Andriantsoa et al. BMC Ecol (2019) 19:8 https://doi.org/10.1186/s12898-019-0224-1 BMC Ecology

RESEARCH ARTICLE Open Access Ecological plasticity and commercial impact of invasive marbled crayfsh populations in Ranja Andriantsoa1, Sina Tönges1, Jörn Panteleit2, Kathrin Theissinger2, Vitor Coutinho Carneiro1, Jeanne Rasamy3 and Frank Lyko1*

Abstract Background: The marbled crayfsh ( virginalis) is a monoclonal, parthenogenetically reproducing fresh- water crayfsh that has formed multiple stable populations worldwide. Madagascar hosts a particularly large and rapidly expanding colony of marbled crayfsh in a unique environment characterized by a very high degree of ecological diversity. Results: Here we provide a detailed characterization of fve marbled crayfsh populations in Madagascar and their habitats. Our data show that the can tolerate a wide range of ecological parameters, consistent with their invasive potential. While we detected marbled crayfsh in sympatry with endemic crayfsh species, we found no evidence for the transmission of the crayfsh plague pathogen, a potentially devastating oomycete. Furthermore, our results also suggest that marbled crayfsh are active predators of the freshwater snails that function as intermedi- ate hosts for human schistosomiasis. Finally, we document fshing, farming and market sales of marbled crayfsh in Madagascar. Conclusions: Our results provide a paradigm for the complex network of factors that promotes the invasive spread of marbled crayfsh. The commercial value of the animals is likely to result in further anthropogenic distribution. Keywords: Marbled crayfsh, Madagascar, Ecology, Habitat diversity, Crayfsh plague, Farming

Background major genetic changes [4, 5], which may have occurred as Te marbled crayfsh Procambarus virginalis [1] is the recently as 25 years ago. Te combination of obligate par- only known decapod that reproduces by thenogenesis and very young evolutionary age has gener- obligate [2]. While the precise origin of ated a population that can be considered a single genetic the animals remains to be identifed, the frst record of clone [6–8]. marbled crayfsh is from the German aquarium trade in According to the Red Queen hypothesis [9], lack of 1995 [1, 2], indicating a very recent evolutionary origin. genetic variation severely curtails the ability of a species Based on morphological characters and genetic data, the to adapt and proliferate [10]. However, the genetically sexually reproducing slough crayfsh from Florida, Pro- homogeneous marbled crayfsh has been described as a cambarus fallax, has been identifed as the most closely successful in various countries [11–18]. related species [2, 3]. Additional evidence strongly sug- Tis is exemplifed by the situation in Madagascar, where gests that marbled crayfsh separated from P. fallax by marbled crayfsh were frst introduced around 2005 [11]. By 2008, the animals had already spread considerably and became widely recognized in the area around the capital *Correspondence: [email protected] 1 Division of Epigenetics, DKFZ-ZMBH Alliance, German Cancer Research city Antananarivo [11, 12]. In 2017, marbled crayfsh had 2 Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, colonized an area of approximately 100,000 km , stretch- Full list of author information is available at the end of the article ing from the highland to the coast [8]. Tis area includes

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Andriantsoa et al. BMC Ecol (2019) 19:8 Page 2 of 10

