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Oral | Posters


Oral Presentations

 

 

Day: 2, Session: 1, Talk: 3

Welcome Address

James W. Fetzner Jr. and Eric Dorfman

Welcome Address

 

 

Day: 2, Session: 1, Talk: 4

HOST COUNTRY LECTURE:
An Overview of U.S. Crayfish Conservation in State Agencies and a Plan to Reverse CRAWnic Neglect

Robert J. DiStefano

Crayfish conservation can be accomplished in the United States (U.S.) by federal government, state governments, universities, non-governmental organizations (i.e., The Nature Conservancy), professional societies (e.g., American Fisheries Society), local governments, and private citizens. State fish and wildlife agencies are charged with protecting and managing each state’s aquatic resources, yet crayfish have historically received little attention from them. This is largely due to state funding formulas that rely heavily on sales of recreational licenses (fishing, hunting, etc.), and perceived responsibility of agencies to be more responsive to that segment of the public who buy licenses. Missouri is unique in having established a state conservation program for crayfish decades ago. The program has produced substantial research data on crayfish species distributions, ecology and threats, especially for imperiled and endemic species. It has yielded several conservation actions (e.g., regulations, education products and activities). I will reference Missouri as one possible model for establishing and maintaining a state government (or regional government elsewhere) crayfish conservation program that involves government staff, but also partners from academia and some stakeholder groups. I will conclude by providing a brief update about states’ involvement in U.S. crayfish conservation.

 

 

Day: 2, Session: 3, Talk: 6

Crayfish Conservation in Southern England

JEN NIGHTINGALE, Grainne McCabe, Gareth Jones and Paul Stebbing

The white-clawed crayfish, Austropotamobius pallipes has suffered severe declines within the south west of England, where the first signal crayfish, Pascifastcus leniusculus farms were established in the 1970s. In response to this decline, The South West Crayfish Partnership (SWCP) was formed in 2008; comprising Bristol Zoological Society, Buglife, Cefas, the Environment Agency, Wildlife Trusts and Associates. The SWCP implements landscape scale, strategic conservation for A. pallipes, in an attempt to safeguard the future of this species in South West England. The conservation effort has four strands: 1. Ark sites: established throughout the south west England, for translocation of the most highly threatened white-clawed crayfish populations and captive-bred reintroductions. 2. Crayfish captive breeding facility: established at Bristol Zoo, which provides plague-free A. pallipes brood stock for ark site release, wild supplementations, research and outreach. 3. Communication strategy: running in tandem with the other three elements, targeting key audiences such as anglers, restaurants, students, school children and zoo visitors. 4. Invasive crayfish control – trialing different control techniques specifically targeting the signal crayfish. The presentation will cover the key elements of this conservation programme, evaluating its success to date and discuss the research elements that run in tandem with all of these conservation strands.

 

 

Day: 2, Session: 3, Talk: 7

Introduced and Then Almost Forgotten: Invasive Crayfish (Procambarus clarkii and Cherax quadricarinatus) in Costa Rica, Central America - Preliminary Results

JUAN CARLOS AZOFEIFA SOLANO, Ingo S. Wehrtmann, Fresia Villalobos-Rojas, Raquel Romero-Chaves and Adrián García-Rodríguez

Costa Rica harbors a remarkable diversity of freshwater native decapods, including 15 crab species (Pseudothelphusidae) and more than 21 species of caridean shrimps (Atyidae and Palaemonidae). Crayfish (Astacoidea) are not part of the native freshwater decapod fauna of the country. However, during the last century, two crayfish species were introduced: Procambarus clarkii in the Reventazón basin (Caribbean slope) in the 1970's, and Cherax quadricarinatus in the Tempisque basin (Pacific slope) in the 1980's. Both species have been recognized as invasive species in many other countries, and there is broad evidence suggesting its negative impact on native species, ecosystem functioning, and even human activities. Despite the potential threat of these crayfish species for the Costa Rican freshwater environments, very limited information is available about their occurrence in the country. Therefore, it is imperative to document the current distribution of both species and to assess their possible effects on the freshwater ecosystems in Costa Rica. We initiated a study aimed to explore the presence of these invasive crayfish species in Costa Rican streams and lakes and to provide information about their feeding ecology. So far, we have collected P. clarkii in two locations, outside the Reventazón basin were it was initially introduced, and belonging to the Pacific slope. More than 160 specimens have been analyzed, ranging in size from 6.5 to 78.1 mm total length. The predominant food item found at the stomachs was unidentified plant detritus, followed by filamentous plants, fragments of chironomid and Ephemeroptera larvae, and plastic. Our collections revealed two new locations for C. quadricarinatus in Costa Rica: one of them a stream in the Caribbean slope, and the other in the Arenal Lake, the largest reservoir in Costa Rica. The size of the collected specimens (n=7) ranged from 45.1 to 92.2 mm total length. Currently, both crayfish species inhabit both Caribbean and Pacific slopes in Costa Rica. It remains to be studied whether these species dispersed naturally or if they were locally introduced by humans. Since both species have been introduced in Costa Rica and the Central American region, the results of the study aim to raise awareness about the possible impacts of these crayfish species on the native flora and fauna of the local freshwater ecosystems, and to develop recommendations for the implementation of management programs.

