Over the past 50 years, populations of California abalone (Haliotis spps.) have dramatically declined due to a suite of factors that include overharvesting, disease, and starvation (Altstatt et al., 1996; Hobday and Tegner, 2000; Karpov et al., 2000; Hobday et al., 2001; Rogers-Bennett et al., 2010), with all seven species being listed as endangered or critically endangered by the International Union for Conservation of Nature (Peters and Rogers-Bennett, 2021a; Peters and Rogers-Bennett, 2021b; Peters and Rogers-Bennett, 2021c; Peters and Rogers-Bennett, 2021d; Peters and Rogers-Bennett, 2021e; Peters et al., 2021). Moreover, black (H. cracherodii) and white abalone (H. sorenseni) were listed under the United States Endangered Species Act in 2009 and 2001, respectively (Federal Register 66 [103]; Federal Register 74 [9]). Current efforts to conserve and restore wild populations of black and white abalone are in place, such as the Final Recovery Plan for Black Abalone (National Marine Fisheries Service, 2020) developed by the Black Abalone Recovery Team and the White Abalone Captive Breeding Program that includes over a dozen partner institutions (Rogers-Bennett et al., 2016). The White Abalone Captive Breeding Program seeks to enhance gametogenesis through the implementation of a wide range of tools that include hormone therapy, molecular biology, nutritional physiology, and other techniques used to increase the reproductive output of this endangered species. Abalone are dioecious, herbivorous gastropods that release sperm and egg into the water column for external fertilization and become reproductive between the age of four and seven years (Hahn, 1989). One of the challenges associated with spawning the limited number of endangered white abalone broodstock used in the captive breeding program, is that gonad score by visual assessment is often an unreliable predictor of spawning success (K. Aquilino and A. Frederick, unpublished data). The lack of predictability in spawning abalone is not specific to white abalone as visual abalone gonad assessment methods have long failed to reliably detect gonad condition and sexually dimorphic traits (Ebert and Houk, 1984).

Ultrasound technology has been used by biologists for decades as a non-lethal technique to assess the reproductive health in finfish conservation (Evans et al., 2004; Swanson et al., 2008; Chiotti et al., 2016; Brizendine et al., 2018; Carim et al., 2021) and for commercial aquaculture purposes (Bonar et al., 1989; Blythe et al., 1994; Colombo et al., 2004; Naeve et al., 2018). While ultrasonography had previously been used in pāua (H. iris) to identify shell lesions that diminish the ability of the adductor muscle to properly adhere to the shell, leading to increased mortality (Nollens et al., 2002), only recently has ultrasound technology been successfully used to characterize abalone reproductive state, specifically red abalone gonad condition (Boles et al., 2022). The goal of this study is to assess the use of non-lethal ultrasonography as a technique to monitor the reproductive condition of endangered black and white abalone and to evaluate the applicability of the technology for use in conservation programs. In this research, we seek to utilize non-lethal ultrasonography to (1) develop a ultrasound scoring index to assess the gonad condition in multiple species of California abalone, (2) characterize the reproductive state of individual endangered captive white abalone during scheduled spawning events during peak reproductive season, and (3) evaluate the reproductive state of rescued wild black abalone brought into captivity following post-fire debris flows. We discuss the application of these methods to support the captive breeding and monitoring of endangered abalone in California.

Ultrasonography provides researchers with a quantitative metric to indirectly assess gonad maturation state in abalone. This project specifically illustrates the applied use of a non-lethal method to monitor the reproductive condition of endangered black and white abalone through the use of high-quality images of the gonad for qualitative and quantitative evaluations. Furthermore, the methodology of maintaining the abalone on plastic sheets successfully avoided injury in endangered abalone thereby reducing handling stress associated with probing and manipulation of the muscular foot.

Ultrasound technology has been successfully used to evaluate the gonad maturation of various species of fish, including those that are cultured or endangered (Moghim et al., 2002; Albers et al., 2013). This non-lethal technique has the potential to enhance abalone husbandry practices by allowing researchers and program managers to monitor the gonad maturation status of abalone over the course of their reproductive cycle. This information can inform broodstock selection and spawning arrangements, which are critical components of captive breeding programs that supplement dwindling populations, restore extirpated areas, or preserve genetic materials (Rytwinski et al., 2021). Successful outcomes from these programmatic goals are determined by metrics for specific management actions. The use of ultrasound technology can help conservation managers avoid relying solely on a spawning success metric, which requires a large sample size, usually with an unknown reproductive state, and with good genetic diversity to achieve a successful outcome for restoration purposes. A spawning event can be particularly challenging for programs that have limited number of abalone in the wild or in captivity, or when the gonad status of the abalone is unknown.