several habitats that are not inhabited by the relatively Madagascar (Ihosy). Sequencing of PCR amplicons from narrowly distributed native crayfsh species of Madagas- three randomly collected animals from each of the fve car [19, 20]. sites showed complete identity with the marbled crayfsh Te invasiveness of marbled crayfsh represents a key reference sequence (Fig. 1b), and thus provided genetic feature to defne their overall impact [13, 21]. However, authentication for the populations analyzed in this study. additional factors can contribute to this picture. For In total, we collected 6641 crayfshes and obtained example, marbled crayfsh may transmit the crayfsh data for 2458 animals (192–817 animals per site, Fig. 1c). plague agent [22], the oomycete Aphanomyces astaci, Catch per hour results difered considerably between which has eradicated major crayfsh populations in individual sites, ranging from 154 animals in Ihosy to < 20 Europe [23]. On the other hand, freshwater crayfsh can animals in Andragnaroa (Fig. 1d). Tese results suggest also function as biocontrol agents for human diseases, that population densities difer between the analyzed such as schistosomiasis [24]. Tis is exemplifed by their sites. Measurements established carapace lengths that efects on the snail populations that function as inter- were often between 10 and 35 mm and total lengths mediate hosts for Schistosoma fatworms and includes between 30 and 80 mm (Fig. 2). weights usually the direct predation of the snails and the consumption of ranged from 1 to 10 g (Fig. 2). Marbled crayfsh were aquatic plants that are used by the snails for shelter, as signifcantly (p < 0.05, Kruskal–Wallis one-way analy- oviposition sites and as food [25]. Lastly, freshwater cray- sis of variance) larger and heavier in the Ihosy river as fsh also represent an increasingly important source of compared to the remaining sites (Fig. 2). Te reasons for nutritional protein for human consumption [26]. these diferences remain to be established. Despite their invasive spread, habitats of marbled cray- Subsequent analyses revealed remarkable diferences fsh have not been analyzed systematically yet. Mada- for several fundamental ecological parameters. For exam- gascar is characterized by a high climate and habitat ple, while marbled crayfsh were initially described to diversity. Te broad range of aquatic ecosystems ren- inhabit the central highland of Madagascar [11], the pop- ders the island ideally suited to better understand mar- ulation in the Ampasimpotsy pond was located almost at bled crayfsh ecology. Furthermore, Madagascar hosts sea level (Fig. 3a). In fact, we detected marbled crayfsh at a unique and diverse fora and fauna, including seven a wide range of altitudes (3–1491 m, Fig. 3b). Additional endemic crayfsh species from the genus [19], examples for the ability of marbled crayfsh to colonize which are potentially threatened by crayfsh plague out- diferent habitats were provided by the Anjingilo rice breaks. Finally, marbled crayfsh have been spreading in felds (Fig. 4a) that are irrigated by thermal water and the Madagascar for the past 10 years [8], but their potential Ranomaimbo lake located in the city center of Antsirabe for human use has not been elucidated. Our study aims (Fig. 4b). Te water of Anjinglio is characterized by a par- to provide a detailed description of the large population ticularly high temperature of 37 °C and elevated Barium of marbled crayfsh in Madagascar. Our results shed light concentrations, which are characteristic of thermal water on the animals’ ability to colonize new environments, (Fig. 4c). Te water of the Ranomaimbo lake was char- their impact on local freshwater ecosystems and their acterized by particularly high conductivity levels and a increasing commercial importance. high concentration of dissolved solids (Fig. 4c), such as Natrium (157 mg/l) and Nitrate (20 mg/l). Tese values Results refect the high levels of pollution that are often associ- After an initial feld survey that determined the distri- ated with urban settlements. Taken together, our fndings bution area of marbled crayfsh in Madagascar [8], we suggest that marbled crayfsh can tolerate substantial var- performed a more detailed follow-up analysis. We inves- iation in ecological parameters. tigated fve aquatic ecosystems consisting of three lentic To explore the potential impact of these populations, and two lotic environments in four out of the fve bio- we frst investigated their infection status with the cray- climatic zones of Madagascar (Fig. 1a, Additional fle 1). fsh plague pathogen A. astaci. Indeed, a previous study Te lentic ecosystems include a randomly selected pond had suggested low levels of A. astaci in marbled crayfsh in the middle of a village on the east coast (Ampasim- populations from Germany [22]. We investigated 100 ani- potsy), a lake on the highlands in the center of a big mals from the fve previously mentioned sites and from city impacted by human activities (Ranomaimbo) and Antananarivo, where marbled crayfsh were frst detected a rice feld (Anjingilo) in a relatively isolated area in the on Madagascar [11]. Quantitative PCR (Fig. 5a) indicated south. Furthermore, two lotic habitats were included: a undetectable (agent level A0 and A1) or very low (A2) slow-fowing highland river located in the Ranomafana levels of A. astaci DNA for the large majority (96/100) National Park rainforest (Andragnaroa) and a slow-fow- of samples. Only four samples presented with somewhat ing lowland river located near a large city in south-central higher levels (A3). However, confrmatory sequencing Andriantsoa et al. BMC Ecol (2019) 19:8 Page 3 of 10

