Inhibiting invasive fish reproduction via germ cell xenotransplantation and hybrid lethality

One of the most important issues in the conservation of aquatic ecosystems is the problem of alien species¹. These are organisms that have been artificially introduced outside their native habitat. The establishment of effective countermeasures against ‘invasive alien species,’ which have various adverse effects on native ecosystems through predation and competition, is a global challenge^2,3.

Eradication by physical capture is commonly used to remove invasive fish⁴. However, eradicating all invasive individuals in large bodies of water through capture eradication alone is technically impossible. Even if established invasive fish populations can be controlled at low densities by physical capture, a significant budget and effort are needed to maintain such low densities. Once the intensity of physical eradication is reduced, populations are known to recover rapidly because of the rebound phenomenon⁵. In other words, a fundamental solution to the problem of invasive fish requires the development of new techniques to eradicate the remaining individuals at low densities after physical capture. One solution to this problem is to use toxic chemicals such as rotenone and antimycin ⁶. However, this method results in the death of all aquatic organisms in the body of water and therefore places a significant burden on the target ecosystem. Therefore, the aim of this study was to develop a new method for eradicating invasive fish using hybrid lethality and surrogate broodstock technology without the use of harmful chemicals and with minimal impact on the native ecosystem (Fig. 1).

Hybrid lethality refers to a reproductive barrier in which interspecific hybridization results in complete mortality of the hybrid offspring, leading to the inviability of the next generation. For example, the next generation resulting from mating of female rainbow trout (Oncorhynchus mykiss) and male brown trout (Salmo trutta) is lethal before it reaches the swim-up stage^7,8. However, interspecies hybridisation is an extremely rare phenomenon under natural conditions, even when closely related heterospecific females and males are sympatrically distributed.

In this study, we attempted to use surrogate broodstock technology to induce hybrid lethality through natural mating. Since the surrogate recipients produced by interspecific transplantation of germline stem cells have all the characteristics of normal wild fish, except that they produce gametes of different species, the surrogate recipients are expected to be able to mate with wild fish of the same species and thus cause interspecific fertilisation of gametes in the natural environment. Thus, if large surpluses of surrogate recipients are released into streams inhabited by invasive fish, stochastic mating between wild female invasive fish and surrogate males can be expected to result in most of the next generation produced by the females being lethal hybrids. Therefore, we predicted that repeated releases of this large surplus of surrogate recipients would make it possible to eradicate nonnative fish.

To establish this methodology, surrogate recipients need to be able to naturally mate with wild fish with high efficiency. However, no examples exist in which surrogate recipients have been released into the natural environment and their reproductive behaviour has been observed. To evaluate the feasibility of this strategy, this study aimed to examine the ability of surrogate males to mate with wild-type females of the same species in an experimental river and whether the resulting next generation would be lethal hybrids (Fig. 1).

Production of rainbow trout surrogate males producing brown trout sperm

Rainbow trout recipients were produced for mating experiments. In this study, brown trout, which generate lethal hybrids when crossed with rainbow trout, were used as donors. Brown trout germ cells were specifically labelled with green fluorescence by Alexa 488-conjugated antibody No. 95 ⁹ (Fig. 2a). This antibody recognises cell surface antigens of live type A spermatogonia (ASGs) in various salmonids¹⁰. Triploid rainbow trout larvae were used as recipients (Fig. 2b, c). On the 14th day after transplantation, donor-derived germ cells were incorporated into the genital ridges of 4 of the 6 recipient larvae examined (Fig. 2d). The average number of incorporated germ cells per recipient was 2.16 ± 0.31 cells (n = 4). The resulting recipients were then raised until they matured (Fig. 2e). Semen was obtained from 6 of the 31 triploid males. PCR-AFLP analyses of sperm DNA revealed that all the sperm samples presented an identical banding pattern to that of the control brown trout without any signs of rainbow trout-derived amplicons (Fig. 2f, Supplementary Fig. 1). In contrast, the nontransplanted control triploids in this study did not produce any sperm. Thus, we succeeded in producing rainbow trout surrogate males that produce only brown trout sperm. In addition, the surrogate recipients were flow acclimated for 125 days under high-flow-rate conditions to improve their swimming abilities.

