Successful artificial incubation and juvenile-rearing of dropped eggs of a critically endangered freshwater crayfish (Cherax tenuimanus)

Introduction

One in three freshwater crayfish species worldwide is at risk of extinction from threats such as water extraction, pollution, habitat loss and fragmentation, and disease (Richman et al. 2015; O’Hea Miller et al. 2024). In Australia, the main threats are climate change, harvesting, agriculture, invasive species and fire (Richman et al. 2015; Kirsch et al. 2024). Although among the most threatened species, Australian freshwater crayfish are seldom adequately conserved in protected areas (Coughran and Furse 2012), in part owing to endemism to small regions and even single rivers (De Graaf et al. 2009; McCormack 2013). The increasing pressure of multiple threatening processes on Australian freshwater crayfish, combined with the lack of adequate protection of most wild populations, means that alternative approaches to long-term conservation are required (Richman et al. 2015).

One alternative approach that has attracted attention for some freshwater crayfish species is captive breeding and ex situ artificial incubation of eggs to increase populations for conservation (Nakata et al. 2004) or to limit transmission of pathogens (Edgerton and Owens 1997; Kozák et al. 2023). Ex situ artificial incubation techniques have been developed for commercial aquaculture of Cherax species, such as yabbies (C. destructor) (Leonard et al. 2001), redclaw (C. quadricarinatus) (King 1993; Shun et al. 2020; Cheng et al. 2022) and smooth marron (C. cainii) (Henryon and Purvis 2000) with promising results. Various bespoke systems and prophylactic treatments have been used and summarised in Table 1, but there is no consensus on a single suitable approach. Furthermore, these studies have little information on post-hatch handling and early-stage juvenile rearing, limiting their value for captive breeding through to reproductively mature adults.

Six species of Cherax are endemic to southern Western Australia (De Graaf et al. 2009). The critically endangered (Austin and Bunn 2010) hairy marron, Cherax tenuimanus Smith has a restricted distribution, being endemic to a single river, the Margaret River (Austin and Ryan 2002). The more widely distributed congener, the smooth marron (C. cainii), is a popular recreationally fished and aquaculture species. It does not naturally occur in Margaret River (Austin and Ryan 2002); however, its presence was detected in samples collected in 1985, which was attributed to its introduction by one or more translocation events (Austin and Ryan 2002; Duffy et al. 2014). Following this introduction, C. tenuimanus has been rapidly replaced by C. cainii throughout Margaret River, owing to reproductive interference and competition (Bunn 2004; De Graaf et al. 2009). Hybridisation and introgression between the two species has caused genetically pure C. tenuimanus to be rare in a mixed population (Bunn et al. 2008; Guildea et al. 2015). A small population persists in the wild (DPIRD, unpubl. data) and a very small captive population has been held at various sites since the early 2000s. Captive breeding programs have had varying levels of success. Semi-intensive production techniques developed for C. cainii have not produced juvenile C. tenuimanus, as a result, the captive population has gradually declined because of natural mortality (DPIRD, unpubl. data). Even though intensive efforts at breeding in recirculating systems have yielded clutches of eggs, no juveniles have been produced because the eggs have consistently been dropped by the ovigerous females (Lette 2020; DPIRD, unpubl. data) and die without maternal care.

Perth zoo holds a small population of C. tenuimanus that have been confirmed, through genetic testing, to be pure C. tenuimanus. In December 2022, a captive female C. tenuimanus held at the zoo mated and produced a clutch of fertilised eggs. When some of these eggs were dropped, we opportunistically recovered the eggs, determined that they were viable and attempted artificial incubation, followed by juvenile rearing, by using techniques applied to other Cherax species (Henryon and Purvis 2000; Cheng et al. 2022; Table 1). Our objective was to develop practical protocols for incubating dropped eggs and then rearing hatchlings beyond a 100-day grow-out period to assess the potential for this approach to conserving C. tenuimanus.

Materials and methods

Obtaining, disinfecting and assessing the dropped eggs and larvae

From the date a clutch was observed (27 November 2022) through to the final release of juveniles from her pleopods (24 March 2023), the berried female C. tenuimanus, originating from the wild in 2016 and currently located at Perth Zoo, was checked daily. Dropped eggs and larvae (Fig. 1a, b) were detected between 29 December 2022 and 10 February 2023 (Fig. 2) and were transferred within 24 h to the West Australian Fisheries and Marine Research Laboratories. During transport, eggs or larvae were placed in a closed 250-mL glass Schott jar containing source water and placed in a small cooler with a frozen ice brick. Temperature of the water ranged between 16 and 20°C.