suggests that marbled crayfsh can act as efective preda- tors of B. pfeiferi. Finally, we also addressed the emerging role of mar- bled crayfsh as a food for human consumption. Marbled crayfsh can be easily caught in rivers and ponds using traditional Malagasy fshing tools (Fig. 6a). Furthermore, the animals are also farmed in larger quantities on rice felds (Fig. 6b). For commercial distribution, 60–80 kg of live animals are packed in large bags (Fig. 6c) and then sold to consumers and/or local vendors. Te measure- ment of 200 arbitrarily sampled animals from commer- cially distributed marbled crayfsh established a median total length of 57 mm (Fig. 6d) and a median weight of 4.8 g (Fig. 6e), thus illustrating the commercial relevance of relatively small animals. Marbled crayfsh currently represent an important component of the animal protein supply on local markets in all areas that were analyzed in this study and are being sold both as live animals (Fig. 6f) and as boiled and processed tail meat (Fig. 6g). Prices ranged from 500 to 1500 MGA per kg for live animals to Fig. 1 Overview of the study. a Map of Madagascar (generated 8000 MGA per kg for tail meat (Fig. 6g), which is compa- by the frst author) indicating the location of the 5 selected sites. rable to the price of rice (approximately 2000 MGA per Bar: 100 km. Area colors indicate bioclimates, see “Methods” for kg, Fig. 6g). Popular marbled crayfsh foods include deep- details. Dark green: humid, light green: sub-humid, grey: montane, beige: dry, brown: sub-arid. b Genetic authentication of 3 randomly fried beignets and rice with marbled crayfsh in tomato selected animals per site. The number of single-nucleotide variants sauce (Fig. 6h). Te increasing acceptance and popularity in the mitochondrial cytochrome B gene was 0 for all animals tested. of marbled crayfsh foods in Madagascar is likely to fur- c Numbers of animals that were analyzed at each site. d Catch ther increase their commercial demand and intentional efciencies (number of catches for 2 persons per hour) for each site propagation.

Discussion and microsatellite analyses could not be carried out due Crayfsh distribution and density is often infuenced by to the low amounts of A. astaci DNA in the tissues. Nota- specifc habitat requirements [29, 30]. However, some bly, we also found marbled crayfsh in natural habitats crayfsh species, such as Procambarus clarkii, are known of two Astacoides species: in the Andragnaroa river, in to tolerate a broader range of environmental factors, sympatry with A. betsileoensis (Fig. 5b) and in a channel such as water temperature, oxygen level and/or aquatic connected to a rice feld in Sahavondronina, in sympatry ecosystem type [31, 32]. Another example is the mother with A. granulimanus. Marbled crayfsh populations in species of the marbled crayfsh, P. fallax, which has been those two locations have been known for at least 2 years, shown to inhabit aquatic ecosystems with various trophic with no indications for crayfsh plague outbreaks. Taken levels, pH levels and water temperatures (summarized in together, these results suggest that marbled crayfsh do [33]). Our fndings are consistent with the notion that the not trigger crayfsh plague outbreaks in Madagascar. marbled crayfsh can tolerate a broad range of ecological We also noticed that the distribution area of marbled parameters [33]. Furthermore, its successful coloniza- crayfsh showed a strong overlap with the freshwater tion of diverse habitats in Madagascar clearly suggests snail Biomphalaria pfeiferi, which acts as the main inter- high plasticity towards physico-chemical parameters and mediate host of the parasitic fatworm Schistosoma man- diverse biocenoses. Additionally, we have shown that alti- soni in Madagascar [27, 28]. However, we could not fnd tude, which is closely linked to the diverse bioclimatic B. pfeiferi at the locations that we analyzed for marbled zones in Madagascar, has a negligible impact on marbled crayfsh, suggesting possible predation. To confrm this crayfsh distribution. Tese fndings signifcantly expand possibility, we performed a laboratory experiment by the suitable habitat of the global marbled crayfsh popu- placing snails of diferent sizes in laboratory boxes with lation [21]. a single marbled crayfsh (Fig. 5c). After the frst night, Adaptation to new environments is often explained by all (N = 28) snails had been eaten by the crayfsh, and the selection of genetic variants. Interestingly, however, only leftover shells (Fig. 5d) were found in the boxes. Tis marbled crayfsh are genetically identical, which suggests Andriantsoa et al. BMC Ecol (2019) 19:8 Page 4 of 10