While abalone stress response was not a focus of the current study, for endangered abalone species, ultrasound technology also represents a potential breakthrough for reducing animal handling stress and broodstock selection. Implementing the use of non-lethal ultrasound image technology in endangered abalone captive breeding and recovery programs increases animal welfare by eliminating the need to sacrifice animals, reducing potential risk of injury from repeated removal and handling, and decreasing incidental stress associated with chemically induced spawning of abalone that are not ready to spawn. Moreover, research on the potential stress response in abalone from prolonged ultrasound exposure should be explored (Barnett et al., 1994; Duck, 2007; Hanson, 2010), in addition to using ultrasound to assess other stressors such as nutrition restriction, (Rogers-Bennett et al., 2010); Meusel et al., 2022), pathogens and parasites (Nollens et al., 2002; Crosson et al., 2014), this new tool should prove beneficial in management and research programs.

Ultrasonography in captive breeding and recovery programs could potentially increase fertilization success by reducing the probability of spawning undersized or immature ova which may contribute to poor survival due to reduced maternally derived nutrients available during the free swimming larval phase or settling period (Moran and McAlister, 2009; Santella et al., 2020). It is currently unknown what the long-term impacts of suboptimal gamete fertilization are on energy reserves (abalone larvae are lecithotrophic), growth, survival, and fitness in juvenile and adult abalone. Clearly, non-lethal tools that aid in determining reproductive condition will be useful for monitoring and managing abalone broodstock conditioning. For instance, the usage of ultrasonography during gametogenic cycles may be critical to understanding broodstock culture and dietary energy mobilization required to improve spawning success and subsequent larval survival.

While white abalone gonad maturation has been demonstrated in captivity through successful annual spawning events, black abalone have historically been difficult to spawn. Fortunately, in this study we were able to evaluate and visualize gonad maturation on a number of black abalone at intake and again prior to release in July when black abalone are said to spawn (Webber and Giese, 1969). We noted several abalone did undergo gonad maturation in captivity with an increase of gonad thickness by 55% (i.e., Black abalone ‘O’: 0.21 cm gonad thickness in March 2021/0.46 cm in July 2021). Whereas individual ‘T’ had an increase in gonad thickness of only 0.2% during the same period. In contrast, some individuals, such as individual ‘I’, displayed a marked decline by 43% in gonad thickness (0.28 cm in March 2021 to 0.16 cm in July 2021). While ultrasonography is sensitive enough to measure small changes in black abalone gonad thickness, without regular assessments, we are not able to verify whether animals that presented with no change or reduced gonad thickness, experienced gonad atresia or spawned in captivity.

Since endangered white abalone used in the current study are involved in a captive breeding program, white abalone were available for repeated ultrasound imaging. Ultrasound imaging was successful in monitoring changes in gonadal thickness in white abalone over a 30-day period ( Figure 4 ). Ultrasound technology also detected changes in male and female white abalone gonad thickness before and after spawning on April 21, 2021 ( Figure 5 ). Male white abalone ‘A’ had a pre-spawn gonad thickness of 0.37 cm (± 0.04) and post-spawn gonad thickness of 0.15 cm (± 0.02), representing a 58.9% decrease in gonad thickness. Female ‘C’ white abalone had a pre-spawn gonad thickness of 1.13 cm (± 0.08) and a post-spawn thickness of 0.92 cm (± 0.07), representing an 18.3% change in gonad thickness. White abalone female ‘L’ had a pre-spawn gonad thickness of 0.96 cm (± 0.02) and a post-spawn gonad thickness of 0.79 cm (± 0.05), also representing an 18.3% decrease in gonad thickness.

In addition to use in black and white abalone culturing programs, these techniques will prove useful when and if other species need conservation efforts. Special consideration should be made when applying this scoring metric based on gonad thickness ( Table 1 ) because globally, abalone vary in size with some adult abalone only reaching 80 mm (Poutiers, 1998). The current multi-species gonad score was created using gonad thickness from large abalone found on the western coast of North America. The red abalone, for example, is the largest of all abalone species and was included in the score determination as red abalone populations, once abundant in northern California, have continued to decline since the recreational fishery was closed in 2017, and have recently been declared an endangered species (Peters et al., 2021). Red abalone starvation has been associated with low gonad scores (Rogers-Bennett et al., 2021), and before future fisheries are reopened, there will need to be evidence of healthy gonad development which should be assessed with ultrasound rather than through lethal sampling. The use of ultrasound should prove beneficial in their assessment as starving red abalone have been shown to require feeding for more than one year before they can spawn (Boles, 2020). Thus, red abalone may also be in need of restoration as their recent decline has been associated with mass die-offs, marine heatwaves, and the extreme sea urchin herbivory of kelp forests (De Wit et al., 2014; Rogers-Bennett and Catton, 2019; McPherson et al., 2021). The recent International Union for Conservation of Nature Red List of Threatened Species reports that 37% of all abalone populations worldwide for which data exists are currently endangered (24%) or vulnerable (13%) for extinction (IUCN, 2022). With impacts from climate change projected to increase in intensity and frequency, abalone conservation tools such as non-lethal ultrasound will become invaluable for restoration practitioners worldwide.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Ethical review and approval was not required for the animal study because the University of California, Davis, does not require ethics approval for gastropod species.

SB and JG designed the research. SB, LR-B, WB, and JG performed the research. SB analyzed the data. SB, LR-B, WB, JB-C, SG, and JG wrote the manuscript. All authors contributed to the manuscript and approve the submitted version.