22–27 °C) with a 12:12 light:dark cycle. Aquariums were filled with dechlorinated tap water and cycled for one week before the addition of crayfish. Aeration and filtration were provided by foam biofilters, and aquariums also contained two unglazed ceramic tiles (10.16 cm × 10.16 cm) and a PVC pipe for shelter. We analyzed water quality from aquariums biweekly with an API Freshwater Master Test Kit (Mars Fishcare North America, Chalfont, Pennsylvania, United States). pH ranged from 7.6–8.4 and ammonia, nitrite, and nitrate levels were all low. We changed one liter of water in each tank weekly. We fed crayfish an alternating diet of half an algae wafer (Hikari Tropical Algae Wafers, Himeji, Japan) or three to four small crab pellets (Hikari Crab Cuisine, Himeji, Japan) every other day.
In Wisconsin, we collected F. immunis from Wild Rice Lake between August 11th and 13th, 2022. We set 49 traps over three nights, collecting only eight F. immunis from one location on the southern shoreline (46.0591, -89.7909) near the inflow of the Trout River. We collected two female, five male form II, and one male form I F. immunis, which had a mean total carapace length of 19.0 mm (range: 12.8–31.0 mm). Faxonius rusticus are occasionally collected from Wild Rice Lake at very low abundances, and consequently F. immunis from this lake would be expected to have some behavioral experience with F. rusticus. We collected F. rusticus from traps throughout Boulder Lake (46.1253, -89.6604), which were recovered on August 12th, 2021 as part of routine crayfish monitoring in Vilas County (
In both Illinois and Wisconsin, crayfish acclimated in the lab for five to seven days prior to the start of behavioral assays to remove effects of previous social interactions (
We compared aggression between F. immunis and F. rusticus using the difference in aggression scores between individuals from each behavioral pair, while simultaneously controlling for other covariates like size or form difference, identity of individual crayfish, and region or source of populations (Illinois and Wisconsin). We first calculated the behavioral score difference for each interacting pair of crayfish by subtracting the mean F. rusticus behavioral score from the mean F. immunis behavioral score. Negative behavioral score differences indicate F. rusticus dominance, whereas positive behavioral score differences indicate F. immunis dominance. We similarly calculated size differences for pairs by subtracting F. rusticus total carapace length from F. immunis total carapace length. Negative values indicate that the F. rusticus individual was larger, whereas positive values indicate that the F. immunis individual was larger. To calculate form difference, we ranked forms by aggression, where form I and form II males are more aggressive than female crayfish but form I males are more aggressive than form II males (
We first analyzed our data with a linear mixed-model where behavioral score difference was the response variable, size and form difference were fixed effects, location (Illinois or Wisconsin) was also a fixed effect, and the identity of individual F. immunis and F. rusticus crayfish included in the pairs represented two random effects to account for repeated observations on the same organisms. Location as a fixed effect was entered as a binary scale with Illinois coded as 1 and Wisconsin coded as 0. We also ran location-specific models for Illinois and Wisconsin separately with the same structure but omitting form difference as a fixed effect for the Illinois model. We ran all linear mixed-models in the lme4 package (
We did not detect F. immunis from the 24 traps set throughout eight different stream and river sites in the Manitowish River watershed during summer 2021 (Table
Neighbor joining phylogenetic tree with 2000 bootstrap replicates including sequences of Calico Crayfish (Faxonius immunis) collected from Illinois (IL) or Wisconsin (WI). We include four additional F. immunis sequences from GenBank, as well as two sequences each of Virile Crayfish (Faxonius virilis), Northern Clearwater Crayfish (Faxonius propinquus) and Rusty Crayfish (Faxonius rusticus) as three known Faxonius crayfish species from Vilas County, Wisconsin. We used two sequences of Red Swamp Crayfish (Procambarus clarkii) as an outgroup. Nodes on the tree represent bootstrap support, and GenBank accession numbers are given at node tips. GenBank accessions for new F. immunis sequences are OQ759592–OQ759606.