Fig. 2 Population structures at diferent locations. Distribution of carapace lengths, total lengths and weights of marbled crayfsh in the Ampasimpotsy pond, the Ranomaimbo lake, the Andragnaroa river, the Ihosy river and the Anjingilo rice feld

a central role of epigenetic mechanisms, such as DNA [34]. Gene expression variability has been suggested to methylation, in their adaptation [8]. A recent study has facilitate adaptation in corals [35], but a functional role of provided a detailed characterization of DNA methyla- DNA methylation in the adaptation of marbled crayfsh tion patterns in marbled crayfsh [34]. Interestingly, more remains to be shown. than 2000 genes were found to be less methylated in Our study provides further insight into the impact of marbled crayfsh than in P. fallax, which was associated marbled crayfsh in Madagascar. Traditionally, the impact with more variable gene expression in marbled crayfsh of invasive crayfsh species is defned by their disruptive Andriantsoa et al. BMC Ecol (2019) 19:8 Page 5 of 10

[37, 38]. It will be important to clarify the infection status of Astacoides in future studies. Our results also shed light on the potential use of mar- bled crayfsh as a biocontrol agent against B. pfeiferi, which functions as intermediate host of Schistosoma fatworms in Madagascar and other African countries [27, 39]. Schistosomiasis remains a major public health problem in Madagascar with infection rates exceeding 50% of the total population [40–42]. Several freshwater have been suggested as biocontrol agents of mollusk intermediate hosts to limit or reduce schistoso- miasis infestation [24, 43–45]. However, the distribution ranges of these species and B. pfeiferi show little over- lap on Madagascar. In contrast, marbled crayfsh are widely distributed in Madagascar and it will be impor- tant to analyze their impact on the local presence and population densities of B. pfeiferi and the prevalence of schistosomiasis. After the frst scientifc survey in Madagascar in 2007– 2008 identifed marbled crayfsh as an ecological threat [11], the Ministry of Agriculture, Livestock and Fisher- ies issued legislation to prohibit the transportation of live marbled crayfsh [46]. In addition, it was claimed that marbled crayfsh consumption might be unhealthy for humans. Together, these factors initially restricted the commercial distribution of the animals. However, Fig. 3 Marbled crayfsh populations inhabit ecological niches at freshwater crayfsh are a popular food in Madagascar and diferent altitudes. a Picture of the Ampasimpotsy coastal pond, the endemic species have been harvested and consumed which is located 3 m above sea level. b Map of altitudinal gradients in large quantities for a long time. While their fshing of Madagascar and altitudes of the 5 selected sites. Picture and map and farming is severely limited by their specifc habitat were produced by the frst author requirements and slow growth [19], marbled crayfsh can grow quickly in diverse aquatic ecosystems. Furthermore, marbled crayfsh are easy to stock, as a single animal can give rise to a new population. As such, they represent an attractive source of income and a cheap alternative efects on ecosystems and their capacity to transmit path- for nutritional protein. By now, marbled crayfsh have ogens to naive hosts [36]. Indeed, the invasive spread of become an abundant and popular food in their distribu- infected marbled crayfsh could potentially have cata- tion area in Madagascar. We expect that this popularity strophic consequences on the seven endemic freshwater will further increase their spread, similar to other inva- crayfsh species of Madagascar. Encouragingly, we found sive species that have extended their distribution range no evidence for crayfsh plague outbreaks. A. astaci levels through commercial networks [47]. were below the limit of detection in 96 of the 100 sam- ples analyzed, with the remaining four samples show- Conclusions ing very low levels of DNA that could not be confrmed Te marbled crayfsh is a newly emerging invasive spe- by sequencing. Also, we have observed two of the seven cies, but very little is known about its ecological interac- endemic crayfsh species in sympatry with marbled cray- tions. Our study provides the frst detailed description fsh, and with no symptoms of the crayfsh plague. Tese of habitats that were successfully colonized by marbled fndings suggest that either the marbled crayfsh in Mad- crayfsh populations in Madagascar. Our fndings sug- agascar do not transmit the crayfsh plague agent or that gest a substantial habitat diversity and thus convincingly the (as yet unidentifed) A. astaci strain is not very viru- establish the ecological plasticity of the animals. Our lent. It is also possible that Astacoides is resistant to the results also provide answers for additional, important disease, similar the American crayfsh species and the questions. For example, we found no evidence for the European freshwater crayfsh Autropotamobius pallipes transmission of the crayfsh plague pathogen by marbled Andriantsoa et al. BMC Ecol (2019) 19:8 Page 6 of 10