 

 

Day: 2, Session: 4, Talk: 1

Illegal Export of Australian Freshwater Crayfish – Intercepted Shipments: A Case of Euastacus

James M. Furse

The unique, and often remarkable, nature of the native flora and fauna of Australia is well known. Many of these native Australian species are highly attractive, desirable and sought-after by private collectors, but also commercial interests known to include the pet/aquarium, and restaurant and gourmet food trades. Live export of native Australian wildlife (i.e. amphibians, birds, mammals, reptiles) for commercial purposes is prohibited under any circumstances. Live export of some invertebrates, fish and plants may be permitted (for commercial purposes), if they originate from approved sources or programs (i.e. captive breeding, aquaculture, or wildlife trade/wildlife trade management plans). For non-commercial purposes live exports of native flora and fauna is permitted (e.g. research, education), but as with any type of native species export, this is strictly regulated. Illegal export of Australian native flora and fauna is a most serious (and Federal) offence: penalties can be very serious indeed. Despite this, it is well established that there is illegal and ongoing "leakage" of native Australian flora and fauna. The freshwater crayfish fauna of Australia is both unique, remarkable, and well known in the aquarium trade. In some regions of the World this fauna is also evidently known in the restaurant and gourmet food trade. Illegal export(s) of native species of freshwater crayfish have previously occurred, and ongoing illegal exports of these animals are suspected, and sadly also expected. This talk will briefly outline the rules and regulations limiting live exports of native Australian flora and fauna, and outline case(s) of apparent illegal exports of Australian freshwater crayfish. A recent case where an illegal shipment of Euastacus was intercepted, and seized, in Australia will be discussed. This discussion will include information on the species that was intercepted, the intended geographical destination, destination-industry and why such activities pose a very serious threat to such species. Other details surrounding the intercepted shipment will be outlined as may be appropriate.

 

 

Day: 2, Session: 4, Talk: 2

Invasive Rusty Crayfish (Orconectes rusticus) Populations in North America are Infected with the Crayfish Plague Disease Agent (Aphanomyces astaci)

LAURA MARTÍN-TORRIJOS, David Buckley, Ignacio Doadrio, Annie Machordom and Javier Diéguez-Uribeondo

European freshwater crayfish are currently included in the IUCN Red list as threatened. In the Iberian Peninsula, the native species (i.e., the white–clawed crayfish, Austropotamobius pallipes) has experienced a drastic decline since 1973. Currently, the implemented management strategies of these species require a better understanding of the patterns of genetic diversity. In this study, we assessed the levels and patterns of the genetic variation by analyzing the largest number of populations of the whole distributional range of the WCC in the Iberian Peninsula. The two ribosomal mitochondrial markers applied (Cytochrome Oxidase subunit I and rDNA 16S genes) indicated high levels of genetic variability, which are significantly geographically structured in three main genetic groups, i.e., two corresponding to Northern and one to Central-Eastern Iberian Peninsula). The diversity found includes new private haplotypes, and reveals WCC populations (i.e., Southern and Central European WCC populations), may be result of the ancient palaeogeographic events, such as geographic barriers, and the Last Maximum Glacial scenario (LMG) (i.e., isolation in glacial refugia). Current conservation and management programs for the WCC in the Iberian Peninsula should take into account these three phylogeographic areas as essential management units in order to preserve the maximum genetic diversity.

 

 

Day: 3, Session: 2, Talk: 1

Development of Mass Production Hatchery Technology for Cherax quadricarinatus

CLIVE JONES and Colin Valverde

Aquaculture production of redclaw (Cherax quadricarinatus) in Australia, has never reached the high level projections made in the 1990’s. This can be attributed to a range of factors, but the most significant is the supply of seedstock. One of the most positive aquaculture characteristics of redclaw is its bearing of offspring in adult form, obviating the requirement for larval rearing, as is necessary for most successfully commercialised aquaculture species. Among other positive characteristics, this was seen as the most significant and methods for pond-based, mass production of juvenile redclaw were devised and implemented by industry. This relied on natural reproduction, and natural productivity in the pond to support the survival and growth of the juveniles. Mean productivity was reported as 63.2 juveniles produced per berried female, and a harvest density of 24.6 juveniles per m2. Under suitable, summer photoperiod and temperature conditions, three consecutive crops of juveniles could be produced per year. Such managed pond production of seed stock for redclaw production was applied by industry with some success, but it soon became evident that productivity was too low and the practice occupied valuable pond space that would be more profitably applied to growout. An alternative production method for the supply of lobster juvenile redclaw was conceived by an innovative redclaw farmer, Colin Valverde, utilising artificial egg incubators, that had been successfully applied to freshwater crayfish species in Europe. The putative advantages of artificial egg incubation include savings of space, water and energy, minimising egg loss, control over the period of embryogenesis, known parentage for genetic selection and prevention of transmission of disease from parent to offspring. The primary advantages were initially for its application to genetic selection and for generating specific pathogen free stock. However, an even greater benefit became apparent, that this approach could support mass production of seedstock. The system that formed the basis of the that developed in Australia for redclaw was based primarily on the Hemputin™ incubator from Finland used for Pacifastacus leniusculus and Astacus astacus. Its design was modified to suit the specific requirements of redclaw and greater production volumes. The procedures and equipment have evolved, providing a foundation for more consistent and greater production of seed. The juvenile crayfish produced by the hatchery have generally moulted twice since hatching and are referred to as stage 3 juveniles or craylings. A hatchery supply of craylings provides the opportunity for farmers to stock an exact quantity of uniform size crayfish into growout ponds, which in turn enables calculation of likely survival, growth and biomass at the end of the production cycle. The Valverde hatchery system can generate tens of thousands of craylings per batch and is scalable. However there are production issues to be resolved including year round supply of eggs to the hatchery and management of egg health. This paper will detail the development of the Valverde hatchery system, and the challenges to see it develop into fully commercial production that can support expansion of the redclaw farming industry.

