AbstractCambarus, which includes Cambarus (Cambarus) carinirostris, Cambarus (Cambarus) bartonii cavatus, Cambarus (Procambarus) robustus and Cambarus (Tubericambarus) thomai. Cambarus (Tubericambarus) thomai is the most prevalent burrowing species occurring along the floodplain. The genus Orconectes consists of two native species, Orconectes (Cambarus) obscurus and Orconectes (Cambarus) sanbornii; and two invasive taxa, Orconectes (Gremicambarus) virilis and Orconectes (Procambarus) rusticus. Orconectes (Cambarus) obscurus has experienced a range extension to the south and occupies streams formerly occupied by Orconectes (Cambarus) sanbornii. Both invasive taxa were allied with anthropogenic habitats and disturbance gradients. The genera Fallicambarus and Procambarus are represented by a single species. Both Fallicambarus (Cambarus) fodiens and Procambarus (Orconectes) acutus are limited to the historic preglacial Marietta River Valley.The crayfish fauna of West Virginia consists of 23 species and several undescribed taxa. Most survey efforts documenting this fauna have been conducted in lotic waterways throughout the Appalachian plateau, Allegheny Mountains, and Ridge and Valley physiographic provinces. Bottomland forests, swamps, and marshes associated with large river floodplain such as the Ohio River floodplain historically have been under-surveyed in the state. These habitats harbor the richest primary burrowing crayfish fauna in West Virginia, and are worthy of survey efforts. In an effort to fill this void, the crayfish fauna of West Virginia’s Ohio River floodplain was surveyed from 2004 through 2009. From this survey, nine species from four genera were documented inhabiting the floodplain. Zoogeography, biology, and conservation status is provided for all nine crayfishes. The dominant genus along the floodplain is  Orconectes (Procericambarus) rusticus  and Orconectes (Gremicambarus) virilis ,can eliminate native species, resulting in lower species diversity and “biotic homogenization” of crayfish faunas (Lodge 1993). Documenting native crayfish communities prior to invasive species infestation or other impact from environmental stressors is important to crayfish conservation  acutus . Orconectes (Faxonius) limosus  in the eastern panhandle of the state. Loughman et al (2009) provided natural history information for 11 of 24 species, while Procambarus (Ortmannicus) zonangulus Hobbs & Hobbs, 1990, as another introduced species.West Virginia crayfishes have received moderate attention in the past century . The firPageBreakThe focus of this study is on the floodplain and stream confluences with the Ohio River mainstem. This area is an ecological system that previous investigators neglected while surveying West Virginia’s crayfish. In addition, several potential conservation threats have occurred in the state since publication of The North American large river floodplain is conducive to crayfish diversity due to the myriad of lentic and lotic habitats associated with these ecosystems . West ViAcer saccharinum, Marsh), red maples  and black willows   . Nutrien, Marsh) . These fActive hydroperiod seasons typically last from January through early June . During PageBreakPageBreakcomplexes and large roadside ditches from Huntington, Cabell County, to Chester, Hancock County, were assessed through trapping, dip netting, or burrow excavation. All large stream confluences were surveyed through trapping in deep water or seining. Headwater streams were evaluated through hand collecting and seining.Both lentic and lotic habitats were surveyed to determine the crayfish diversity on the floodplain Figures 2. All marCollapsible minnow traps were the chief collecting method used for this study. Collapsible minnow traps were preferred over classic metal minnow traps because of their ease of storage and manipulation in the field, larger entrance portals, and rate of degradation by natural predators  in the event of trap loss . Entrance portal diameter has been shown to bias capture rates for various crayfish, sizes warranting the use of collapsible traps with larger portals . Traps wTraps were placed in water bodies during mid- to late-January during 2004 and 2005, and checked biweekly January through April. During this sampling period, surface activity of primary and secondary burrowing crayfishes increase; making crayfish community analysis more efficient than in other seasons for these behavioral groups . This meBurrowers were also collected by excavation. Burrow activity was determined by the presence of chimneys or fresh mud pellets at burrow portals. Active burrows were excavated with trowels and shovels until enlarged ”resting chambers” were reached . Burrow morphology data were collected on burrows containing crayfish that were not destroyed during the excavation process; data collected included central shaft depth , resting chamber width and height, terminal burrow depth , and burrow contents. All measurements were in centimetersProcambarus acutus. This species is a secondary burrower that is active in ephemeral surface waters prior to drawdown  a minimum of five seine haul efforts (a single seine haul = one seining effort) and maximum of 10 seine hauls were performed. Effort was increased with increasing stream size and habitat complexity.The primary collection method used for stream species were seines. Seines were setup at the terminal ends of riffles, runs, and glides in first through sixth order streams. ByLeaf packs were surveyed in stream pools using long-handled, sturdy bait well dip nets. These were used to “shovel” leaf packs onto a minnow seine that was spread out on the stream bank. Crayfish were then picked from the collected leaf pack on shore. After they were removed from the leaf pack it was returned upstream of its original location so the contents could again be used by the stream’s benthos. All crayfish life stages utilized leaf packs, making this method extremely important for determining reproductive success and recruitment.PageBreakPageBreakPageBreakData sheets and field jar labels were completed for each site surveyed . VoucherAll conservation ranks were determined following Nature Serve’s conservation ranking criteria .The following section provides accounts for each species encountered along the West Virginia Ohio River floodplain. Descriptions, morphometrics, natural history and habitat, distribution, and conservation are discussed for each taxon. A description of the information content emphasized for each subheading is explained below.PageBreakThe diagnosis section describes morphological characters for each of the species. Characters and information content that uniquely identify each species are included. Specific color patterns and geographic morphs unique to the Ohio River floodplain are provided.Morphometric data specific to animals captured during the survey are discussed. Total carapace lengths (TCL) for the largest male and female of each species are indicated. Morphometric tables are presented for each species and contain mean, range, and standard deviation for carapace length, palm length, areola width, and areola length for all specimens.Distribution of each taxon encountered in the floodplain study area is discussed relative to previous survey efforts. Most of this discussion is a comparison of results observed by Ecological observations for each species, including burrowing ability and habitat preferences, are described for each taxon. For primary and secondary burrowers, specific burrow usage and burrow morphology and architecture, as well as surface water usage, are described. Lentic and lotic habitats used by stream species are noted and specific microhabitats utilized are identified. Seasonal shifts in habitat usage and ontogenetic niche shifts are also described in this section. All observed species-specific behaviors are identified and discussed.Current conservation standing and potential mechanisms of imperilment are identified and discussed following Master et al. (2009). Recommendations are made for taxa in need of conservation efforts and future monitoring.PageBreak bartoni carinirostris Cambarus (Cambarus) bartonii carinirostris Cambarus (Cambarus) carinirostris Cambarus carinirostris rd tubercle on mesial surface of fixed finger of propodus; first form gonopods contiguous at base, with 2 terminal elements bent 90° to base; central projection with subapical notch; total length of central projection equal to length of mesial process; mesial process bulbous, truncating distally; second form gonopod non-corneous and blunt; annulus ventralis rhomboid in shape, embedded shallowly in sternum and movable.Rostrum broad, margins thickened and parallel, terminating in a 90° angle cephalically to form acumen; acumen consisting of a single upturned spiniform tubercle; median carina absent in floodplain populations; postorbital ridges truncate, cephalic margin with a weak tubercle; cephalothorax slightly flattened dorsoventrally in profile; 2–5 punctations across narrowest region of areola; branchiostegal region moderately punctate, with small tubercles; chelae broad and robust; mesial surface of palm with a single row of 5–7 adpressed tubercles; two prominent subpalmar tubercles present; enlarged 3Carapace dorsally brown, beige, or pink; rostrum margins red to reddish brown; chelae olivaceous green to brown; dactyl and propodus tubercles cream or yellow; pereiopods white, cream, or yellowish gray, rarely light blue; abdomen terga dorsally brown or beige, bordered in crimson; ventral surfaces cream or white.Cambarus carinirostris were collected from four counties at eight locations. Localities and demographics are listed below.BROOKE COUNTY: Cross Creek at entrance to Bruin Drive adjacent to Brooke High School, 40.306442 -80.5997; 4 September 2005 – (WLU 05090401), 2 II♂. RT 2 crossing of nameless tributary 2.27 km (1.41 mi) S of Beech Bottom, 40.23163 N / 80.6523 W; 28 June 2005 – (WLU 05072801), 2 II♂. HANCOCK COUNTY: Hardin Run 0.81 km (0.5 mi) from CR 2-7/RT 2 intersection on CR 2-7, PageBreak40.533314 -80.60326; 23 August 2005 – (WLU 0508230), 1 I♂, 2 II♂, 2 ♀. MARSHALL COUNTY: Boggs Run at RT 2 crossing, 40.02481 N / 80.72577 W; 28 July 2005 – (WLU 05072801), 1 II♂, 1♀. Long Run at Long Run/Fish Creek confluence, 39.805878 -80.8052; 20 July 2005 – (WLU 05072002), 3 ♀. Nameless tributary at RT 2 crossing adjacent to Columbia Chemical operations, 39.85933 -80.79305; 28 July 2005 – (WLU 05072803), 6 I♂, 1 ♀. WETZEL COUNTY: Proctor Creek at RT 2 crossing, 39.70037 -80.81791; 9 July 2008 – (WLUPageBreak 08070901), 5 II♂, 1 ♀. RT 2 crossing of nameless tributary at Marshall/Wetzel County line, 39.720192 -80.82281; 20 July 2005 – (WLU 05072001), 1 II♂.Cambarus carinirostris ranges from central West Virginia north through the Monongahela River system in West Virginia and