Fig. 4 Marbled crayfsh population habitats. a Picture of the Anjingilo rice feld and b the Ranomaimbo lake. c Specifc water parameters of the fve habitats analyzed. All pictures were produced by the frst author

Fig. 5 Biological impact of marbled crayfsh on Madagascar. a Real-time PCR detection of the crayfsh plague agent Aphanomyces astacii in animals that were collected in various locations in Madagascar. Numbers of animals analyzed per site are indicated by circle size (small 10, = intermediate 20, large 30). Agent levels are indicated by section colors. b Sympatry of marbled crayfsh and Astacoides betsileoensis in the = = Andragnaroa river. c Laboratory experiment to test the predation of Biomphalaria pfeiferi snails by marbled crayfsh. Bar: 2 cm. d Leftover B. pfeiferi shells, found after overnight co-housing with marbled crayfsh. Bar: 2 mm. Map and pictures were produced by the frst author Andriantsoa et al. BMC Ecol (2019) 19:8 Page 7 of 10

Fig. 6 Commercial impact of marbled crayfsh on Madagascar. a Marbled crayfsh fshing in the Andragnaroa river, using the traditional Malagasy fshing tool (“tandroho”). b Farming on marbled crayfsh on rice felds in Ihosy. c Commercial distribution of live marbled crayfsh in large bags. d Length and e weight of 200 arbitrarily sampled animals from a commercial distribution bag. f Market sale of live marbled crayfsh in Antsirabe. g Market sale of processed marbled crayfsh tail meat in Antananarivo. h Representative prices (in MGA/kg) for various quantities and qualities of marbled crayfsh. i Typical marbled crayfsh-based food: rice with marbled crayfsh in tomato sauce. All pictures were produced by the frst author Andriantsoa et al. BMC Ecol (2019) 19:8 Page 8 of 10