 

 

Day: 3, Session: 2, Talk: 2

The Louisiana Crawfish History Summary, The Last Fifty Years

JAY V. HUNER, Robert P. Romaire, C. Greg Lutz, Albert P. Gaude III., James W. Avault Jr., W. Ray McClain and Mark G. Shirley

The Louisiana USA crawfish industry has two commercial sources of crawfish, Procambarus spp., a wild harvest, mostly from the Atchafalaya River Basin (ARB) in the south-central area of the state, and a cultivated harvest from aquaculture ponds, located primarily in the southwestern area of the state. The combined harvest in 2016 was 135.2 MT with aquaculture accounting for 91%. The modern crawfish aquaculture industry originated in the early 1960s when low water during the ARB spring flood resulted in a poor harvest. State biologists worked with farmers in the Mississippi River alluvial valley and the southwestern prairie area to cultivate crawfish in purpose built impoundments and rice field impoundments. Area in crawfish culture increased from 4,050 ha in 1968 to 90,050 ha in 2017. Aquaculture accounted for 40-60% of annual harvest until the year 2000 and thereafter accounted for 70-95%. Changes in the hydrology of the ARB have largely accounted for declining wild crawfish harvests. The dominant species is Procambarus clarkii, the red swamp crawfish. Some Procambarus zonangulus, the southern white river crawfish, are harvested. Three major events impacted the Louisiana crawfish industry. First, import of crawfish products from the Peoples’ Republic of China beginning in the mid-1990s lowered prices. Second, an insecticide applied to rice fields used to cultivate crawfish in 1999-2001, combined with historical record summer drought which negatively impacted reproduction, led to widespread crop failure. Third, White Spot Shrimp Virus (WSSV), highly lethal to Procambarus spp. became widely distributed in both wild and cultured crawfish crops. Initially, competition from Chinese crawfish products had a negative impact on the Louisiana crawfish industry. However, over time imports led to development of new domestic markets and industry wide quality control practices. The highest crawfish production cost is harvesting – labor and bait. Crawfish are harvested in mesh traps that must be tended manually. Mechanized harvest boats have reduced the time necessary to tend traps but trapping is still highly inefficient. Initially, cut rough fish was the common bait, and later grain-based manufactured baits were developed but are relatively ineffective during colder months of the November/December – May/June seasons. A short-lived soft-shelled crawfish industry involving as many as 150 farmers developed in the 1980s. Despite good acceptance of the product, profits were low and entrepreneurs left the trade. No more than three producers remain today. Crawfish are cultivated by simulating the cool season wet, warm season dry hydrology of Louisiana. This works so well with rice farming that 75% of crawfish area is integrated with rice. The agricultural community refers to this landscape as working wetlands. The food rich system attracts predaceous, omnivorous, and vegetarian species of birds in great numbers. As a result, the National Audubon Society has designated the southwestern Louisiana region as an Important Bird Area of Global Importance. However, concerns about bird impacts on crawfish crops remain.

 

 

Day: 3, Session: 2, Talk: 3

Crayfish, Conservation, and the Coalfields: A Case Study in the Initiation of a Crayfish Conservation Effort in North America

Zachary J. Loughman

Crayfishes have been recognized as one of the most imperiled animal groups on the planet internationally now for more than a decade. Conservation efforts in Europe and Australia have been many, and respective governments on both continents and their associated conservation agencies have been quick to recognize and protect pockets of diversity and specific highly imperiled species. In North America, most recent efforts have occurred via state level conservation agencies, which when said agencies have effective, forceful legal power, garner extensive protection for crayfish. Not all state level agencies are created equal, and when agencies lack enforcement power, crayfishes and other imperiled species can be eliminated in the names of progress. In these situations, listing as a federally imperiled species becomes paramount for protection of said taxa and their associated current and potential habitat. Cambarus callainus (Big Sandy Crayfish) and Cambarus veteranus (Guyandotte River Crayfish) are two Central Appalachian endemic species of tertiary burrowing crayfish whose populations have experienced drastic declines due to excess sedimentation and other stressors allied with extractive industries and development of riparian corridors. In 2014, a status assessment was written by the United State Fish and Wildlife Service (USFWS) based on work completed by astacological workers prior to that time, which indicated that water quality threats associated with coal mining, development, and off-road vehicle tourism were likely pressures to both species ability to persist in the coal fields of Kentucky, Virginia, and West Virginia, and in the case of C. veteranus, West Virginia only. In 2015, a range-wide assessment was completed for both species that involved visiting every historical location as well as over 100 new sample locations. Resultant of this work, current data was acquired that identified the principal current threats to both taxa had not changed, and possibly had gotten worse, which ultimately was used in addition to previous workers data to justify listing both species federally. On April 7th, 2016 C. callainus was listed as threatened and C. veteranus was listed as endangered. Beginning in the spring of 2017, an aggressive conservation campaign was initiated involving federal, nonprofit, and state level conservation agencies to gather data that ultimately will be used to conserve and protect both species. Captive rearing protocols, life history studies of C. callainus and C. veteranus as well as their crayfish associates, telemetry studies, and crayfish community analysis in addition to the creation of a response team for spills associated with extractive industry was created. In addition to research efforts, development of educational workshops for biologists working in the coalfields was also undertaken and initiated in the spring of 2016. Here in a review and case study will be provided detailing this process and the hope for its ultimate utilization in the preservation and future conservation of these highly imperiled central Appalachian endemics.