Pennsylvania and the Allegheny River system in Pennsylvania and New York  bartonii cavatus PageBreakHay, 1902 replaces this species in Fishing Creek. The distribution of Cambarus carinirostris is the same as reported by New York .The western Ohio . Cambaruio North . Within Cambarus carinirostris is a moderate sized crayfish. Mean TCL was 29.1 mm . The largest individual was a form I male with a TCL of 39.4 mm collected from Holbert Run in Hancock County. The largest female was also collected from Holbert Run, and had a TCL of 32.1 mm. Morphometric data for Cambarus carinirostris is presented in Cambarus carinirostris , and Cambarus robustus Girard, 1852, both of which were collected with Cambarus carinirostris. Seasonal data for Cambarus carinirostris are presented in Cambarus carinirostris populations along the floodplain are stable and do not warrant special conservation action.PageBreakHay, 1902Cambarus bartonii cavatus Cambarus (Bartonius) bartonii Cambarus (Cambarus) bartoni cavatus Cambarus (Cambarus) bartonii cavatus PageBreakprojection with shallow subapical notch; total length of central projection equal to mesial process length; second form gonopod non-corneous and blunt; mesial process bulbous, truncating distally; annulus ventralis rhomboid in shape, embedded shallowly in sternum and movable.Rostrum broad; margins reduced, subparallel, terminating cephalically in a gentle angle to form acumen; anterior region of rostrum excavated; acumen consisting of a single upturned spiniform tubercle; postorbital ridges truncated, cephalic margin with weak tubercle; cephalothorax oval shaped and slightly dorsoventrally flattened in profile; 2–3 punctations across narrowest region of areola; branchiostegal region moderately punctate, with small tubercles; chelae broad and robust; mesial surface of palm consisting of two rows of defined tubercles; first row with 5–8 rounded tubercles; second with 3–4 tubercles; two prominent subpalmar tubercles present; first form gonopods contiguous at base, with 2 terminal elements bent 90° to the base; central Carapace dorsally olivaceous brown, beige, or tan; rostrum margins chestnut brown to brown; chelae olivaceous green to brown; dactyl and propodus denticles cream or yellow; pereiopods tan, light green, cream, or gray; abdomen terga dorsally brown or beige, bordered in gray; ventral surfaces cream or white.Cambarus bartonii cavatus were collected from six counties at 15 locations. Collection locales and demographics are listed below.JACKSON COUNTY: Flooded field adjacent to RT 33 S, 9.72 air km (6.04 mi) N of Ravenswood, 39.04274 -81.7827; 3 April 2005 – (WLU 05040301), 3♀. Little Sandy Creek, at intersection of RT 68/CR 8, 38.991497 -81.761765; 21 July 2006 – (WLU 06012103), 1 II ♂. Vernal pool complex adjacent to railroad tracks 3.56 km (2.21 mi) N of Ravenswood, 39.09015 -81.79469; 3 April 2005 – (WLU 05040302), 1♀. West Creek, at intersection of CR 12 PageBreak/CR 10, 38.924362 -81.94200; 20 July 2006 – (WLU 06072001), 1 II ♂, 1♀. MASON COUNTY: Pin oak swamp adjacent to Point Pleasant Moose Lodge in Wagner, 38.833603 -82.12227; 6 June 2005 – (WLU 05060601), 2 ♀. Roadside ditch adjacent to RT 2, 9.17 km (5.7 mi) S of Point Pleasant, 38.80469 -82.18821; 4 March 2005 – (WLU 05030401), 2 I♂, 1 ♀. Roadside ditch adjacent to RT 2 N, 2.91 km (1.81 mi) N of Clover, 38.589428 -82.19548; 4 March 2005 – (WLU 05030402), 1 ♀. Roadside ditch adjacent to RT 2 N, 1.93 km (1.2 mi) N of Glenwood, 38.58816 -82.201004; 3 April 2005 – (WLU 05040305), 1 I♂, 2 ♀. Roadside ditch adjacent to RT 2 N 0.90 air km (0.56 mi.) N of Hogsett, 38.70056 -82.17708; 17 March 2005 – (WLU 05031707), 1 ♀. PLEASANTS COUNTY: Middle Island Creek at RT 2 crossing, 39.40328 -81.197624; 21 July 2006 – (WLU 06072104), 1 ♀. TYLER COUNTY: Nameless tributary at RT 2 crossing in Friendly, 39.513386 -81.06285; 28 July 2005 – (WLU 05072806), 1 I♂, 1 ♀. WETZEL COUNTY: Doolins Run at RT 2 crossing, 39.639576 -80.85607; 28 July 2005 – (WLU 05072805), 1 II♂. WOOD COUNTY: Big Run at CR 21-1 crossing, 39.364048 -81.45656; 21 July 2004 – (WLU 06072101), 1 ♀. Boaz Swamp Wildlife Management Area, 39.462868 -81.10855; 12 April 2004 – (WLU 04042101), 1 ♀. Nameless tributary crossing 3.54 km (2.2 mi) S of Parkersburg, 39.05142 -81.742836; 21 July 2006 – (WLU 06072106) 1 II♂, 1 ♀.Cambarus bartonii cavatus ranges from northern Georgia and Tennessee through eastern Kentucky, east central Ohio and western Virginia  survey in the late 1980’s.Virginia . In Westoodplain . It is rCambarus bartonii cavatus is a medium to large crayfish. The largest individual collected was a female with a 51.6 mm TCL taken from an ephemeral pool complex 3.6 km north of Ravenswood, Jackson County. The largest male collected was a form I collected from a roadside ditch 1.9 km north of Glenwood, Mason County, with a TCL of 45.6 mm. Mean Cambarus bartonii cavatus carapace length was 34.4 mm  . Morphometric data for Cambarus bartonii cavatus is presented in Cambarus bartonii cavatus , were the width of the crayfish’s carapace at the widest point. Cambarus bartonii cavatus burrows did not follow this same pattern and, usually, were wide and oblong. Anecdotally, Cambarus bartonii cavatus burrows were readily identified by the presence of these structural components, but this method of identification was not used to definitively verify Cambarus bartonii cavatus presence at a site.The central shaft and central chamber of other floodplain burrowing species , Orconectes virilis and Procambarus acutus. Seasonal data for Cambarus bartonii cavatus are presented in In late winter females comprised 66% of trap captures. Those captured in late winter/early spring all possessed active glair glands. This condition has been used in previous studies to indicate future egg extrusion and is likely the explanation for this increase in female activity . No ovigCambarus bartonii cavatus populations along the floodplain are stable and do not warrant special attention.Girard, 1852Cambarus robustus  Girard 1852:90. Cambarus Bartonii robustus Cambarus bartonii robustus Cambarus (Bartonius) bartoni robustus Cambarus (Cambarus) bartonii robustus Cambarus (Cambarus) bartonii robustus Cambarus (Bartonius) robustus Cambarus (Puncticambarus) robustus PageBreakspiniform tubercle; postorbital ridges prominent, cephalic margin with tubercle; cephalothorax dorsoventrally flattened in profile, anterior portion weakly vaulted; 2–5 punctations across narrowest region of areola; branchiostegal region moderately punctate, with small tubercles; chelae robust; mesial surface of palm consisting of two rows of defined tubercles; first row with 7–9 rounded tubercles; second with 5–7 smaller tubercles; additional tubercles scattered over dorsal region of palm; three prominent subpalmar tubercles present; first form gonopods contiguous at base, with 2 terminal elements bent 90° to base; central projection with distinct subapical notch; total length of central projection equal to mesial process length; mesial process bulbous, truncating distally; second form gonopod non-corneous and and blunt; annulus ventralis rhomboid in shape, embedded shallowly in sternum and movable.Rostrum narrow to slightly broad, margins reduced and parallel, terminating in gentle angle cephalically to form acumen terminating in a single upturned Carapace dorsally brown; cephalic region reddish brown, branchial region pinkish brown to light brown; cervical groove black; rostrum margins orange or red; chelae olivaceous green to green; tubercles on chelae yellow or orange; dactyl and fixed finger denticles cream or yellow; perieopods green or light blue; abdomen terga bodies dorsally brown or olivaceous brown; bordered in red, ventral surfaces cream or white.Cambarus robustus were collected from five counties at seven localities, as listed below.HANCOCK COUNTY: Kings Creek at RT 2 crossing, 40.435715 -80.592514; 17 October 2005 – (WLU 05101701), 4 I♂, 2 II♂, 1 ♀. MARSHALL COUNTY: Long Run at Long Run/Fish Creek confluence, 39.805878 -80.8052; 30 October 2005 – (WLU 05103002), 2 ♀. Fish Creek at RT 2 crossing, 39.808643 -80.81616; 30 October 2005 – (WLU 05103002), 1 I♂, 2 ♀. PLEASANTS COUNTY: Ben’s Run at RT 2 crossing, 39.46337 -81.08457; 28 July 2005 – (WLU 05072802), 3 ♀. TYLER COUNTY: Cow House Run at RT 2 crossing, 39.551327 -81.01001; 28 July 2005 – (WLU 05072804), 1 II♂. WETZEL COUNTY: Fishing Creek at RT 2 crossing, 39.63576 N/ -80.85848 W; 20 July 2005 – (WLU 05072001), 1 I♂, 1 ♀. Proctor Creek at RT 2 crossing, 39.70037 N/ -80.81791 W; 9 July 2008 – (WLU 08070901), 2 II♂, 1 ♀.Cambarus robustus has an extensive distribution, ranging from southern Ontario and central New York south to North Carolina and Virginia, and west to Illinois .Illinois .Given thh basins , but hasPageBreakCambarus robustus was likely under surveyed during this effort. This species prefers free-flowing streams more similar to mainstem rivers rather than habitats associated with big river confluences like those sampled in this survey.Cambarus robustus TCL was 33.0 mm . Morphometric data are presented in The largest individual collected was a 46.6 mm TCL form II male collected in Kings Creek, Hancock County. The largest female was also taken there and was 36.0 mm TCL. Mean Cambarus robustus  diogenes Cambarus (Lacunicambarus) diogenes diogenes Cambarus (Lacunicambarus) diogenes Cambarus (Tubericambarus) thomai Cambarus thomai Rostrum slightly broad, margins converging to form acumen terminating in single reduced, upturned tubercle; postorbital ridges reduced, rarely terminating in small tubercle; cephalothorax dorsolaterally compressed in profile and vaulted; areola obliterated; branchiostegal region devoid of tubercles; chelae robust and diamond shaped; mesial surface of palm with disorganized prominent tubercles, mesialmost tubercles serrate; basiodactyl row consisting of 5–9 reduced rounded tubercles; first form male gonopods contiguous, with 2 terminal elements bent 90° to the shaft; central projection truncated distally and lacking sub-apical notch; total length of central projection equal to mesial process length; mesial process short, truncating distally; second form gonopod non-corneous and blunt; annulus ventralis rhomboid in shape with deep “S” shaped sinus, embedded shallowly in sternum, and movable.Carapace dorsally brown, light green, olive, light blue, or blue grey; rostrum margins orange or red; chelae body light green, light brown, or blue; propodus light blue or light green; dactyl and propodus denticles cream or yellow; pereiopods tan, light green, cream, or gray; abdomen body light green, light blue gray or brown; tubercles covering chelae light yellow, cream, or orange; two light dorsal stripes present on dorsal surface of abdomen; ventral surface cream or white.Cambarus thomai were collected from eight counties at 26 localities, listed