crayfsh. We also show that the animals are active preda- 25 °C. Neighboring vegetation and animals co-collected tors of the intermediate hosts for human schistosomia- in the fshing tools were recorded for each site (Addi- sis. Finally, we provide the frst documentation for the tional fle 1). Finally, water samples were collected at each commercial exploitation of marbled crayfsh for human site, and stored in a cold and dark place. Barium, Natrium consumption. Altogether, our study thus identifes key and Nitrate levels were determined by Raifeisen- factors for the ecological assessment of this new invasive Laborservice (Ormont, Germany). species. Genetic authentication of marbled crayfsh Methods Genomic DNA was isolated and purifed from 100 mg Field work abdominal musculature using a Tissue Ruptor (Qiagen), Te study was carried out from October 2017 to March followed by proteinase K digestion and ethanol precipita- 2018 in Madagascar. Details about collection sites are tion. Genotyping was performed by sequencing of a PCR provided in Additional fle 1. Pictures of habitats were amplicon from the mitochondrial cytochrome b gene, as taken using a Canon PowerShot D30 digital camera described before [8]. Te complete sequencing results are or a Samsung Galaxy S6 camera. For each habitat, we provided in Additional fle 2. chose sampling stations of 20 to 150 cm depth and 50 to 1000 cm width. Collections were done in the morn- Crayfsh plague analysis ing from 8:00 to 11:00 for 5 to 7 days. One efort unit is Te crayfsh tissue samples were dried and pulverized defned as 2 persons per site per hour on total surface area with a Tissue Lyser II (Qiagen, Germany). DNA was of 100 m2 represented either by a transect or a quadrat. extracted with the E.Z.N.A. Insect DNA Kit (omega bio- Crayfsh were caught without release, either by the tra- tek, USA) according to the manufacturer’s instructions. ditional fshing tool “tandroho” (50 cm × 30 cm × 30 cm) Te qPCR protocol to detect A. astaci was identical to or with a net (200 × 400 cm) or manually in burrows. the one used in [49] with an increased annealing tem- Carapace length and total length were measured using a perature and decreased annealing time [50]. Te results manual caliper and weight was recorded using a portable of the qPCR are categorized in semi-quantitative levels scale with 0.1 g precision. Female sex was morphologi- called agent levels, with increasing amounts of A. astaci cally confrmed by the presence of the gonopores on the DNA. Agent levels A0 and A1 are generally considered base of the third pair of legs and the ovaries under the negative, while A3 to A7 are considered positive (A2 is carapace. Abdominal musculature samples from three considered ambiguous). animals per site were preserved in ethanol for genotyp- ing, while soft cuticles, uropods and legs were preserved B. pfeiferi predation for the molecular detection of the crayfsh plague agent Biomphalaria pfeiferi snails (N = 28, 1–4 mm, a kind Aphanomyces astaci. After data collection, all animals gift from Nelia Luviano and Christoph Grunau, Univer- were sacrifced according to current Malagasy legisla- sity of Perpignan) were divided into seven groups of four tion, which prohibits the release or live transportation and placed in closed laboratory boxes with a single mar- of marbled crayfsh. Statistical comparisons of body size bled crayfsh (TL 6–8 cm) that had been starved for 24 to structure among the sites were performed by the non- 48 h. Te boxes were checked every hour, left unattended parametric test of Kruskal–Wallis. All maps were drawn overnight from 18:00, and checked again on the next with QGIS (Open Source Geospatial Foundation Project) morning at 9:00. Representative images were acquired on version 2.18.7. Te shapefles used to draw the 5 biocli- a Stereomicroscope (Olympus SZX10). matic boundaries [48] were downloaded from http:// www.mobot​.org/MOBOT​/Resea​rch/madag​ascar​/gazet​ Market and trade investigations teer/. Madagascar boundaries and altitudinal gradients Marbled crayfsh value on the market was evaluated shapefles were downloaded from the Free Spatial Data in among three main groups: chiefs of locations (n = 11), DIVA-GIS (http://www.diva-gis.org). fshermen and/or crayfsh collectors (n = 27) and local vendors (n = 17). Chiefs were interviewed frst to evalu- Habitat parameters ate the status of marbled crayfsh in the area, such as fsh- For each sampling site, bottom sediments (mud, clay, ing activities, locations of populations and markets. Tey sand) were visually identifed and water temperature was also identifed local collaborators (e.g. fshermen and/or recorded with a multiparameter device (Hanna Instru- crayfsh collectors) that could provide further informa- ments HI991300) 10 cm below the surface between 8:00 tion about crayfsh locations, farming and prices on the and 10:00 in the morning. Te same device was used to markets. Local vendors were interviewed about the main record the conductivity and the pH with a calibration at Andriantsoa et al. BMC Ecol (2019) 19:8 Page 9 of 10

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