Fig. 1.

Stages of C. tenuimanus during artificial incubation (a) egg, (b) first-stage larva with egg casing, (c) second-stage larva, and (d) juvenile.

Fig. 2.

Gantt chart of dropped C. tenuimanus egg development. Day 0 is 29 December 2022. The study was terminated after 180 days. RAS, recirculating aquaculture system.

On arrival at the research laboratory, eggs were disinfected with 75% ethanol solution for 60 s. This was repeated every second day for 21 days. This procedure was then changed to a salt water bath (100 g L⁻¹ solution, API aquarium salt) for a period of 5 min every second day for 14 days. During each disinfection event, the egg was observed under a stereo microscope (Nikon SMZ 745T) to determine egg development. Each egg was tracked with a development schedule reported for C. destructor (Sandeman and Sandeman 1991), with particular focus on the changing proportion of yolk and presence of a heartbeat observed in the egg. Dead eggs were removed using forceps, preserved in formalin solution and sent off for disease diagnostic testing. The disinfection process was continued for the first and second larval stages, through which Cherax spp. progress prior to reaching the first juvenile instar.

Artificial incubation of eggs

We incubated eggs by using Ziss Aqua Egg Tumbler suspension upwellers (ZET-55, Fig. 3) because these had been trialled successfully by the authors with Cherax cainii eggs, as well as C. quadricarinatus by Shun et al. (2020). A maximum of five eggs were placed in each of the 12 incubators used. Eggs were incubated with those dropped on the same or a similar date. Artificial incubators were distributed among four 16-L glass aquaria (36 × 26 × 22 cm), aerated at a rate sufficient to keep the eggs continuously but gently moving. Incubators and aquaria were disinfected with a 12.5% sodium hypochlorite solution prior to use.

Fig. 3.

Ziss Aqua ZET-55 Egg Tumbler suspension upweller.

The trial was undertaken in a climate-controlled room set at 18–20°C, with a photoperiod that changed to replicate the natural diel cycle at that time of year. For biosecurity, the room was restricted access, with a Virkon footbath and handwashing facilities on entry; laboratory coats were mandatory. Aquaria were filled with tap water that had been filtered through a carbon filter (Puretec EC011) and aerated for at least 24 h prior to use. This tap water did not contain chloramines, as confirmed by test strips (AquaChek). Water quality was monitored daily for pH, ammonia, nitrate, nitrite and hardness (API test kits). If ammonia or nitrite concentrations exceeded 0.1 mg L⁻¹ or nitrate concentrations exceeded 10 mg L⁻¹, half the water in the aquaria was changed. Salinity was also tested daily by using a Eutech EC-PCSTestr35 Multi-Purpose Meter and maintained close to 1 g L⁻¹ by using aquarium salt (API).

Rearing juveniles (hatchlings) in a recirculating aquaculture system

As soon as second-stage larvae had moulted to the first juvenile instar (Fig. 1c, d), they were placed into one of two purpose-built recirculating aquaculture systems (RAS, Fig. 4a). Each RAS consisted of a 200-L sump, Fluval FX6 canister filter, ZKSJ Blue70-12000 DC pump and shelving, each with fifty-four 5-L clear plastic tubs. Juveniles were randomly assigned to a tub and housed individually with a ‘hide’ (Fig. 4b) made of 12-mm diameter black plastic hose, which was enlarged as the animals grew. Each system was filled with carbon-filtered (PureTec EC011) tap water and a probiotic (0.25 g, Sanolife MIC) was added biweekly. The system was cycled for 14 days to establish suitable biological filtration capacity prior to addition of animals. Nitrate, nitrite, ammonium, carbonate hardness, general hardness, pH, calcium, free chlorine, chlorine and salinity were tested daily (Table 2), and salinity was maintained close to 1 g L⁻¹ by using aquarium salt (API).

Fig. 4.

(a) Recirculating aquaculture system (RAS). (b) Juvenile C. tenuimanus in a hide in RAS.

Table 2.Water parameters measured in the RAS during the study.

Ammonia (mg L−1)	General hardness (mg L−1)	Carbonate hardness (mg L−1)	pH	Nitrite (mg L−1)	Nitrate (mg L−1)	Salinity (g L−1)	Calcium (mg L−1)	Chlorine (mg L−1)	Free chlorine (mg L−1)
0–0.25	125.3–214.8	107.4–161.1	7.8–8.2	0–0.25	0–10	0.81–1.17	60–120	0	0

Juveniles were randomly assigned feed types and fed either frozen peas, corn or crushed lupin, renewed as needed. Sinking spirulina algae wafers, Aquatopia freshwater crayfish pellets, Repashy gel premix and AquaOne frozen bloodworms were also provided on different days, according to a weekly schedule. Each morning, faeces and other waste were manually siphoned from the tubs, and water was changed twice weekly. Filters and pumps were cleaned periodically to maintain suitable water quality.