below.CABELL COUNTY: Green Bottom Swamp at Hoeft Marsh Wildlife Management Area, 38.58616 N / 82.24878 W; 2 April 2009 – (WLU 09040201), 2 I♂. HANCOCK COUNTY: Tomlinson Run backwater at RT 2 crossing, 40.54026 -80.628075; 30 March 2006 – (WLU 06033001), 3 I♂. JACKSON COUNTY: PageBreakFlooded field adjacent to RT 33 S, 9.72 air km (6.04 mi) N of Ravenswood, 39.04274 -81.7827; 18 March 2005 – (WLU 05031803), 7 I♂, 3 O♀. Vernal pool complex adjacent to railroad tracks 3.56 km (2.21 mi) N of Ravenswood, 39.09015 -81.79469; 3 April 2005 – (WLU 05040302), 3 I♂, 1 II♂. MASON COUNTY: Ditch adjacent to RT 2 N, 1.77 km (1.10 mi) N of Rt 2 railroad crossing in Ashton, 38.63165 -82.16464; 18 March 2005 – (WLU 05031805), 3 I♂, 1 II♂. Krodel Park marsh adjacent to Fort Randolph reproduction, 38.785404 -82.12209; 5 March 2005 – (WLU 05030502), 4 I♂. Maple swamp adjacent to RT 2 railroad crossing in Ashton, 38.622005 -82.16758; 26 March 2004 – (WLU 04032601), 18 I♂, 2 ♀; 30 March 2004 – (WLU 04033001), 6 I♂, 3 O♀, 1 ♀; 28 April 2004 – (WLU 04042801), 4 I♂,1 O♀, 2 ♀; 18 March 2005 – (WLU 05031805), 2 I♂. Pin oak swamp adjacent to Point Pleasant Moose Lodge in Wagner, 38.833603 -82.12227; 26 March 2004 – (WLU 04032601), 3 I♂; 12 April 2004 – (WLU 04041205), 3 I♂, 2 O♀. Red-Osier Dogwood swamp adjacent to RT 33 S in Hartford, 39.008915 -81.99847; 5 March 2005 – (WLU 05030504), 1 I♂. Roadside ditch adjacent to RT 2 N, 2.91 km (1.81 mi) N of Clover, 38.589428 -82.19548; 4 March 2005 – (WLU 05030402), 3 I♂. Roadside ditch adjacent to RT 2, 9.17 km (5.7 mi) S of Point Pleasant, 38.80469 -82.18821; 12 April 2004 – (WLU 04041206), 6 I♂. Roadside ditch adjacent to RT 2 N, 0.22 air km (0.14 mi) N of Hogsett, 38.694496 -82.1765; 17 March 2005 – (WLU 05031707), 5 I♂. Roadside ditch adjacent to RT 2 N 0.90 air km (0.56 mi.) N of Hogsett, 38.70056 -82.17708; 17 March 2005 – (WLU 05031707), 4 I♂, 1 II♂. Roadside ditch adjacent to RT 2 N 1.93 km (1.2 mi) N of Glenwood, 38.58816 -82.201004; 3 April 2005 – (WLU 05040305), 1 I♂. Roadside ditch adjacent to RT 62 N, 0.34 km (0.21 mi) N of Hallwood, 38.97562 -82.081314; 17 March 2005 – (WLU 05031706), 1 I♂. Roadside ditch adjacent to RT 62 S at Mountaineer Power Plant, 38.974934 -81.94418; 5 March 2009 – (WLU 05030509), 2 I♂. Slough adjacent to RT 33 N in Mason, 39.00983 -82.03899; 5 March 2005 – (WLU 05030505), 1 I♂; 17 March 2005 – (WLU 05031704), 6 I♂, 4 ♀. Vernal pool complex at RT 2/Lighthouse Gospel Church Road intersection, 38.82201 -82.13136; 17 March 2005 – (WLU 05031707), 2 I♂, 2 O♀. PLEASANTS COUNTY: Ohio River embayment 4.03 air km (2.52 mi) S of St. Mary’s, 39.397575 -81.202415; 12 April 2004 – (WLU 05030506), 1 II♂, 1 OF. Vernal pool adjacent to RT 2 N across from Cytec Community Fishing Area, 39.347824 -81.32024; 5 March 2005 – (WLU 04041203), 2 I♂. TYLER COUNTY: Maple swamp adjacent to RT 2 S in Friendly, 39.50822 -81.06736; 20 March 2004 – (WLU 04032001), 2 I♂. WETZEL COUNTY: Maple Swamp adjacent to RT 2 S in New Martinsville, 39.32582 -80.866234; 2 April 2004 – (WLU 04040201), 8 I♂, 2 ♀; 21 March 2006 – (WLU 06032104), 3 I♂, 1 II♂. (23.) Ohio River backwater at Marshall/Wetzel County line, 39.717846 -80.514959; 2 April 2004 – (WLU 04040203), 2 I♂; 21 March 2006 – (WLU 06032101), 3 I♂. WOOD COUNTY: Bellville Wildlife Management Area 4.03 km (2.50 mi) S of Bellville, 39.132915 -81.730865; 5 March 2005 - (WLU 05030507), 6 I♂. Boaz Swamp Wildlife Management Area,PageBreak 39.462868 -81.10855; 25 March 2004 – (WLU 04032501), 1 I♂; 12 April 2004 – (WLU 04041203), 2 I♂, 1 O♀; 5 March 2005 – (WLU 05030509), 1 I♂. Lee Creek at CR 11 crossing, 39.153275 -81.73507; 2 April 2004 – (WLU 04040203), 2 I♂.Cambarus thomai is the largest burrowing crayfish occurring in West Virginia, and the most frequently collected species in this study. The largest individual collected was a form I male, 53.6 mm TCL from Bellville, Wood County. The largest female measured 38.6 mm TCL and was collected from a flooded field 1.1 km north of Ravenswood, Jackson County. Mean Cambarus thomai carapace length was 37.0 mm . Morphometric data for Cambarus thomai are presented in Cambarus thomai distribution includes western Pennsylvania, central and eastern Ohio, central and western West Virginia and eastern Kentucky  in Brooke and Hancock counties, stating that populations persisting in both counties were stable. Cambarus diogenes. Jezerinac described Cambarus thomai in 1993 based on material from West Virginia in his description  sampled in the county. Populations decline northPageBreak of these basins. The most substantial northern population occurs in New Martinsville, Wetzel County. In the Middle Ohio North, Middle Ohio South, and Lower Ohio basins, Cambarus thomai  also used surface waters, with 50% of females captured at this time carrying eggs. Linear regression analysis of ovigerous females indicates there is not a strong relationship between carapace length and the number of pleopodal eggs  and were the only demographic observed at this time. Dip netting yielded large numbers of young-of-the-year in July and August; however, whether neonates remain in surface waters may depend on water availablility throughout the fall into winter. During drawdown in several sites in Mason County, juveniles were observed burrowing.Cambarus thomai utilizing surface waters rather than relying on burrows. On several occasions individuals would seek cover under substrate debris in surface waters although burrows were readily available.Neonate utilization of surface waters may be a dispersal mechanism to enable colonization and equally distribute individuals throughout wetlands or redistribute individuals into areas of high productivity. Nocturnal searches found Cambarus thomai populations are stable within the Middle Ohio North, Middle Ohio South and Lower Ohio basin. Additional survey efforts are needed in the Upper Ohio North and Upper Ohio South basins to determine the status of northern populations.Astacus fodiens Cambarus argillicola Cambarus (Bartonius) argillicola Bartonius argillicola  Wiliamson 1907:749.Cambarus (Cambarus) fodiens Cambarus fodiens Cambarus (Bartonius) fodiens Fallicambarus (Creaserinus) fodiens Fallicambarus fodiens PageBreak of 3–6 circular tubercles; basiodactyl row consisting of 5–7 punctations; opposable surface of dactyl with distinct basal notch; junction of dactyl and propodus setiferous; first form gonopods basally contiguous, with 2 terminal elements bent 90° to shaft; central projection of populations on the floodplain possessing distinct subapical notch; total length of central projection equal to mesial process length; mesial process bulbous, truncating distally; second form gonopod non-corneous and blunt; subapical notch absent in second form gonopod; annulus ventralis rhomboid in shape with deep S-shaped sinus and C-shaped fossa; embedded shallowly in sternum, and movable.Rostrum slightly broad and moderately excavated, deflected ventrally; margins converging to form acumen cephalically with reduced upturned tubercle; postorbital ridge reduced, not terminating in tubercle; cephalothorax dorsolaterally compressed in profile and vaulted; areola obliterated; branchiostegal region devoid of tubercles; chelae diamond shaped; mesial surface of palm with 2 distinct rows of tubercles; dorsalmost row consisting of 6–9 serrate tubercles; second row consistingCarapace dorsally and laterally tan, brown, reddish brown, or gray; cephalic and branchial region mottled with black or deep grey spots; chelae tan, deep gray, or gray brown; tubercles on chelae cream or light gray; distal region of dactyl and propodus increasingly orange; perieopods green or light grey; abdomen grey or olivaceous brown, with 2 distinct dorsal stripes; ventral surfaces cream or white.Fallicambarus fodiens were collected from two counties at three locations in the current study, as listed below.CABELL COUNTY: Green Bottom Swamp at Hoeft Marsh Wildlife Management Area, 38.58616 -82.24878; 2 April 2009 – (WLU 09040201), 2 I♂, 2 II♂, 1 O♀, 4♀. MASON COUNTY: Vernal pool complex at RT 2/Lighthouse Gospel Church Road intersection, 38.82201 -82.13136; 17 March 2005 – (WLU 05031707), 1 I♂, 1 ♀; 28 March 2005 – (WLU 05032801), 1 I♂, 3 ♀. Pin oak swamp adjacent to Point Pleasant Moose Lodge in Wagner, 38.833603 -82.12227; 26 February 2004 – (WLU 04022601), 1 II♂, 1 O♀; 26, March 2004 – (WLU 04032601), 3 I♂, 2 ♀; 30 March 2004 – (WLU 04033001), 1 I♂; 12 April 2004 – (WLU 04041202), 1 I♂; 28 April 2004 – (WLU 04042801), 1 II♂, 2 ♀.Fallicambarus fodiens is a wide-ranging species occurring along the Atlantic Slope in Maryland, Virginia, and North Carolina east to the Ohio and Mississippi river valleys excluding the majority of the Appalachian Mountains. Northern populations occur along the Great Lakes and in southern Ontario . Morphometrics data for Fallicambarus fodiens  were collected carrying instars and eggs in February and March 2004 and 2005  were resting or grazing at their burrow entrances on periphyton that had colonized submerged canary grass . Within the colony burrows were flooded by 15–30 cm of standing water. Instars were observed leaving burrow entrances to graze. Upon provocation, disturbed females stopped moving long enough to allow instars to reattach to their abdomens, then retreated to their burrows. Seasonal data for Fallicambarus fodiens is presented in Fallicambarus fodiens include Cambarus bartonii cavatus, Cambarus thomai, Orconectes virilis, and Procambarus acutus acutus.Fallicambarus fodiens warrants conservation attention and is deserving of S1 status. Surveys are needed along the Kanawha River in wetland habitats to determine if this species persists there. Along the floodplain threats to this species’ survival include land use practices and their associated pollutants, hard surface run off, and destruction of bottomland forests.Cambarus obscurus Cambarus propinquusobscurus  var. Cambarus propinquus obscurus Cambarus (Faxonius) obscurus Faxonius obscurus  Creaser 1933:5.Faxonius (Faxonius) obscurus Orconectes obscurus Orconectes (Orconectes) obscurus Orconectes (Crockerinus) obscurus PageBreakwide; basiopodite spine of antenna well developed. Ischiopodite of antenna without spine. Chelae smooth, broad and robust, length 91% of TCL; mesial surface of palm consisting of two well developed rows of tubercles; mesialmost row consisting of 7–11; dorsolateral row with 5–11; lateral margin of propodus smooth, dorsal surfaces of both dactyl and fixed finger of propodus with weak dorsolateral ridges; some elongate setae at base of fixed finger. First form gonopods short, comprising 33% of TCL, with two terminal elements about equal length; corneous central projection comprising 23% of pleopod length, tapering distally to point; mesial process non-corneous, spatulate, partially surrounding central projection; cephalic base of