 

 

Day: 3, Session: 3, Talk: 3

Multi-method Inference of Temperature Tolerance and Preference for a Native and an Invasive Crayfish

JACOB T. WESTHOFF, Chris Rice, Hisham Abdelrahman and James A. Stoeckel

Conservation and management of crayfishes can be informed through a greater understanding of crayfish thermal ecology, especially as it relates to the suitability of thermal habitats for native and invasive crayfish. We used a combination of behavioral and enzymatic endpoints to estimate temperature preference, optimal respiratory enzyme tolerance (ORET), and critical thermal maximum (CTM) for the imperiled native Coldwater Crayfish (Faxonius eupunctus) and the invasive Ringed Crayfish (Faxonius neglectus). Significant differences in these parameters would allow for thermal partitioning of space and thus enhance the probability of coexistence. Crayfish used in CTM and preference tests were acclimated at one of four temperatures (10, 15, 20, 25°C) for two weeks prior to testing, whereas ETS assays used crayfish acclimated at 21°C. Estimates of CTM were 33.9°C for F. eupunctus and 33.2°C for F. neglectus. Mixed linear model analysis of CTM data showed no difference between species or genders, but a strong effect of acclimation temperature (p-value < 0.01). Mixed linear model analysis using likelihood ratio tests indicated F. eupunctus preferred slightly colder water (19.6°C) than did F. neglectus (21.3°C; p-value = 0.03). That analysis also identified a significant difference (p-value = 0.01) between males (20.3°C) and females (21.0°C), but acclimation temperature and the interaction between gender and species were not significant. Mean ORET did not differ between F. eupunctus (28.4°C) and F. neglectus (28.5°C), but did differ from a third congener, Faxonius marchandi (29.7°C), based on two-way ANOVA results. For all species, ORET was higher than organismal thermal preference estimates, but lower than CTM, suggesting it may provide a useful breakpoint for managers. Management strategies should target temperature regimes that approach but do not exceed OET to increase the frequency of optimal temperature occurrences while minimizing the risk of exposing crayfish to their thermal maxima. Across all estimated metrics, F. eupunctus and F. neglectus differed by less than 2°C, indicating that significant biological difference is unlikely. Thus, habitats suitable for the native F. eupunctus will also be thermally available to the invasive F. neglectus, thereby increasing the opportunity for interaction and negative population effects.

 

 

Day: 3, Session: 3, Talk: 4

Normal Biochemistry of the Murray Crayfish Euastacus armatus (Parastacidae)

Martin Asmus, Shane Raidal and MAGGIE J. WATSON

Haemolymph samples were collected from wild and captive held adult male and female Murray Crayfish Euastacus armatus. Haematological analyses were performed in order to determine reference values for this species including protein, albumin, globulin, creatine kinase, aspartate transaminase, glutamate dehydrogenase, glucose, gamma-glutamyltransferase, potassium, sodium, calcium, phosphate, chloride, uric acid, cholesterol, amylase and bile acids. Additionally, protocols for measurements of phenoloxidase and prophenoloxidase (part of the non-specific immune system in crayfish which leads to the melanisation and sclerotisation in stressed animals) are being trialled. Alterations from these reference values can be used to determine stress and disease state of the crayfish. These tests are being used to monitor the health and stress levels of Murray Crayfish intended for use in a large-scale translocation of crayfish from healthy populations to areas of the Murray River that no longer support crayfish. Murray crayfish populations in affected parts of the river dropped by 81% in 2010–11 due to hypoxic water events.

 

 

Day: 3, Session: 3, Talk: 5

Hunting Missouri’s Rarest Crayfish Using eDNA and Visual Surveys: A First Look at the Natural History of the Caney Mountain Cave Crayfish