Results

Discussion

Our work indicated that dropped eggs of the critically endangered Cherax tenuimanus can be successfully artificially incubated with a high level of survival. From 41 eggs dropped by a single berried C. tenuimanus, 35 (85%) were hatched using ZISS incubators. Of 42 larvae (DPIRD hatched animals and a subsequent 7 larvae from the zoo), 31 (74%) were reared through first and second larval stages in the same ZISS incubators and then survived as juveniles in a RAS.

Artificial incubation of dropped eggs and rearing of juveniles in a RAS has the potential to increase captive populations of C. tenuimanus. In captivity, this species has demonstrated regular clutch failure and dropped eggs, severely hampering conservation efforts to build an adequate captive population (Lette 2020; DPIRD, unpubl. data). This method also offers a suitable alternative to maternal incubation, provided that eggs can be obtained from the female without negative effects. Freshwater crayfish embryos develop outside the females’ body and therefore the eggs are suited to artificial incubation (McLay and Van den Brink 2016). Suspension incubators can imitate the environment provided in a female’s brood chamber to provide conditions for successful development. The constant flow inside the incubators mimics the female’s oscillations of her pleopods to ‘fan’ her eggs, providing freshwater, oxygen and preventing microbial establishment (Leonard et al. 2001). Brooding females groom and clean eggs (Bauer 2013) and are known to discard unhealthy or dead embryos (Fernández et al. 2020).

During the incubation period, the issue of greatest concern was the presence of potentially undesirable organisms as freshwater crayfish eggs are susceptible to infection and disease (Romero and Jiménez 1997). Epistylis (caused by a ciliated protozoan) and temnocephalan flatworms were visible under the microscope on multiple eggs throughout the experiment, albeit at low levels. Various antifungal treatments have been previously used during artificial incubation of freshwater crayfish eggs (Henryon and Purvis 2000; Carral et al. 2009; Shun et al. 2020). However, effective agents such as formaldehyde and malachite green show concerning health impacts toward humans and the aquatic environment (Sapkota et al. 2008; Carral et al. 2009). Salt solution is a suitable non-toxic alternative, assisting the treatment of parasites and improving survival of early-stripped eggs (Seemann et al. 2014; Rida et al. 2021). Previous studies have used salt solution at concentrations of up to 15–16 g L⁻¹ (Seemann et al. 2014). However, on the basis of advice from a commercial producer (Dr Lisa Elliott, pers. comm., Australian Redclaw Crayfish Hatchery), who successfully trialled the dosage on C. quadricarinatus, we demonstrated that C. tenuimanus eggs and first-stage larvae can be treated with 100 g L⁻¹ salt water for up to 5 min, with 100% survival and highly effective results.

The previous lack of success in captive breeding of C. tenuimanus (Lette 2020; DPIRD, unpubl. data) begs the following question: should artificial incubation be prioritised over maternal care for conservation of the critically endangered C. tenuimanus? Our study was highly successful; however, we acknowledge the lack of replication of ovigerous females. We counter this criticism with the observations that this species is so rare that it is unethical to harvest females from the wild for a trial, nor could we access dropped eggs from other females because no other breeding in captivity occurred at the time of the study.

This study entailed intensive work on comparatively few eggs and hatchlings. To use our methods to incubate a whole clutch of potentially hundreds of eggs and yet maintain low densities of eggs per incubator would require substantial resources. This may be warranted for a species such as C. tenuimanus, where even a small increase in numbers represents a substantial increase in population. Future work should look at increasing densities in incubators (Shun et al. 2020; Cheng et al. 2022) as well as developing a protocol for successful grow-out to mature adults.

In conclusion, our work indicated that dropped eggs of this critically endangered species can be successfully artificially incubated and reared, and that the protocols and facilities described show promise for arresting the mortality of eggs dropped in captivity. Despite the success and potential benefits of this approach to ensure the persistence of this species, the main limiting factor remains the low number of successful pairings in captivity over multiple decades. Nonetheless, for threatened crayfish species such as C. tenuimanus where each egg or larvae represent a substantial addition to the population, captive breeding as described may provide a crucial tool for maintaining and enhancing the population.