central projection with right angle shoulder. Form two male gonopod non-corneous, blunt, shoulder not prominent or absent. Female annulus ventralis deeply embedded in sternum, moveable, wider than long, cephalolateral prominences well developed, distinctly separated by a trough; fossa rather deep, sinus sinuate in caudal 67% of annulus.Rostrum with slightly converging margins, not thickened, with marginal spines or tubercles; median carina absent; postorbital ridges possessing a sharp spine. Cephalothorax ovoid, slightly, dorsoventrally compressed, without setae. Areola 3.7–6.6 times longer than wide, comprising 27–39% of TCL, with 2–3 rows of punctations across narrowest region; cervical groove interrupted just above cervical spine; lacking hepatic spines; suborbital angle obsolete. Antennal scale about 1.5 times as long as Carapace, abdomen and dorsal surface of chelae brown; rostral margins, caudal edge of carapace, and anterodorsal surface of terga dark brown; tips of chelae and knob at base of dactyl orange; tubercles on mesial and lateral margins of dactyl, mesial margin of palm, and mesial margin of propodus, and two spines or tubercles on anterodorsal surface of carpus yellow; reddish stripe on lateral margin of chelae; ventral margins beige.Orconectes obscurus were collected from six counties at 16 locations in the current study, as listed below.BROOKE COUNTY: Buffalo Creek at RT 2 crossing in Wellsburg, 40.261375 -80.61508; 4 September 2005 – (WLU 05090402), 2 I♂, 3 ♀. Cross Creek at entrance to Bruin Drive adjacent to Brooke High School, 40.306442 -80.5997; 28 June 2005 – (WLU 05062803), 1 ♀; 4 September 2005 – (WLU 05090403), 2 I♂, 1 II♂, 2 ♀. (3.) RT 2 crossing of nameless tributary 2.27 km (1.41 mi) S of Beech Bottom, 40.23163 -80.6523; 28 June 2005 – (WLU 05062801), 4 II♂. RT 2 crossing of nameless tributary in Beech Bottom proper, 40.306442 -80.5997; 28 June 2005 – (WLU 05062801), 1 ♀. HANCOCK COUNTY: Hardin Run 0.81 km (0.5 mi) from CR 2-7/RT 2 intersection on CR 2-7, 40.533314 -80.60326; 23 August 2005 – (WLU 05082302), 2 I♂, 1 ♀. Holbert Run 1.61 km (1.0 mi) from CR 2-8/ RT 2 intersection adjacent to CR 2-8, 40.474045 -80.58584; 23 August 2005 – (WLU 05082303), 1 I♂, 1 II♂, 1 ♀. Kings Creek at RT 2 crossing, 40.435715 N / 80.592514 W; 17 October 2005 – (WLU 05101701); 3 I♂, 4 ♀. Tomlinson Run backwater at RT 2 crossing, 40.54026 -80.628075; 8, March 2005 – (WLU 06030801), 9 I♂; 30 March 2005 – (WLU 06033001), 1 I♂. MARSHALL COUNTY: Big Grave Creek at Ohio River confluence in Moundsville, 39.9046 -80.75731; 20 July 2005 – , 2 I♂, 1 II♂. Fish Creek at RT 2 crossing, 39.808643 -80.81616; 30 October 2005 – (WLU 05103002), 7 I♂, 2 ♀; 30 April 2006 – (WLU 06043001), 8 O♀. Little Grave Creek at RT 2 crossing in Moundsville, 39.920944 -80.748566; 28 July 2007 – (WLU 05072807), 1 I♂, 1 II♂, 4 ♀. Nameless tributary at RT 2 crossing adjacent to Columbia Chemical operations, 39.85933 -80.79305; 28PageBreak July 2007 – (WLU 05072809), 2 I♂. OHIO COUNTY: Short Creek at RT 2 crossing, 40.18312 -80.676865; 4 September 2005 – (WLU 05090404), 2 I♂, 2 ♀. PLEASANTS COUNTY: Ben’s Run at RT 2 crossing, 39.46337 -81.08457; 28 July 2005 – (WLU 05072802), 1 I♂, 2♀. WETZEL COUNTY: Fishing Creek at RT 2 crossing, 39.63576 N/ -80.85848; 20 July 2005 – (WLU 05072001), 2 II♂, 1 ♀. Proctor Creek at RT 2 crossing, 39.70037 N/ -80.81791 W; 20 July 2005 – (WLU 05072001), 2 II♂, 3 ♀.Orconectes obscurus occurs in north-west New York south through western Pennsylvania and north-central West Virginia, east to Maryland’s portion of the Youghiogheny River system and west to the Flushing Escarpment of Ohio . Sexual dimorphism is displayed in this species, with form I and form II male chelae significantly larger (t(345) = 6.8201, p = 0.0001) than female chelae. Morphometric data for Orconectes obscurus is presented in The largest individual was a 39.0 mm TCL form I male collected in Tomlinson Run backwater in Hancock County. The largest female was collected from the confluence of Little Grave Creek and the Ohio River in Marshall County and possessed a 37.4 mm TCL . Mean TCL for the species was 29.2 mm .obscurus  occupy sOrconectes obscurus is also a tertiary burrower, creating minimal burrows under substrate items. Small gravel and cobble piles usually were present along margins of slab boulders harboring Orconectes obscurus. One significant behavioral difference between the genera Orconectes and Cambarus along the floodplain is the difference in expressed territoriality. Orconectes obscurus and other orconectids displayed limited territorality. In one instance in Cross Creek, Brooke County, 11 individuals were collected from a single slab boulder.Orconectes obscurus were collected from two ephemeral streams. This habitat has not previously been reported for the species, and is rarely reported forany Orconectes species with a mean egg diameter of 1.7 mm. There was no correlation between pleopodal egg number and TCL . Beginning in late June of both 2005 and 2006, young of the year were frequently captured, indicating their release from female’s pleopods. Males in late July and early August underwent a late summer molt into first form condition Cambarus Sanbornii Cambarus propinquus sanbornii Cambarus propinquussanbornii var. Cambarus propinquussanbornii  var. PageBreakCambarus propinquus sanbornii Cambarus propinquus sanborni Cambarus (Faxonius) propinquus sanbornii Cambarus obscurus sanbornii Faxonius sanbornii Faxonius (Faxonius) sanbornii Orconectes propinquus sanbornii Orconectes (Orconectes) propinquus sanbornii Faxonius sanborni sanborni  Creaser 1962:2.Orconectes sanbornii sanbornii Orconectes sanbornii  Stevenson 1967:208. Orconectes sanbornii sanbornii Orconectes (Crockerinus) sanbornii sanbornii PageBreakOrconectes (Crockerinus) sanbornii Rostrum with slghtly converging margins, not thickened, with marginal spines or tubercles; median carina absent. Cephalothorax ovoid, slightly, dorsoventrally compressed, without setae. Areola 3.4–9.3 times longer than wide, comprising 31–37% of TCL, with 2–3 rows of punctations across narrowest region; cervical groove interrupted just above cervical spine; lacking hepatic spines; suborbital angle obsolete. Antennal scale about 1.5 times as long as wide; basiopodite spine of antenna well developed; ischiopodite of antenna without spine. Chelae smooth, broad and robust, length 85% of TCL; mesial surface of palm with two well developed rows of tubercles; mesialmost row consisting of 7–11 tubercles; dorsolateral row with 7–11; lateral margin of propodus smooth, dorsal surfaces of both dactyl and fixed finger of propodus with weak dorsolateral ridges; some elongate setae at base of fixed finger. First form male gonopods short, comprising 30% of TCL, with two terminal elements about subequal length; corneous central projection comprising 16% of pleopod length, tapering distally to point; mesial process non-corneous, spatulate, partially surrounding central projection; cephalic base of central projection sloping, without right angle shoulder. Form two male gonopod non-corneous, blunt, shoulder not prominent or absent. Female annulus ventralis deeply embedded in sternum, moveable, wider than long, cephalolateral prominences flattened; fossa and sulcus shallow; sinus straight.PageBreak mesial margin of palm, and mesial margin of fixed finger of propodus, and two spines or tubercles on anterodorsal surface of carpus, yellow; reddish stripe on lateral margin of chelae; ventral margins beige.Carapace, abdomen and dorsal surface of chelae brown; rostral margins, caudal edge of carapace, and central surface of terga dark brown; tips of chelae and tubercles at dactyl base orange; tubercles on mesial and lateral margins of dactyl,Orconectes sanbornii were collected from four counties at seven locations in the current study, as listed below.JACKSON COUNTY: Little Mill Creek at crossing of RT 33 N 9.43 air km (5.86 mi) N of Ravenswood, 38.86171 -81.85407; 20 July 2006 – (WLU 06072001), 1 II♂, 2 ♀. Little Sandy Creek at intersection of RT 68/CR 8, 38.991497 -81.761765; 21 July 2006 – (WLU 06072103), 1 I♂, 1 II♂, 11 ♀, 4 Juv. MASON COUNTY: Sliding Creek at intersection of CR 4/RT 33, 38.999382 -81.987686; 20 July 2006 – (WLU 06072002), 2 I♂, 1 ♀. PLEASANTS COUNTY: Middle Island Creek at RT 2 crossing, 39.40328 -81.197624; 21 July 2006 – (WLU 06072104), 1 II♂, 2 ♀. WOOD COUNTY: Big Run at CR 21-1 crossing, 39.364048 -81.45656; 21 July 2006 – (WLU 06072106), 3 II♂, 5 ♀, 1 Juv. Lee Creek at CR 11 crossing, 39.153275 -81.73507; 21 July 2006 – (WLU 06072102), 1 II♂, 2 ♀. Nameless tributary crossing 3.54 km (2.2 mi) S of Parkersburg, 39.05142 -81.742836; 21 July 2006 – (WLU 05072105), 1 II♂, 1 ♀.Orconectes sanbornii occurs throughout the Middle Ohio river drainage in Ohio, West Virginia and Kentucky . Currently, Orconectes sanbornii inhabits the Middle Ohio North, PageBreakMiddle Ohio South, and Lower Ohio basins . Morphometric data for Orconectes sanbornii is presented in The largest individual was a female with a 35.5 mm TCL from West Creek, Jackson County. The largest male was a form I with a TCL of 34.3 from West Creek. The mean TCL for Orconectes sanbornii Cambarus virilis Cambarus wisonsinensis Cambarus debilis Cambarus cousii Cambarus Cousei Cambarus wisconsiensis Cambarus cousei Cambarus viridis Cambarus (Faxonius) virilis Faxonius virilis Faxonius (Faxonius) virilis Orconectes virilis PageBreakOrconectes (Orconectes) virilis Orconectes (Gremicambarus) virilis Rostrum with straight margins, not thickened or possessing spines or tubercles; median carina absent; postorbital ridges terminating cephalically with spine or tubercle. Branchiostegal spine reduced; hepatic spine absent. Cephalothorax oval shaped and slightly dorsoventrally flattened in profile; without setae; suborbital angle obsolete. Areola 7.1–19.0 times longer than wide, comprising 34–39% of TCL, with 1–2 rows of punctations across narrowest region. Chelae smooth, broad and robust; mesial surface of palm with two rows of defined tubercles; first row with 6–8 rounded tubercles; second with 5–8 tubercles; lateral margin of propodus smooth; dorsal surfaces of both dactyl and fixed finger of propodus with prominent well developed longitudinal ridges; elongate plumose setae at base of fixed finger of propodus. First form male gonopods long, comprising 42% of TCL, with 2 terminal elements, both bent and curving at about 30° to the base; central projection corneous, comprising 24% of gonopod length, cephalic base without shoulder. Form two male gonopod noncorneus, gently curving caudally; mesial process subequal in length to central projection, blunt. Female annulus ventralis rhomboid, fossa large, sulcus wide, cephalolateral prominences weak, sinus only evident on caudal surface.Carapace and abdomen dorsally olivaceus or brown; rostral margins darker