ROBERT J. DiSTEFANO, David C. Ashley, Shannon K. Brewer and Joshua B. Mouser

Stygobitic (cave) crayfishes are regarded as the most imperiled crayfishes in the U.S. and Canada. The Caney Mountain Cave Crayfish (Faxonius stygocaneyi), discovered in 1998, is known from only a single population in Mud Cave in the Caney Mountain Conservation Area (CMCA) of southern Missouri. It is listed as “critically imperiled” by the state of Missouri and “threatened” by the American Fisheries Society. We conducted the first study of F. stygocaneyi to gather preliminary data on its natural history and population, and to locate possible additional populations. We visited Mud Cave on 14 occasions (at least once each season) between 2014 and 2018. Visual surveys along a transect of inundated (wet) and non-inundated (mud) habitat were conducted during most visits except late April-May 2017 when the cave was flooded. Supplemental baited trapping was also performed intermittently. Water samples (2 L at two Mud Cave locations) were taken on each of 7 visits in 2017 and 2018. Water and air temperatures were recoded for most of 2014-2016. We captured a total of 23 (carapace length, 15.0-47.0 mm, average: 27.2 mm) and observed an additional 42 F. stygocaneyi, including the first-ever records of juveniles (August 2016 and 2017) and an ovigerous female (August 2016). Multiple visual searches of the known five other caves and three springs at CMCA detected no F. stygocaneyi. In addition to water samples from Mud Cave, we also collected and filtered multiple water samples from Onyx, and Bear Hollow caves, and three springs thought to be in the same drainage in 2017-2018. We developed primers and probes to amplify F. stygocaneyi DNA from those samples. Study results were limited by use of non-invasive sampling methods, high turbidity, flooding on some occasions, and what appears to be a small population. Yet we observed four times more crayfish compared to the previous 17 years since the species’ discovery, and collected the first biological data. Visual and eDNA sampling of adjacent caves and springs suggest the species could be endemic to only Mud Cave.

 

 

Day: 5, Session: 2, Talk: 1

Crayfish Chimney Function: Airflow, Oxygen, and Pheromones

JAMES A. STOECKEL, Hisham Abdelrahman, Mary Szoka, David Blersch and Jeremiah Davis

Most burrowing crayfish build chimneys. However, chimneys are often present only seasonally, located only over a single burrow opening, or completely absent, suggesting that they are periodically constructed for specific function and purpose(s). In this study we use a combination of field and wind-tunnel experiments to investigate function and purpose of Cambarus c.f. polychromatus chimneys. Field trials with smoke bombs and natural burrows showed that air flow is a major outcome of chimney construction. In a typical burrow complex, smoke was drawn in through three non-chimney openings, traveled ~0.5 m underground to the groundwater level, and then back up to the surface and exited through the chimney. Smoke was not drawn through the burrow when the chimney was removed. Chimneys appear to drive airflow through burrows by creating temperature differentials with non-chimney openings, and/or pressure differentials related to wind blowing across chimney and non-chimney entrances (Bernouli’s Principle). Wind tunnel trials with model burrows showed that air velocity through burrows was highest when chimneys were upwind of chimneyless openings. Air velocity through burrows also increased with increasing chimney height and increasing wind speed. Ongoing field studies suggest that need for increased oxygen is not likely the primary purpose driving crayfish to build chimneys to draw air through burrows. Individuals that were freshly molted or brooding, representing two life-history stages that require the most oxygen, were only found in plugged and chimneyless burrows, respectively. Alternatively, based on results of this and previous studies, we hypothesize that an important outcome of increased airflow through and out the burrows is dispersal of pheromones to facilitate intraspecific communication between adults during the reproductive season, and subsequent recruitment of young to conspecific burrow colonies.

 

 

Day: 5, Session: 2, Talk: 5

Invasive Rusty Crayfish (Orconectes rusticus) Populations in North America are Infected with the Crayfish Plague Disease Agent (Aphanomyces astaci)

JÖRN PANTELEIT, Thomas Horvath, Japo Jussila, Jenny Makkonen, William Perry, Ralf Schulz, Kathrin Theissinger and Anne Schrimpf

The American rusty crayfish, Orconectes rusticus, is an invasive species in various parts of North America, where it displaces resident crayfish species. While the influence of the crayfish plague disease agent, Aphanomyces astaci, has been studied extensively in Europe, the impact of A. astaci on the invasion success of crayfish within North America has so far received no attention. As a first approach to the question, whether A. astaci might play a role in the invasion success of O. rusticus within North America, we tested 84 O. rusticus samples for infection with A. astaci from 10 different locations in the Midwest, which are outside of the O. rusticus native distribution range. We used quantitative real-time PCR (qPCR) to assess the infection prevalence and determined the mitochondrial haplotypes and multilocus microsatellite genotypes where this was possible. With qPCR, we detected A. astaci DNA in 4 out of 10 locations. The results were confirmed by isolation of A. astaci. Analyses of the pure culture isolates and the crayfish tissue samples by haplotyping and genotyping revealed a novel microsatellite genotype. Our results clearly identify O. rusticus as a vector of A. astaci in North America for the first time. The threat caused by these novel strains to endangered crayfish species in North America still remains unknown, but conservation efforts should consider A. astaci infections when developing and implementing invasive species management plans.

 

 

Day: 5, Session: 2, Talk: 6

Simultaneous eDNA Monitoring of the Host-pathogen Complex Pacifastacus leniusculus and Aphanomyces astaci Under Varying Environmental Conditions

JOHANNES C. RUSCH, David A. Strand, Charlotte Laurendz, Stein I. Johnsen, Lennart Edsman and Trude Vrålstad

