Virome Analysis of Signal Crayfish (Pacifastacus leniusculus) along Its Invasion Range Reveals Diverse and Divergent RNA Viruses

Crayfish are a keystone species of freshwater ecosystems and a successful invasive species. However, their pathogens, including viruses, remain understudied. The aim of this study was to analyze the virome of the invasive signal crayfish (Pacifastacus leniusculus) and to elucidate the potential differences in viral composition and abundance along its invasion range in the Korana River, Croatia. By the high-throughput sequencing of ribosomal RNA, depleted total RNA isolated from the crayfish hepatopancreas, and subsequent sequence data analysis, we identified novel and divergent RNA viruses, including signal crayfish-associated reo-like, hepe-like, toti-like, and picorna-like viruses, phylogenetically related to viruses previously associated with crustacean hosts. The patterns of reads abundance and calculated nucleotide diversities of the detected viral sequences varied along the invasion range. This could indicate the possible influence of different factors and processes on signal crayfish virome composition: e.g., the differences in signal crayfish population density, the non-random dispersal of host individuals from the core to the invasion fronts, and the transfer of viruses from the native co-occurring and phylogenetically related crayfish species. The study reveals a high, previously undiscovered diversity of divergent RNA viruses associated with signal crayfish, and sets foundations for understanding the potential risk of virus transmissions as a result of this invader’s dispersal.

Eroded Swimmeret Syndrome: Update of the Current Knowledge

Eroded swimmeret syndrome (ESS) was first described in 2014 from Swedish signal crayfish (Pacifastacus leniusculus (Dana)), and later also from Finland, with gross symptoms and disease agent candidates identified and described by 2015. The ESS was first discovered affecting alien signal crayfish in Fennoscandia. The ESS is caused by a multiple infection involving Aphanomyces astaci (Schikora) and Fusarium species complex (SC). The ESS symptoms include first melanised spots in swimmerets, then partial swimmeret erosion and finally loss of a swimmeret. There could be a total loss of all swimmerets in the most severe cases. Both females and males can be affected by the ESS. In females, the ESS lowers reproductive success while in males the ESS often causes erosion of the gonopods and thus possible partial failure in mating. The ESS is more frequent among mature females that have reproduced once compared to immature females or those that are mature but have not yet reproduced. The proportion of females with ESS has ranged from 10 to 50% among Lake Saimaa signal crayfish in Finland and in a wider survey from Sweden the range was from 0 to 38%. Among Lake Saimaa male signal crayfish, the ESS proportion has been less than 10%, while it was only 0.6% in the Swedish data. The ESS has also been observed among alien signal crayfish in Switzerland. There are recent observations of ESS affecting narrow-clawed crayfish, Pontastacus leptodactylus (Eschscholtz), in Croatia and Romania (i.e., among native European crayfish stocks). Here, we summarise current knowledge about the ESS and speculate on a few potentially crucial impacts of this syndrome.

SI on economic costs of invasions - Feeling the pinch: global economic costs of crayfish invasions and comparison with other aquatic crustaceans

Despite voluminous literature identifying invasive species impacts, understandings of monetary costs remain limited. Recently, profound impacts have been attributed to invasive crustaceans, but associated monetary costs lack synthesis. Here, we analyse globally reported costs of invasive freshwater crayfish across taxonomic, spatial and temporal descriptors. Moreover, we compare their cost magnitude to other invasive crustaceans — crabs, amphipods and lobsters. Between 2000 and 2020, crayfish caused US$ 1.28 billion in reported costs; the vast majority (95%) attributed to Astacidae (principally the signal crayfish Pacifastacus leniusculus) and the remainder to Cambaridae. According to reports, crayfish costs mostly impacted European economies (US$ 1.23 billion), followed by costs reported for North America and Asia. Despite well-known damages caused by invasive crayfish, costs were unreported elsewhere, highlighting knowledge gaps and challenges in cost quantifications. Invasive crayfish costs increased exponentially in the last two decades, averaging at US$ 61 million per-annum. Invasive crabs caused costs of similar magnitude (US$ 1.25 billion; US$ 53 million per-annum) but were mostly confined to North America (95%). Damage-related costs dominated for both crayfish (83%) and crabs (99%), with management spending lacking. Reported economic impacts from amphipods (US$ 178.8 thousand) and lobsters (US$ 44.6 thousand) were considerably lower. We identify burgeoning economic costs from these invasive groups yet highlight pervasive knowledge gaps at multiple scales. Further cost reporting is required to better-ascertain the true scale of monetary costs caused by invasive aquatic crustaceans.

Getting rid of rain and stars: mitigating inhibition effects in ddPCR assays, the case of the invasive crayfish Pacifastacus leniusculus in the streams of Luxembourg

ddPCR is getting more and more popular in the field of eDNA-based aquatic monitoring. Even if emulsion PCR used in ddPCR confers a partial resistance to inhibition due to the high number of reactions for the same sample (between 10K and 20K), it is not impervious to it. Inhibition impacts the fluorescence amplitude of positive droplets, affecting both their dispersion and their position relatively to the negative droplets cloud. This fluctuation could jeopardize the use of a shared threshold among several samples and thus the objective assignation of the positive droplets. This is even more critical for low concentration samples such as eDNA samples: the positive droplets are scarce and it is thus crucial to objectively discriminate if they can be counted as positive by establishing an appropriate threshold. Another issue is the artifactual generation of high fluorescence droplets that could be counted as positive with a single threshold solution. Here we propose a double threshold method to take both high fluorescence droplets and PCR inhibition impact into account allowing for an objective sorting of the positive and negative droplets in ddPCR assays.