brown to black; postorbital ridges and caudal margins of cephalic portion of carapace along cervical groove brown; two rows of blotches on dorsal surface of abdomen; dorsal surface of chelae emerald green; tips of propodus and dactyl darker green; all knobs on chelipeds beige or tan; ventral surfaces cream or white.Orconectes virilis were collected in Mason and Pleasants counties at three locations in the current study, as listed below.MASON COUNTY: Krodel Park marsh adjacent to Fort Randolph reproduction, 38.785404 -82.12209; 26 March 2004 – (WLU 04032601), 2 I♂, 1 ♀; 28 April 2004 – (WLU 04042801), 1 II♂, 1 ♀; 17 March 2005 – (WLU 05031703), 1 I♂; 28 March 2005 – (WLU 05032802), 7 I♂; 5 May 2005 – (WLU 05050501), 5 II♂, 1 ♀. Pin oak swamp adjacent to Point Pleasant Moose Lodge in Wagner, 38.833603 -82.12227; 27 February 2005 – (WLU 05022701), 1 I♂. PLEASANTS COUNTY: Ohio River embayment 4.03 air km (2.52 mi) S of St. Mary’s, 39.397575 -81.202415; 30 March 2004 – (WLU 04033002), 2 II♂.Orconectes virilis native range includes Saskatchewan south through Montana and Utah east to Ontario, and throughout northern portions of the Mississippi River system. Several disjunct populations persist in Ohio and throughout the northeast.West Virginia Orconectes virilis populations are invasive . This species was the largest crayfish collected in this study. Morphometrics for Orconectes virilis are presented in The largest observed individual was a 52.4 mm TCL form I male collected from Krodel Park Lake, Mason County. The largest female was 43.3 TCL, also from Krodel Park. The mean TCL for Orconectes virilis Cambarus rusticus  Girard 1852:88. Cambarus juvenilis Cambarus (Faxonius) rusticus Faxonius rusticus Cambarus (Faxonius) rusticus rusticus Cambarus (Faxonius) juvenilis Faxonius (Faxonius) rusticus rusticus Orconectes rusticus rusticus PageBreakOrconectes (Orconectes) juvenilis Orconectes rusticus Procambarus rusticus  Huner 1978:4.Orconectes (Procericambarus) rusticus Rostrum with concave margins, not thickened, with spines or tubercles; median carina absent; mandible with smooth cutting edge. Cephalothorax oval, slightly dorsoventrally compressed in profile. Areola 4.6–19.4 times longer than wide, comprising 36–39% of TCL, with 2–3 rows of punctations across narrowest region; branchiostegal spine poorly developed; suborbital angle obsolete or poorly developed. Chelae robust; mesial surface of palm with two rows of defined tubercles, first row with 5–9 tubercles; second row with 4–9 tubercles of smaller diameter. First form male gonopods long, comprising 26% of TCL, with 2 straight terminal elements; central projection comprising 56% of gonopod length; well developed shoulder at cephalic base of central projection. Second form male gonopod non-corneous, straight, mesial process slightly subequal in length to central projection, blunt, shoulder not evident. Annulus ventralis rhomboid in shape, fossa moderately large, cephlolateral prominences well developed, trough narrow, sinus evident on caudal surface.Carapace and abdomen dark green or brown; large rusty lateral blotch present on each side of posterior margin of carapace; fixed finger of propodus and dactyl with red tips and black band; ventral surfaces cream or white.Orconectes rusticus were only collected from Marshall County at two locations in the current study, as listed below.MARSHALL COUNTY: PPG Wildlife Management Area adjacent to RT 2 S, 39.736244 -80.84638; 21 March 2006 – (WLU 06032103), 1 I♂; 18 April 2006 – (WLU 06041802) 4 I♂. WETZEL COUNTY: Ohio River backwater at Marshall/Wetzel County line, 39.717846 -80.514959; 2 April 2004 – (WLU 04040203), 4 I♂, 3 O♀; 11 April 2004 – (WLU 04041101), 3 I♂, 3 O♀; 21 March 2006 – (WLU 06032101), 10 I♂, 1 O♀; 18 April 2006 – (WLU 06041801) 9 I♂, 3 ♀.Orconectes rusticus .PageBreakrusticus is nativerusticus ., and isrusticus  and are Orconectes rusticus populations. Bayer Chemical Plant and Pittsburgh Paint and Glass (PPG) Chemical Plant both possess embayments connected to the Ohio River mainstem that contain Orconectes rusticus populations. The PPG population is present in a “pond” with a connection to the Ohio River mainstem. The Bayer population is present in a series of backwaters with mainstem connections. Trapping results show that the Bayer population has higher densities than the PPG population.The Upper Ohio South basin is the only basin within the floodplain that currently harbors Orconectes rusticus is limited to these two backwaters despite its presence in the Ohio River mainstem needs furthur investigation. Populations present in the mainstem could operate as sources for future invasions into new watersheds. The extent of the range of Orconectes rusticus within the mainstem is also in need of future work. Given the Ohio River’s manipulation into a series of non-contiguous pools, investigations into those pools that harbor Orconectes rusticus populations and those pools that do not is a proactive move to understand basins at risk of future invasions.Why Orconectes rusticus were collected from two sites. The largest individual was an ovigerous female 44.1 mm TCL. The largest male was a 38.4 mm TCL form I male from PPG Wildlife Management Lake. Mean Orconectes rusticus TCL was 31.0 mm . Nine females were ovigerous and had a mean carapace length of 30.1 mm. Morphometrics data for Orconectes rusticus is presented in Forty-four Orconectes rusticus inhabits two Ohio River back- waters Cambarus acutus  Girard 1852:91.Astacus Blandigii  Leconte 1956:400.Cambarus acutus  var. A Cambarus acutus  var. B Cambarus stygius Cambarus Stygius Cambarus blandingii acutus Cambarus blandingi acutus Cambarus (Cambarus) blandingi acutus Ortmannicus blandingi acutus Procambarus (Ortmannicus) acutus acutus Procambarus acutus Procambarus (Ortmannicus) acutus Rostrum slightly broad and triangular; width of rostrum margins reduced; margins converging terminating in 2 marginal spines; acumen with distal rostral spine; postorbital ridges prominent, cephalic margin with tubercle; cephalothorax dorsolaterally compressed in profile, anterior portion vaulted; areola obliterated at narrowest point; branchiostegal region moderately punctate, with small tubercles; small cervical spine present; chelae elongate and lance shaped; mesial surface of palm with single dorsal row of 7–9 pronounced tubercles; additional tubercles scattered over dorsal surface of palm. Bases of first form male gonopods contiguous; gonopod with 4 terminal elements, covered by dense setae; central projection pointed and corneous; caudal process short; mesial process pointed and straight in profile; cephalic process elongated and pointed; second form gonopod annulus ventralis circular in shape, embedded deeply in sternum, and movable.PageBreakCarapace, chelae, and pereiopods dorsally and laterally red-gray, gray-purple, burgundy, or red; branchial region of smaller individuals mottled with black spots; tubercles on chelae cream, red-brown, red, or black; dorsal surface of abdomen with a distinct black wedge.Procambarus acutus were collected in Mason County at three locations, listed below.MASON COUNTY: Roadside ditch adjacent to RT 2, 9.17 km (5.7 mi) S of Point Pleasant, 38.80469 -82.18821; 12 April 2004 – (WLU 04041206), 1 I♂, 3 ♀; 13 April 2004 – (WLU 04041301), 1 II♂; 28 April 2004 – (WLU 04042802), 2 I♂, 1 II♂; 17 March 2005 – (WLU 05031701), 2 I♂; 18 March 2005 – (WLU 05031802), 2 II♂, 2 ♀. Krodel Park marsh adjacent to Fort Randolph reproduction, 38.785404 -82.12209; 5 March 2005 – (WLU 05030502), 1 I♂, 2 ♀; 17 March 2005 – (WLU 05031703), 8 I♂; 22 March 2005 – (WLU 05032201), 2 I♂, 2 II♂, 2 O♀, 8 ♀, 1 Juv. Pin oak swamp adjacent to Point Pleasant Moose Lodge in Wagner, 38.833603 -82.12227; 26 February 2004 – (WLU 04022601), 4 I♂, 2 ♀; 26 March 2004 – (WLU 04032601), 3 ♀, 2 Juv.; 12 April 2004 – (WLU 04041205), 4 I♂, 2 ♀, 2 Juv.; 28 April 2004 – (WLU 04042801), 9 II♂, 2 ♀; 21 March 2005 – (WLU 05032101), 1 ♀; 10 April 2005 – (WLU 05041001), 1 I♂, 1♀.Procambarus acutus is a wide-ranging species associated with wetlands present throughout the central and eastern United State excluding the majority of the Appalachian Mountains , and Fallicambarus fodiens, are found in the Lower Ohio and Lower Kanawha drainages in West Virginia. They are theorized to be pre-glacial Marietta River relicts . Morphometrics data for Procambarus acutus are presented in The largest Procambarus acutus  between carapace length and total number of pleopodal instars. Procambarus acutus PageBreakpopulations appear to have six distinct size cohorts although these data need to be interpreted with caution due to low sample size  from early May 2005 through mid-July 2005. Crayfish associates collected with Procambarus acutus included Cambarus thomai, Cambarus bartonii cavatus, Fallicambarus fodiens and Orconectes virilis.Amplexus was observed in the field on 5 May 2005 at 22:00 h. Two amplexing pairs were observed resting on pond substrate adjacent to fallen logs. No amplexing pairs were observed away from cover objects. Several specimens also amplexed in collecting buckets within minutes of being introduced on this same day. An interspecies amplexus was observed on 5 May 2005, when a large form I male Procambarus acutus is a native species and should be given protection. Future investigations need to focus on the Kanawha River Floodplain between Point Pleasant and St. Albans. The Moose Lodge wetland is located on the Kanawha River and is the most diverse site surveyed in this study. All major habitats along the Ohio River floodplain that have been surveyed extensively in Mason County did not yield any additional Procambarus acutus populations. When present at a site, Procambarus acutus was the dominant surface water crayfish. Conservation efforts for this species should focus on preserving habitat. The Moose Lodge wetland should be conserved for protection and monitoring, not only for Procambarus acutus populations, but also for the myriad of Marietta River relicts occurring in the wetland.PageBreakBased on the Marietta River Valley theory, we believe that (Jugicambarus) monongalensis Ortmann, 1905Cambarus Allegheny Blue MudbugCambarus monongalensis Ortmann, 1905  dubius Faxon, 1884Cambarus Upland Burrowing CrayfishCambarus dubius Faxon, 1884  to a southern fauna (Orconectes sanbornii and Cambarus bartonii cavatus).The Middle Ohio North basin crayfish fauna is composed of six native and one invasive species . NortherCambarus carinirostris occupies Proctor Creek in extreme northern areas, while Cambarus bartonii cavatus dominates in the remaining stream systems. Both species occur in headwater streams. Cambarus carinirostris occupies larger streams within the northern regions of the basin than does Cambarus bartonii cavatus, with the latter limited to first through third order streams.This is the only