In 2016 the North-American signal crayfish Pacifastacus leniusculus was included in the list of Invasive Alien Species of concern to the European Union. In Norway the species is black-listed and rated as a high-risk species, and eradication has been carried out whenever feasible. This is because it is a chronic carrier of the oomycete Aphanomyces astaci, which is lethal to all European freshwater crayfish species and listed among the 100 worst invasive species. Environmental DNA (eDNA) methodology is now being used on a wide variety of target species and on many different platforms including targeted PCR and broad spectred sequencing methods. Detection and monitoring of invasive, endangered and elusive species is commonly performed using species specific quantitative real-time PCR (qPCR) or droplet digital PCR (ddPCR), and has proved useful for targeting species of specific concern. Recently eDNA monitoring of A. astaci has been implemented in the national crayfish plague surveillance in Norway. For the carrier of the crayfish plague, the signal crayfish, a qPCR assay for eDNA detection has also been published recently. In this study, we present simultaneous eDNA monitoring of the host-pathogen complex P. leniusculus - A. astaci under varying environmental conditions both in aquarium and field experiments. We used the published qPCR assays for both species and redesigned them so they can be run as a duplex ddPCR assay. We compared water samples by means of qPCR and ddPCR from two lakes in Sweden and Norway with different signal crayfish population densities and A. astaci prevalence in the population. We also studied eDNA emission from the host-pathogen couple in aquarium-experiments with A. astaci positive signal crayfish held at different densities, temperatures and feeding regimes. Samples were obtained by filtering water (1 & 5 L) on-site through glass fibre filters. These were subsequently analysed using the species-specific qPCR and ddPCR assays for the respective targets. The concentrations of detectable eDNA copies of the two targets are influenced differently and by several factors, including population density of the crayfish, pathogen prevalence, temperature, and turbidity including microbiological activity in the water. They are therefore subject to significant fluctuation. Thus, there seems to be no straightforward correlation between eDNA copy-number and crayfish density and the probability of detecting one target rather than the other varies according to different conditions. Therefore, for a reliable monitoring of crayfish plague (A. astaci) and signal crayfish alone or together, the simultaneous monitoring concept for both targets is recommended.

 

 

Day: 5, Session: 3, Talk: 3

Observations of Chelae Injury in Two Crayfish Species in Three Sinuous Rills

CHESTER R. FIGIEL JR.

Crayfish often lose or autotomize limbs (e.g., chelae) during agonistic conflicts. This defense mechanism increases the probability of surviving the encounter, however can result in long-term functional and energetic costs. For example, chela autotomy influences crayfish competitive ability, foraging time, capacity to obtain mates, and modifies crayfish distribution and behavior. Biotic factors (e.g., population density or predators), as well as abiotic factors (e.g., refugia or habitat complexity) can influence the frequency of injury. In this study, I investigated the prevalence of chela injury (loss or partially regenerated chelae) in two crayfish species (Cambarus striatus and Procambarus spiculifer) that were collected in three sinuous rills (Mountain Creek (MC), Liberty Bell Creek (LB), and Cascade Branch (CB)) in west central Georgia, USA over a four year period. My objectives were to determine if injury was similar among sites, among crayfish species, or varied with time of year. Additionally, within a species my objectives were to examine whether injury differed in crayfish size classes or sex. Cambarus striatus were collected in each of the three streams with the percent injured as follows: MC 15.5%, n = 579; LB 16.3%, n = 1555; and CB 21.3%, n = 1159. There were significantly more injuries from crayfish collected at CB than at the other two streams (p < 0.05). This was most likely driven by the greater percent of injuries in the juvenile size classes (6.0 mm to 15.9 mm carapace length (CL)): CB (24.5%) compared to the similar size classes at LB (16.6%) and MC (15.0%). The frequency of injury between males (18.2%), females (20.0%), or juveniles (19.0%) did not differ significantly nor were there significant differences between Form I (15.6%) and Form II (18.9) males (p > 0.05). Of the 590 C. striatus crayfish having an injury, approximately 13% of these crayfish had both chelae injured. Procambarus spiculifer were collected in each of the three streams (however only 3 individuals were collected at LB so data were not used for that site). The percent injured for P. spiculifer differed significantly between MC 12.1%, n = 727; and CB 23.7%, n = 169 (p < 0.05). There were a greater percent of injuries in the larger adult size classes (> 31.0 mm CL = 26.4%) compared to smaller size classes (< 31.0 mm CL = 9.9%). The frequency of injury among males (13.8%) and females (15.9%) did not differ significantly, however differed from the percent of injured juvenile crayfish (7.3%). Of the 126 P. spiculifer crayfish having an injury, approximately 12% of these crayfish had both chelae injured. The percent of injured crayfish captured varied widely from month to month for both species and no discernable pattern emerged through time. Given the level of injury in these populations, it is vital to understand how injury influences population dynamics of these two species with differing ecological needs and lifestyles.

 

 

Day: 5, Session: 3, Talk: 5

A New Technique for Determining Crayfish Population Demographics

JOSHUA MOUSER, Jason Glover and Shannon K. Brewer

Aging organisms provides crucial population demographic information such as growth, recruitment, and mortality. Crustaceans are typically aged via indirect techniques, such as length-frequency histograms and mark recapture. Indirect techniques are limited to the populations and specific study period, do not provide an actual age, and histograms are inaccurate for older age classes with fewer individuals. In contrast, direct techniques rely on calcified structures, but often the individual must be sacrificed. Recent work has demonstrated that the gastric mill, located in the stomach of crustaceans, may be useful for obtaining direct age estimates. Therefore, our objective was to determine if a common North American crayfish species could be reliably aged using gastric mill ossicles. We collected approximately 100 adult and 300 age-0 (<13 mm carapace length) ringed crayfish Faxonius neglectus from streams in the Ozark Highlands ecoregion. Gastric mills were extracted from the crayfish, separated, cleaned, sectioned, and mounted on a microscope slide. Each crayfish was aged independently by three different readers and a consensus age was reached if there were discrepancies. For a subset of individuals, two readers aged each ossicle independently to determine which ossicle provided the most consistent age estimates. We compared length-frequency histograms from field collections to our direct age estimates. All three ossicles showed consistent growth bands, but the zygocardiac and mesocardiac ossicles had superior readability. Crayfish age estimates ranged from zero to ten years, whereas the length-frequency histograms suggested eight year classes. Our age estimates did not match length-frequency histograms well, especially after year 4. Independent age estimates from each of the paired zygocardiac ossicles were similar. Our results suggest that ringed crayfish may live to be much older than five years as generally accepted, and aging crayfish directly may improve the age bias reflected in histograms. Future work will focus on validating that bands correspond to one year of growth, environmental effects on band deposition, and if daily bands are deposited in age-0 crayfish.