Faxonius rusticus was significantly more aggressive than F. immunis from our combined location model, as evidenced by an intercept that was significantly lower than zero (Intercept = -1.375, df = 14.202, t = -5.817, P < 0.001). However, we found no significant effect of size difference (Coefficient = 0.050, df = 37.242, t = 1.539, P = 0.132), form difference (Coefficient = 0.230, df = 39.520, t = 0.924, P = 0.361), or location (Coefficient = 0.022, df = 14.273, t = 0.092, P = 0.928) on crayfish behavior. The two location-specific models were highly similar to each other (Figure
Linear mixed models with 95% confidence intervals between size difference and behavioral score difference of Rusty Crayfish (Faxonius rusticus) and Calico Crayfish (Faxonius immunis) pairs from location-specific models of our two study regions, Illinois (IL) and Wisconsin (WI). Negative size difference indicates F. rusticus was larger than F. immunis. Negative score difference indicates F. rusticus behaved more aggressively than F. immunis. Both models include random effects for individual crayfish used in pairs to account for repeated observations on the same organisms, and the Wisconsin model includes a non-significant fixed effect for reproductive form difference that is not plotted.
Our study confirmed an F. immunis population in Wild Rice Lake, Vilas County, Wisconsin, which had not been documented since the 1970s (
We found F. rusticus to be consistently dominant over F. immunis despite using individuals from different regions (Illinois or Wisconsin), invasion histories (native or non-native populations), and methods of collection (trapped or seined). This contradicts some recent research that has found region of origin and invasion history of populations to affect behavioral outcomes between crayfish species pairs (
We sampled streams and rivers of the Manitowish River watershed in summer of 2021 to survey for F. immunis populations outside of lake ecosystems commonly monitored in Vilas County, Wisconsin. We did not detect F. immunis from any of these stream or river sites, including relatively intensive trapping of the Trout River flowage downstream of Wild Rice Lake. This suggests that F. immunis is not spreading out of Wild Rice Lake into downstream ecosystems at present, although more widespread, intensive sampling focused on habitats neglected for crayfish monitoring in Wisconsin may reveal other F. immunis populations (
Species identification challenges could also account for some uncertainty around F. immunis distributions in northern Wisconsin and elsewhere, as evidenced by our misidentification of four F. virilis individuals from Douglas Creek, Illinois as F. immunis prior to mtDNA barcoding. Crayfishes of the family Cambaridae should be identified using male form I gonopods (e.g.,
Our study could be interpreted to support F. immunis as an innocuous non-native species, rather than a harmful invasive species, in Wild Rice Lake. Invasive species spread in new regions (
Alternatively, time lags between the introduction and establishment of a non-native species and subsequent spread or negative impacts as an invasive species can complicate management decisions and policy priorities (
Lakes of northern Wisconsin have experienced a sequence of natural, post-glacial colonization and human-assisted invasion by crayfishes that will likely continue following declines of F. rusticus populations.
Funding was provided by a University of Illinois ASPIRE fellowship to EKS and United States Department of Agriculture Hatch Project ILLU-875-984 to ERL.
Crayfish were collected under fishing licenses consistent with Illinois and Wisconsin regulations.
EKS: Funding Acquisition, Conceptualization, Investigation, Data Curation, Formal Analysis, Writing - Original Draft, Writing - Review and Editing; JHH: Methodology, Data Curation, Formal Analysis, Writing - Review and Editing; DKS: Conceptualization, Investigation, Writing - Review and Editing; ERL: Funding Acquisition, Conceptualization, Investigation, Writing - Review and Editing, Supervision
The University of Wisconsin Trout Lake Station provided housing and equipment in Wisconsin, and Mark A. Davis provided genomic laboratory space at the Illinois Natural History Survey at the University of Illinois. Joe Reinhofer assisted with collecting crayfish. This manuscript was improved by comments from Alison M. Bell, Christoph Chucholl, Rebecca C. Fuller, Joseph J. Parkos III, and two anonymous reviewers.