basin where two secondary burrowing stream forms occur but not syntopically. Orconectes obscurus and Orconectes sanbornii are both tertiary burrowers, which occupy the Middle Ohio North basin. Orconectes obscurus populations are shifting south and occupy stream systems where Orconectes sanbornii historically occurred. Cambarus robustus, another tertiary burrower, is also found but in reduced numbers relative to the orconectids.PageBreak Within the Middle Ohio North basin, Cambarus robustus inhabits smaller streams than occupied by populations in the Upper Ohio North and Upper Ohio South basins. Orconectes virilis, an invasive species, occurs in one river embayment. This population could be a potential source for future invasions.Cambarus thomai is the sole primary burrowing crayfish species inhabiting the Middle Ohio North floodplain. Populations in New Martinsville, Wetzel County, are the most abundant and there are healthy northern populations along the floodplain. Large populations of the species are present at the Ben’s Run/Ohio River confluence , silver maple (Acer saccharinum L.), and black gum (Nyssa sylvatica Marsh). Bottomland forest sites, such as Moose Lodge wetland, represent an ecological dynamic unique to ephemeral wetlands. Forest canopies and the allochthonous energy contained within them are important forage for crayfishes and provide cover in ephemeral pools , while sites without buffer zones that lacked canopies comprised less stable populations with reduced numbers of individuals  and improve site condition, providing more stable, diverse, and increased numbers of crayfish populations than those without buffers . These bividuals . Sites wividuals . SimilarCambarus thomai, which was the most prevalent burrowing species along the least-disturbed portions of the floodplain, were particularly impacted and reduced at sites associated with agricultural activities Agricultural impacts caused the most extreme anthropogenic stressors, including the clearing of forests and riparian buffer zones, increased usage of chemicals , and compacted soils. tivities . At agriWith the conservation goal of preserving diverse floodplain crayfish populations, protection of remaining bottomland forest habitat in the Ohio River riparian corridor must occur. Less than 10 tracts of bottomland forest remain that are 100 hectares or larger in size. Sites such as Boaz Swamp, Greenbottom Swamp, McClintock Wildlife Management Area, and the Moose Lodge wetland are critical areas in need of protection. This action will preserve both crayfish diversity and multiple floral and faunal communities unique to this habitat.Orconectes rusticus and Orconectes virilis, were discovered along the Ohio River floodplain. Both species occur throughout the mid-Atlantic region and are responsible for native crayfish declines and extirpation  and had direct connections to the Ohio River; however, stream confluences within 1 km north and south of these sites did not harbor Orconectes rusticus populations. Orconectes rusticus reached high densities in these backwater habitats, feeding on the abundance of macrophytes and periphyton. Backwater microhabitats utilized by Orconectes rusticus include shallow, littoral regions. This microhabitat provides abundant forage and refugia along large river backwaters. The invasion dispersal occurs through smaller stream confluences with the Ohio River. Understanding the dispersal relationship between invasive crayfish and large river system corridors and smaller stream confluence connections is an important research need.Successful conservation of imperiled species is directly correlated with a fundamental understanding of life history parameters and species ecological needs . CrayfisOrganisms respond differentially to environmental stressors, and in the case of genetic lineages, different approaches to ecological adaptation can result in the creation of unique forms. Life history models . For instance, Details of Ohio River floodplain crayfish natural history may provide linkages to the specific mechanisms causing species decline. Once specific mechanisms are identified, conservation actions can be formulated. Information can be shared among astacologists and government agencies responsible for protecting non-game wildlife. These data can assist in the creation of management plans and their implementation; thus ensuring a rich floodplain crayfish fauna for future generations.


Drebrin, a side binding protein of actin filaments and well known for spine formation, was identified as a phosphorylated protein. Drebrin has two isoforms, an embryonic form drebrin E and an adult type long isoform drebrin A. Ser142 was identified as a common phosphorylation site to drebrin E and A and Ser342 as a drebrin A-specific site. Phosphorylated drebrin is localized at the distal area of total drebrin in the growth cone of cultured primary neurons. By expressing nonphosphorylatable or phosphorylation mimicking mutants in developing neurons in utero, the reversible phosphorylation/dephosphorylation reaction of drebrin was shown to be involved in radial migration of cortical neurons. These results suggest that Cdk5-p35 regulates neuronal migration through phosphorylation of drebrin in growth cone processes.Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase which plays a key role in neuronal migration, neurite outgrowth, and spine formation during brain development. Dynamic remodeling of cytoskeletons is required for all of these processes. Cdk5-p35 phosphorylates many cytoskeletal proteins, but it is not fully understood how Cdk5-p35 regulates cytoskeletal reorganization associated with neuronal migration. Since actin filaments are critical for the neuronal movement and process formation, we aimed to find Cdk5 substrates among actin-binding proteins. In this study, we isolated actin gels from mouse brain extracts, which contain many actin-binding proteins, and phosphorylated them by Cdk5-p35  Mammalian neurons display active cellular movements and dynamic morphological changes during brain development Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/Thr kinase that is activated by the neuron-specific regulatory subunits p35 and p39 In vitro characterization of drebrin has been previously described but its regulation has yet to be investigated. Phosphorylation of drebrin is indicated in non-neural cells Drebrin is a side-binding protein of actin filaments In this study, we found that drebrin is phosphorylated by Cdk5-p35. We additionally identified Ser142 and Ser342 in drebrin at Cdk5-phosphorylation sites and identified the role of neuronal migration in the embryonic cortex.All animal experiments were performed according to the guidelines for animal experimentation of Tokyo Metropolitan University. The study was approved by the Research Ethics Committee of Tokyo Metropolitan University . All efforts were made to reduce the suffering of animals used.Monoclonal anti-drebrin (M2F6) and monoclonal anti-GFP were purchased from MBL . Monoclonal anti-neuron-specific class III β-tubulin (Tuj1) was obtained from Genzyme-Techne , anti-actin was obtained from Sigma-Aldrich , and monoclonal anti-myc (4A6) was obtained from Millipore . Anti-Homer2a was used as described previously Phospho-Ser142 peptide C-LARLS(pS)PVHR and non-phospho-Ser142 peptide C-LARLSSPVHR were obtained from BEX . The keyhole limpet hemocyanin (KLH)-conjugated phospho-Ser142 peptide was immunized into 15-weeks-old female rabbits  using TiterMax Gold  as adjuvant. The anti-phospho-Ser142 antibody (pS142) was purified from antiserum using a phospho-Ser142-bound column after removing non-phospho-antibody with a Ser142 peptide-bound column.HA-Cdk5, kinase-negative HA-Cdk5 (Cdk5-D144N), and p35-myc have been described previously 5′-GATCTCGAGGCATGGCCGGCGTCAGC-3′ and 5′-TTGGATCCCTACAGTCTGGGAGCAGACTCC-3′ as forward and reverse primers, respectively, for the N-terminal fragment, 5′-TAACTCGAGGCATGGACGGTGAAGAGGTCTGC-3′ and 5′-GTGGATCCCTAATCACCACCCTCGAA-3′ for the C-terminal fragment. Nonphosphorylation Ala and phosphorylation-mimic Asp mutants of drebrins were constructed by site-directed mutagenesis using pEGFP-drebrin A, pEGFP-drebrin E, pEGFP-drebrin A-NT, pEGFP-drebrin-CT, pEGFP-ins2 and pET19b-drebrin A and pET19b-drebrin E as templates. The primers used were as follows:5′-TCGGCTCTCTGCCCCAGTGCTGC-3′ and 5′-GCAGCACTGGGGCAGAGAGCCGA-3′ for S142A, 5′-CTTCCTCCTCTGCCCCTCCACG-3′ and 5′-CGTGGAGGGGCAGAGGAGGAAG for S342A, 5′-CCTCCACGGGCTCCCTTTCCC-3′ and 5′-GGGAAAGGGAGCCCGTGGAGG-3′ for T346A, 5′-CCTGCCACCGCGCCCCAAACCT-3′ and 5′-AGGTTTGGGGCGCGGTGGCAGG-3′ for T356A, 5′-ATGGCGCCCGCTCCCATTCCCA-3′ and 5′-TGGGAATGGGAGCGGGCGCCAT-3′ for T377A, 5′-ATTCCCACCCGGGCCCCATCTGATT-3′ and 5′-AATCAGATGGGGCCCGGGTGGGAAT-3′ for S383A, 5′-ACAGCCTCCGCCCCCATCACGGA-3′ and 5′-TCCGTGATGGGGGCGGAGGCTGT-3′ for T392A, 5′-TCGGCTCTCTGACCCAGTGCTGC-3′ and 5′-GCAGCACTGGGTCAGAGAGCCGA-3′ for S142D, 5′-CTTCCTCCTCTGACCCTCCACG-3′ and 5′-CGTGGAGGGTCAGAGGAGGAAG-3′ for S342D. pEGFP-drebrin A-WT, S142A/S342A, and S142D/S342D were subcloned into BamHI site of pCAG-GFP-MCS2 XhoI site of pCAGGS. Mutations were confirmed by DNA sequences.Escherichia coli BL21-CodonPlus (DE3)-RP cells and obtained as a heat-stable supernatant of the cell extracts pET19b-drebrin A, pET19b-drebrin E, and their Ala mutants were expressed in et al. 2, 0.1 mM EDTA, 0.1 mM EGTA, 0.5% Nonidet P-40 (NP-40). The actin gel fraction was collected and used for in vitro phosphorylation.Actin gels were prepared from mouse brains according to the method described previously by Taguchi Protein bands were stained using the ProteoSilver Plus Silver Stain Kit  according to the manufacturer’s protocol and excised from the polyacrylamide gel. After washing, the gels were digested by incubation in buffer with trypsin 32P]ATP. Phosphorylation was detected by autoradiography after 10% polyacrylamide gel SDS-PAGE, and the extent of phosphorylation was quantified using a FLA7000 bioimage analyzer .Cdk5-p35 was expressed and purified from Sf9 cells  infected by Baculovirus encoding Cdk5 and p35, as described previously 4 cells/cm2 for immunoblotting or at a density of 1.5×104 cells/cm2 on coverslips for immunofluorescent staining. Cells were transfected with the indicated plasmids using PolyFect Transfection Reagent  or Lipofectamine 2000  according to the manufacturer’s instructions.COS-7 and Neuro2A cells were obtained from Japanese Collection of Research Bioresources  and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. Cells were plated at a density of 3×105 cells/cm2 for immunoblotting or 1.3–7.8×104 cells/cm2 for immunofluorescence staining in DMEM and HAM F-12 (1∶1) supplemented with 5% fetal bovine serum and 5% horse serum. At 4 h after plating, culture medium was changed to Neurobasal Medium containing B27 Supplement and 0.5 mM L-glutamine. Half of