 

Poster Presentations

 

 

POSTER 56

Assessing Rarity Patterns in Crayfish at Multiple Spatial Scales Using Scale-area Curves

JOHN W. JOHANSEN, Hayden T. Mattingly, Christopher A. Taylor and Guenter A. Schuster

Identification of at-risk species often relies mostly on range size, particularly for poorly studied species. Although this provides a relatively efficient method for identifying species of conservation concern, it may lead to an inaccurate assignment of conservation status. For example, many species occupy small native ranges but are locally abundant and temporally stable. Additionally, extinction processes operate at different spatial-scales. Scale-area curves provide a framework that examines rarity at multiple spatial scales, and thus, can lead to development of more impactful conservation strategies. Using a well-vetted database of Alabama crayfish collections, we used measures of area of occupancy to construct scale-area curves and assess rarity patterns for lotic crayfishes at two spatial scales: 1 km2 and 100 km2. Area of occupancy (AOO) is a measure of range size that varies depending on the spatial-scale of interest. For each species, AOO was estimated by counting the number of occupied cells in nested grids at increasing user-defined areas. In addition to AOO, the degree of range fragmentation was determined for each species from the slope of the scale-area curve at each spatial scale. Principle components analysis was used to identify scale-specific patterns of rarity. For example, at the 1 km2 spatial scale, we identified 5 groups of species based on AOO and degree of range fragmentation while at the 100 km2 scale six groups were identified. At the 1 km2 scale, several state imperiled species (S2) had higher levels of fragmentation than many critically imperiled species (S1). This indicates the lower ranked (S2) species may actually be more susceptible to loss of local populations due to increased range fragmentation. Accordingly, understanding metapopulation dynamics and maintaining habitat connectivity should be a priority for this subset of state imperiled (S2) species. This demonstrates the need to examine multiple variables and spatial scales in prioritizing species of conservation concern, particularly for those species that lack basic biological and ecological data beyond range size.

 

 

POSTER 59

Survey Says: U.S. State- and Canadian Provincial-Level Natural Resource Agencies Focus on Crayfish Conservation

Cheyenne E. Stratton and ROBERT J. DISTEFANO

Taylor et al. (1996) issued a "warning shot" about a crayfish imperilment plight, and "neglect" of the fauna by natural resources agencies. In the ensuing decades some highly imperiled aquatic faunal groups, such as unionid mussels and crayfish, have received moderately increased attention by U.S. and Canadian natural resources agencies. Such attention appears to have translated to increased funding for work on crayfish, possibly due to concern for individual species' imperilment, or resource problems caused by them (e.g., invasive crayfishes). We wondered 1) how perceived increased agency attention to crayfish might be reflected in numbers and types of staff assigned to work on crayfish conservation and management?, 2) where (topically) these staff are directing their efforts?, and 3) what are agencies' major constraints/impediments to and needs for crayfish conservation and management? We conducted a two-part telephone survey in 2017 and 2018 to learn about natural resources agencies' level of involvement and direction in crayfish conservation and management. In Part I (2017) we called natural resource agencies in all 50 U.S. states and 13 Canadian provinces/territories (63 "jurisdictions") to determine the number who employed or contracted staff to work on crayfish, where these jurisdictions were located (regionally), and in what topical/subject areas they were working. In Part II (2018) we made follow-up calls to only jurisdictions that had reported doing crayfish work in Part I of the survey. We asked them about their agencies' prioritization of crayfish, impediments to crayfish work, and information they believed most useful to help them conserve/manage crayfish (data needs). Part I results indicated nearly half of jurisdictions are conducting crayfish work, mostly in the Southeastern U.S., and concentrating on determining species' distributions and conservation status, or on threats (i.e., invasive species). Part II suggested that more than half of agencies working on crayfish consider them a priority faunal group, with the largest impediment being insufficient funding. Jurisdictions' most commonly cited information needs were species compositions (native and introduced), distributions, conservation status assessments, ecology, and threats. Our survey results suggest an encouraging but limited increase in U.S. state and Canadian provincial/territorial natural resources agencies working on crayfish since Taylor et al.'s (1996) challenge.

 

 

POSTER 62

Habitat Associations of Endemic Crayfishes in the Meramec River Drainage: The Freckled Crayfish (Cambarus maculatus) and Belted Crayfish (Faxonius harrisonii).