culture medium was changed every 2 days.ICR mice were purchased from Sankyo Labo. Mice were housed in a temperature-controlled room under a 12-h light/12-h dark cycle with free access to food and water. Brains were dissected from embryos at E14–16. Cortical and hippocampal neurons were prepared as described previously in vitro) by electroporation using an Amaxa Nucleofector  and the number and length of axon and neurites was measured as described previously Plasmid vectors were introduced into neurons at 0 DIV .2. Proteins were transferred to PVDF membranes using a submarine blotting apparatus. Immunodetection was carried out using an enhanced chemiluminescence system  or Millipore Immobilon western chemiluminescent HRP substrate (Millipore).Laemmli’s SDS-PAGE was carried out using 7.5% polyacrylamide gels. Proteins were transferred to PVDF  membranes using a semi-dry blotting apparatus. Phos-tag SDS-PAGE was performed with 5% or 7.5% polyacrylamide gels containing 60 αM Phos-tag acrylamide and 90 αM MnClImmunoreaction was visualized as digital images by scanning X-ray film, and band intensities were measured using Image J software. GFP images of cultured cells expressing EGFP-drebrin were acquired with Zeiss 510 Exciter or 710 confocal microscopes. Axonal length and neurite number were measured using ZEN imaging software (Zeiss). The longest process of primary neurons at 3 DIV was defined as an axon and its length was measured. Other processes longer than 20 μm were defined as neurites and their number (per neuron) was measured. Protrusions with length of 0.5 αm to ∼5 αm on dendrites of neurons at 21 DIV were defined as dendritic spines, and the length, width, and number per 30∼50 αm primary dendrite were measured. All quantitative data were represented as mean ± SEM and were subjected to two-tailed unpaired Student’s t-test for single comparison, or one-way ANOVA analysis, followed by Tukey’s or Tukey-Kramer’s post-hoc test, for the multiple comparison. Values of p<0.05 were considered to be statistically significant.in vitro in the presence of [γ-32P]ATP P site of drebrin A-NT and ins2 and expressed them in Neuro2A cells with or without Cdk5-p35. There are three (S/T)P sites in ins2 with an SPPR sequence as a preferred consensus for Cdk5 . Their aTo confirm the phosphorylation of these sites in full-length drebrin, we mutated Ser142 in drebrin E and Ser142 and Ser342 in drebrin A to Ala . Introduin vitro phosphorylation by incubating recombinant drebrin with purified Cdk5-p35 in the presence of [α-32P]ATP. Drebrin E-WT was phosphorylated by Cdk5-p35 and its phosphorylation was completely abolished with the S142A mutant  by immunoblotting . Their migration to cortical plate was examined at P2. Most of neurons expressing GFP or GFP-drebrin WT migrated to upper cortical plate ; therefore, we examined the effect of phosphorylation on the migration of embryonic cortical neurons using For quantification, we divided the cerebral cortex into three zones: upper cortical plate (UCP), lower cortical plate (LCP), and intermediate zone (IZ), as shown in the right side of Here, we demonstrated that the phosphorylation of drebrin occurs at Ser142 and Ser342 by Cdk5, and also described the roles of Ser142 and Ser342 in neuronal migration. We first identified drebrin as a protein in actin gels phosphorylated by Cdk5 by mass spectrometric analysis, and then further determined Ser142 as a phosphorylation site common in drebrin E and A, and Ser342 as a drebrin A-specific site by Phos-tag analysis in combination with Ala mutants. Phosphorylation was confirmed in neurons and detected in a subset of drebrin present at the distal region of total drebrin in the transition zone of the growth cone, which is involved in migration of embryonic cortical neurons. These results indicate that Cdk5 regulates neuronal migration through phosphorylation of drebrin in growth cone of processes in migrating neurons.Phosphorylation of drebrin has previously been described et al., During the preparation of this manuscript, phosphorylation of drebrin at Ser142 by Cdk5 was reported Phosphorylation at Ser142 was detected in a fraction of drebrin in the growth cone. The growth cone is the motile structure at the tip of neurites which lead the extension and navigate the direction, and is divided into three parts: the central domain, which includes microtubules elongating from neurite shaft, the peripheral domain, composed primarily of filopodia and lamellipodia, in which actin filaments are organized in parallel bundles and a mesh-like network, respectively, and the transitional domain at the interface of peripheral and central domains where actin filaments accumulate. Depending on the outgrowth stage, the growth cone shows two different images, the filopodial growth cone or the lamellipodial growth cone et al. A similar localization of phosphorylated drebrin at Ser142 was shown by Worth p35 binds to and activates Cdk5 but also determines the cellular localization of the active Cdk5 complex et al.Overexpression or knockdown of drebrin has been shown to suppress and stimulate neurite outgrowth, respectively in utero electroporation of shRNA inhibited the migration of cortical neurons . Based on these data, we examined the effect of drebrin phosphorylation on neuronal migration. We introduced 2A or 2D mutant or WT drebrin A into differentiating neurons at the ventricular zone of the embryonic mouse brain. From the distribution pattern of phospho-drebrin in the growth cone, we initially hypothesized that phospho-mimic drebrin would inhibit migration; however, drebrin A-2A, as well as -2D, decreased migration compared to drebrin A-WT, whose overexpression did not affect the migration. These results suggest the reversible phosphorylation and dephosphorylation of drebrin is important for neuronal migration.It was recently reported that drebrin is required for the correct migration of oculomotor neurons in chick embryonic brains by growth cone formation and navigation Mice lacking Cdk5 or p35 have inverted neuronal layers in the neocortex et al. et al., 2013). Drebrin could be another target of Cdk5 in the growth cone of neurons at the time as the multipolar-bipolar transition. The results that neurons expressing drebrin A-2A or -2D showed impairment in neuronal migration support this hypothesis.Cdk5 is involved in multipolar–bipolar transition Cdk5 has previously been described as a direct regulator of the microtubule cytoskeleton in radial neuronal migration


The cellular defense to infection depends on accurate activation of transcription factors and expression of select innate immunity genes. Interferon regulatory factor 5 (IRF5), a risk factor for systemic lupus erythematosus, is activated in response to pathogen recognition receptor engagement and downstream effector molecules. We find the nucleotide-binding oligomerization domain containing protein 2 (NOD2) receptor to be a significant activator of IRF5. Phosphorylation is key to the regulation of IRF5, but the precise phosphorylation sites in IRF5 remained to be identified. We used mass spectrometry to identify for the first time specific residues that are phosphorylated in response to TANK-binding kinase-1 (TBK-1), tumor necrosis factor receptor-associated factor 6 (TRAF6), or receptor interacting protein 2 (RIP2). RIP2, a kinase known to function downstream of NOD2, was the most effective activator of IRF5-regulated gene expression. To determine if the phosphorylated residues are required or sufficient for IRF5 activity, aspartic acid phosphomimetic substitutions or inactivating alanine substitutions were tested. Phosphorylation of carboxyl serines 451 and 462 appear the primary trigger of IRF5 function in nuclear accumulation, transcription, and apoptosis. Results indicate polyubiquitination of IRF5 does not play a major role in its transcriptional activity, and that ubiquitination and phosphorylation are independent modifications. Interferon regulatory factor 5 (IRF5) is an autoimmune susceptibility factor associated with increased risk of human systemic lupus erythematosus (SLE) IRF5 is a latent transcription factor with constitutive expression in lymphocytes, macrophages and dendritic cells Although phosphorylation appears to be key for IRF5 activation, the specific phosphorylated amino acids that lead to IRF5 activation remained to be determined. Mass spectrometry was not used prior to this study to directly identify IRF5 phosphorylation sites. Indications that serine clusters at the carboxyl terminus were important for activity were suggested based on homology with IRF3 and targeted mutations The response of cells to various ligands of pattern recognition receptors can be complex, involving cross-talk of diverse downstream signals. Thus in order to evaluate the impact of specific molecules on IRF5 transcriptional activity, we examined the effect of TBK-1, TRAF6, or RIP2 co-expression with IRF5 on a responsive reporter gene. IRF5 is required for induction of a number of cytokines including interleukin-12, and for this reason we used a luciferase gene reporter assay regulated by the promoter of the interleukin-12 (IL-12) p40 subunit gene The IL-12 p40 gene is a bona fide target of IRF5, but there is also evidence that IRF5 plays a role in the induction of a subset of type I interferon (IFN) genes Phosphorylation of IRF5 appears to activate its ability to induce transcription of target genes. Since phosphorylation can change the mobility of proteins during gel electrophoresis, we evaluated the effect of activators on the migration of IRF5 through SDS-PAGE. IRF5 was immunoprecipitated from cells co-expressing TBK-1, TRAF6, or RIP2, separated by gel electrophoresis, and detected by silver staining . There win vitro phosphorylation of bacterially expressed IRF5 by immunocomplexes containing TBK-1, and in vivo phosphorylation of IRF5 by co-expression with TBK-1 in tissue culture cells. Modified forms of IRF5 were separated by SDS-PAGE and submitted for analysis. Two phosphoserines were identified, S158 and S309  IRF5 wt and phosphorylation site mutants were tagged with GFP to enable direct visualization by fluorescence microscopy. Cellular localization was observed with or without LMB treatment to determine the impact of mutations on nuclear import. The effect of LMB on latent wt IRF5 can be seen to result in nuclear accumulation, indicating a functional constitutive NLS . AlthougMutations of the other phosphoserines identified by mass spectrometry were also evaluated for nuclear localization. The alanine or aspartic acid substitutions for S309, S317, and S462 had no notable effect on IRF5 localization, but S451D resulted in detectable nuclear IRF5 in 20–30% of the cells. The reason for the selective nuclear appearance of S451D in a subset of cells remains to be determined. The double mutant SS451,462DD that demonstrated significant transcriptional activation of the IL12p40 gene gained prominent nuclear accumulation independent of any activator.IRF5 was reported to be polyubiquitinated via a K63 ubiquitin linkage in response to TLR signaling It remained to be determined whether specific phosphorylation of IRF5 was required for its ubiquitination, or whether ubiquitination was sufficient for IRF5 activation. To evaluate these relationships we analyzed the IRF5 SS451,462AA (C′-2A) mutation that cannot be phosphorylated on the carboxyl serines, and the IRF5 SS451,462DD (C′-2D) mutation that is transcriptionally active . TRAF6 wTo further investigate the interplay between phosphorylation and ubiquitination of IRF5, we tested the effect of the ubiquitin-editing enzyme A20, known to possesses K63-deubiquitinating activity Another line of evidence indicated that direct ubiquitination of IRF5 is not necessary for its transcriptional function. Specific lysine residues of IRF5 were previously reported to be required for K63-ubiquitination  Studies by our group and others have linked expression of IRF5 with another cellular function, the promotion of apoptosis Engagement of pattern recognition receptors (PRRs) stimulates innate immune responses that promote clearance of microbial infections, but in addition these responses can contribute to inflammation and autoimmunity We investigated the effects of several potential activators of IRF5; TBK-1, TRAF6, and RIP2. TBK-1 was previously identified as a kinase that phosphorylates the carboxyl terminus of the related transcription factor, IRF3, in response to viral infection Although the gene induction by IRF5 in the presence of TBK-1 was modest, phosphorylation by TBK-1 produced a significant shift in the mobility of IRF5 in SDS-PAGE. For this reason we identified the residues that are phosphorylated on IRF5 by TBK-1 with mass spectrometry. Ser-158 and Ser-309 were found to be phosphorylated, and mutations of these serines eliminated specific shifts in IRF5 mobility by SDS-PAGE. These residues did not appear to be modified by TRAF6 or RIP2. In addition, these residues did not reside in the carboxyl terminus of IRF5, a region that was expected to contribute to its modification and activation TRAF6 and especially RIP2 were far more effective activators of IRF5 than TBK-1 , and masSLT), and so the result suggests that the serine phosphorylation impairs nuclear export of IRF5.As a transcription factor IRF5 must gain entrance to the nucleus, and nuclear transport is one mode of regulation. Although IRF5 has two reported NLSs and is transported to the nucleus continually, it also has a dominant NES Since K-63 polyubiquitination was reported to be necessary for IRF5 activation, we investigated the relationship between IRF5 phosphorylation and ubiquitination We used two other approaches to evaluate ubiquitination and transcriptional activity of IRF5 . A20 is Since another property of IRF5 is its ability to promote apoptosis, we tested the effects of substitution mutants on this function . ExpressPhosphorylation of IRF3, IRF5, and IRF7 triggers a conformational change that promotes dimerization and binding to CBP/p300 In this study our objective was to elucidate the molecular modifications that regulate IRF5 transition from latency to an active transcription factor. For the first time specific phosphorylation sites of IRF5 have been identified by mass spectrometry, and their contributions to gene induction and apoptosis have been evaluated. In addition, the effectiveness of RIP2 as an upstream activator of IRF5 suggests that IRF5 plays a preferential role in NOD-like receptor signaling. This knowledge advances our understanding of the molecular mechanisms that trigger IRF5 activity in health and disease.Human HEK293, HT1080, HeLa and murine RAW264.7 cells were obtained from ATCC. Cells were grown in Dulbecco's modified Eagle's medium with 8% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) (Invitrogen). To measure the effect of NOD2 signaling, 50 µg/ml of muramyl dipeptide (MDP) or 15 µg/ml insoluble peptidoglycan (InvivoGen) was added to RAW264.7 cultures. Leptomycin B (LMB) was used at 10 ng/ml .Plasmids T7-His-tagged pcDNA3 vector, T7-His-tagged IRF5v.5, GFP-IRF5, and FLAG-TBK-1 have been described TransIt-LT1 transfection reagent . The luciferase activity was measured by a Lumat model LB 9507 luminometer using Dual-Luciferase Reporter Assay System (Promega). Results were normalized to co-transfected pRLTK reporter gene . Values are means of three to six independent experiments, and bars show one standard error of the mean, and are expressed as the activity relative to pcDNA3 alone.Transfection of RAW264.7 cells was performed by electroporation . HEK293, HT1080 and HeLa were transfected using HT1080 cells on coverslips were transfected with GFP-IRF5 constructs and 24 hours later treated with leptomycin B for 1 hour. Cells were fixed in 3.7% formaldehyde/PBS and stained with 2 µg/ml of Hoechst 33342 at room temperature for 15 minutes. Coverslips were washed and mounted in Vectashield antifade solution . GFP-tagged proteins were observed with Zeiss Axiovert 200M and Axiovision Version 4.5 and images captured with Adobephotoshop.HeLa cells were transfected with GFP-IRF5 constructs, washed with media six hours post-transfection, and cell death was measured 1, 2 or 3 days post-transfection by propidium iodide staining and evaluation with a FACSCalibur flow cytometer  Antibodies used included anti-IRF5 , anti-T7 (Novagen), anti-RIP2 , anti-omni , anti-c-Myc , anti-HA , anti-FLAG , and secondary anti-mouse (Rockland) and anti-rabbit (Invitrogen) antibodies for Western blot analysis with Odyssey Imager (Li-COR Biosciences). For immunoprecipitation, cells were lysed in 50 mM Tris (pH 7.5), 400 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 50 mM sodium fluoride, 10% glycerol, 10 mM β-glycerolphosphate, 1 mM sodium vanadate, 1 mM PMSF and protease inhibitor mixture (Sigma-Aldrich). Lysates were clarified by centrifugation at 12,000 g for 10 min prior to antibody addition. Immunocomplexes were collected with protein-G beads, eluted, and separated on 8.5% SDS-PAGE. Proteins were transferred to Immobilon-P (Millipore) for Western blotting and reactive signals were detected with the Odyssey Imager (Li-COR Biosciences) and analyzed using Image J software (NIH). Alternatively, secondary antibodies linked to HRP were used  and the membrane was incubated in enhanced chemiluminescence reagents and exposed to film. Proteins visualized without Western blotting were detected by silver staining HEK293 cells were co-transfected with plasmids encoding His-tagged IRF5 and c-myc-tagged TBK-1 or c-myc-tagged TRAF6 or HA-tagged RIP2 with wild type HA-tagged ubiquitin or HA-tagged K0R63K ubiquitin. 48 hours post-transfection cells were harvested and His-tagged IRF5 proteins were isolated on Ni-NTA agarose beads (Qiagen). Elutes from the Ni-NTA agarose beads were subjected to 8.5% SDS-PAGE analysis and Western blot.in vitro by FLAG-TBK-1 immunoprecipitated from mammalian HEK293 cells. Proteins were separated on 7.5% SDS-PAGE and detected by Coomassie brilliant blue staining. A slower mobility IRF5 protein band was provided to the University of Massachusetts Medical School Proteomics Lab for in gel protein cleavage with trypsin and analysis by LS/MS/MS. Their data interpretation indicated phosphorylation of serine 156 or 158 and we confirmed 158 was modified by mobility shift analyses  were used to collect T7-IRF5 immunocomplexes from cell lysates. IRF5 was visualized in SDS-PAGE by staining with SimplyBlue (Invitrogen), and slow mobility IRF5 protein band was eluted, treated with iodoacetamide, and submitted for analysis to ProtTech Inc. . The sample was digested with trypsin and chymotrypsin to generate peptides that were reconstituted in 2% acetynitrile, 100 mM fumic acid pH 3.0, and analyzed by nano LC-MS/MS system for sequencing. A high-pressure liquid chromoatography C18 column was coupled with an ion-trap mass spectometer. The MS/MS data were analyzed with Protech's proprietary software. Peptide containing IRF5 serine 309 was identified by LS/MS/MS to be phosphorylated in the presence of TBK-1 in vivo. Additional in vivo phosphorylation analyses were performed by co-transfection of T7-His-IRF5 with either myc-TBK-1, myc-TRAF6, or HA-RIP2 in HEK293 cells. IRF5 was collected on T7 antibody agarose beads from the individual transfections, eluted, and subsequently pooled. Proteins were separated by SDS-PAGE, visualized with Commassie brilliant blue stain, treated with iodoacetamide, and submitted for analysis to ProtTech Inc. as described. Peptide analyses by LS/MS/MS identified additional phosphorylated amino acids corresponded to threonine 10, serine 309, serine 451, and serine 462. The detectable b-type and y-type fragment ions for the phosphopeptides were annotated in Analysis of IRF5 phosphorylated amino acids by TBK-1 was performed with two approaches. Bacterially expressed and purified His-tagged ΔNIRF5 was phosphorylated analyses . In vivoFigure S1Transfection controls for protein expression in . Cell lysates from a transfection shown (TIF)Click here for additional data file.Figure S2IRF5 phosphorylated amino acids identified by LC-MS/MS. Phosphopeptides identified are shown with specific phosphorylated threonine or serine. a–e) Identification of in vivo phosphorylated amino acids, threonine 10, serine 309, serine 317, serine 451, and serine 462 by ProtTech, Inc. following IRF5 immunoprecipitation from HEK293 cells expressing TBK1, TRAF6, and RIP. (see Methods). f) Identification of serine 158 phosphorylation by in vitro kinase reaction with TBK-1. Analysis by University of Massachusetts Proteomics Lab.(TIF)Click here for additional data file.Figure S3Transfection controls for IRF5 expression in . Expression levels of wt IRF5 and IRF5 mutations in absence or presence of omini-tagged RIP2 are similar in luciferase reporter studies shown in a) and 3d (b). Western blots with anti-IRF5, anti-omni or anti-tubulin antibodies.(TIF)Click here for additional data file.Figure S4Polyubiquitination of IRF5 via ubiquitin lysine 63. Assay performed as described in (TIF)Click here for additional data file.Figure S5Expression controls for IRF5 in . Expression levels are similar for wt IRF5, IRF5 KK427,428RR (KK/RR), and IRF5 KK427,428RR, SS451,462DD . Proteins from an experiment of (TIF)Click here for additional data file.Figure S6Controls for IRF5 expression in apoptosis studies of . Western blot of cell lysates from experiment shown in (TIF)Click here for additional data file.Table S1Oligonucleotide primers for plasmid generation. Single stranded forward primer DNA sequences are shown (5′ to 3′) that were used to introduce mutations in the IRF5 DNA sequence.(PDF)Click here for additional data file.