Joe Chilton, Amanda E. Rosenberger and ROBERT J. DiSTEFANO

Understanding the habitat associations of rare species is important to make informed management and policy decisions. The Freckled Crayfish (Cambarus maculatus) and Belted Crayfish (Faxonius harrisonii) are two of Missouri’s rare and endemic crayfish species. Both species are listed as vulnerable on Missouri’s list of species and communities of conservation concern due to their limited range. Their native range is limited to the Meramec River drainage in eastern Missouri. We sampled 60 sites throughout the two species’ known range for presence and habitat variables. Replication was performed spatially within sites using kick-seines, drag seines, and visual timed-searches. Local- and landscape-scale habitat variables were evaluated for possible associations with the crayfishes through occupancy modeling with the R package “unmarked”. We found boulders and Strahler stream order were positive estimators of occupancy, while percent agriculture was negatively associated with the Freckled Crayfish. Belted Crayfish were associated with larger substrate size, increased embeddedness of substrate, and aquatic vegetation. This information will guide conservation managers in future projects and policy decisions regarding these two species.

 

 

POSTER 67

Another Cautionary Tale of Numts: Multiple Different Copies of the COI Gene in the Camp Shelby Burrowing Crayfish (Fallicambarus gordoni)

JAMES W. FETZNER JR.

During a preliminary phylogeographic study conducted several years ago involving the Camp Shelby Burrowing Crayfish (Fallicambarus gordoni) it was noted that most of the generated COI barcode sequences were “messy” (i.e., contained multiple peaks at many sites along the length of the sequence), suggesting some sort of contamination was being co-amplified. This messy data was so prevalent (almost every specimen) that it ultimately eroded confidence in the base calls for the region sequenced and led to the termination of the project due to a very limited sequencing budget that had already been expended. In order to investigate the source of this contamination, COI PCR products from eight F. gordoni individuals (one from each sampled site), plus one specimen of another Fallicambarus species, were subsequently cloned. Eight individual colonies per individual were picked, amplified, and sequenced to see if the source of the contamination could be identified (i.e., as bacterial, human, pseudogene, or other). Checking the sequences using Genbank BLAST searches revealed that the sequences were most closely related to COI genes from the genus Fallicambarus, suggesting they are additional copies present within the genome, rather than contaminant DNA from an external source (i.e., bacterial). The results suggested that multiple different copies of the COI gene appear to be present within the genome of F. gordoni individuals, often containing multiple point mutations and/or length differences (=indels), which directly resulted in the messy sequences seen during the original project. In some cases, the sequences appeared to be pseudogenes because they often contained multiple stop codons. This study provides another cautionary tale about numts (nuclear copies of mitochondrial genes) and making sure they are accounted for when analyzing mitochondrial datasets in phylogeographic and systematic studies of freshwater crayfish.

 

 

POSTER 68

Gene Expression in the Crayfish Endocuticle

JERONIMO REYES-OLMEDO, Christian Kim, Trevor Dacus and Paul R. Cabe

Few genomic resources exist for any crayfish families and species despite their high species diversity, importance in freshwater ecosystems, and economic importance in aquiculture. The lack of such resources limits many areas of study, including phylogenetic relationships, local adaptation, and gene expression. We report on an exploratory study of transcripts abundantly expressed in the endocuticle tissues of Cambarus crayfish. For this work, mRNA was extracted from endocuticle tissues and copied to cDNA using reverse-transcriptase PCR. This pool of PCR products was fragmented and prepared for Illumina sequencing, yielding more than seven million paired end reads (150 base pairs each end). The sequence reads were assembled into putative transcripts using the Trinity software pipeline, and the transcripts ranked by abundance in the cDNA sample using both Sailfish and Salmon software tools. The most abundant transcripts were identified using DNA and/or protein BLAST searching. The transcripts include both well-known and unidentified gene sequences.

 

 

POSTER 77

The Crayfish Morphology Database: Developing an Online Platform for Maintaining and Sharing Specimen Data and Images Used in the Descriptions of New Crayfish Species

JAMES W. FETZNER JR.

A new online database, and associated website, are under development with the goal of capturing, maintaining and sharing a standard suite of morphological measurement data and specimen images used in the description of new freshwater crayfish species. The website is password protected, allowing authors to secure their project data under a user account that only they can access. Once published, their data could become available to the community. The site consists of eight tabbed web forms that capture information on field collections, geographic locality, and specimen-level morphological measurements broken out by major feature (e.g., Carapace, Rostrum, Chela, Gonopod, and ‘Miscellaneous’). A tab is also provided to upload specimen images captured from a variety of views. The database currently is set up for capturing data on North American taxa, but if there is interest from the community, this could be expanded to include other crayfish groups from around the globe. Measurement data can be entered automatically into the web form via a digital caliper connected to a computer, or entered manually. A standard set of photographic images can also be captured and annotated, and then uploaded to the project, making them available for side-by-side viewing, allowing for multiple specimen comparisons of various morphological features. Features such as simple statistical analyses of the data (counts, frequencies, etc.) still need to be implemented, but could be generated and output as a summary report. The full project data can also be output to Excel format to allow for more detailed statistical analyses. Most of the data presented in new species descriptions are just estimates of the mean and/or the range of values measured. Thus, all of the underlying data captured for each individual specimen in a study are often lost to science, unless the specimens have been designated as types. Having a repository for these types of data will help to make comparisons among species a lot easier in the future and will reduce the duplication of effort when making comparisons among multiple species.


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