Natural mating between surrogate males and wild-type females

We next verified the ability of the surrogate recipients to mate with wild-type females in a natural environment. Six mature surrogate males and 17 mature wild-type females were released into artificial spawning grounds (Fig. 3a,b. As a result, wild-type female No. 2 first showed probing behaviour using her anal fin, surrogate male A042 immediately approached, and they began oviposition and ejaculation (Fig. 4a–c, c’), Supplementary Movie 1). In addition, the wild-type female performed nest-covering behaviour after the spawning event. During the 7-day observation period, surrogate male A042 mated 9 times, surrogate male A053 mated once, and surrogate male A100 mated once, for a total of 11 mating events.

Analyses of offspring produced by the natural mating of surrogate recipients

After confirming the mating of the surrogate recipients, the locations of the spawning redds were identified using underwater camera images and visual observations of the spawning grounds (Fig. 4d). By excavating these spawning redds, we retrieved eggs (Fig. 4e). Eggs laid by various females at the same site on the same day were indistinguishable and were collected together. Egg group #9 had a fertilisation rate of 0%, whereas the other egg groups had a fertilisation rate similar to that of the control group (Table 1). Furthermore, by rearing the fertilised eggs, we confirmed that the eyed rate was less than 68%, the hatch rate was less than 36%, and all individuals in all four egg groups except egg group #8 (see below) died by the swim-up stage (Table 1, Fig. 5a, a’). Next, the genetic origins of the embryos that died before swimming-up were analysed to verify whether the five groups of fertilised eggs underwent interspecific hybridisation. As a result, amplification of rainbow trout-derived amplicons and brown trout-derived amplicons was confirmed in all egg groups (Fig. 5b). Polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis of the D-loop regions of the mitochondrial genomes confirmed the amplification of rainbow trout female-derived fragments in all egg groups (Fig. 5c). These results indicate that the surrogate male recipients induced interspecific fertilisation through natural mating and produced lethal hybrids in all the groups besides group #8.

Egg group #8 contained two normal surviving individuals (Table 1). Therefore, we performed the same species-identification analysis for egg group #8 and found that seven individuals, including the two surviving individuals, were pure rainbow trout (Supplementary Fig. 4b). In addition, the mating video of egg group 8 revealed that a small wild-type male that could not be removed from the experimental river participated as a sneaker during the mating between the surrogate male and the wild-type female (Supplementary Fig. 4a, Supplementary Movie 2).

The results showed that male surrogate rainbow trout that produce gametes of different species can mate naturally with wild-type females in the natural environment. Furthermore, hybrid lethality can be induced in the resulting offspring through natural mating with surrogate males. This novel method for controlling the reproduction of invasive fish species could serve as a useful approach for reducing or eventually eliminating alien fish species.

The methodology developed in this study can theoretically be applied not only to species distributed in rivers and streams but also to those in still water. The physical capture method, such as the use of an electric shocker, has been shown to reduce the population of invasive fish to a low level. However, the capture efficiency decreases as the population density decreases, and it is inevitable that few individuals remain, making it difficult to eradicate invasive species. Conversely, according to the methodology developed in this study, by releasing enough surrogate recipients to mate with the invasive fish, most of the next generation derived from the eggs produced by wild females can be expected to be lethal hybrids. This methodology can be the most effective when used as a final step towards eradication after most of the alien population has been eliminated to a minimum level using conventional physical capture methods.

In a methodology for releasing sterile insects, the ratio of the number of sterile individuals to the number of wild individuals is called the “overflooding ratio”¹¹. Calculating an appropriate overflooding ratio requires data on the population dynamics of the wild population, including migratory capacity, longevity, distributional bias, and rate of increase¹². Theoretically, the overflooding ratio required to eradicate a wild population is at least 10^13,14. However, some actual eradication projects have required overflooding ratios of 100 to 1,000 because of the reduced ability of sterile insects to migrate and mate and the distribution bias of wild populations¹⁵. Conversely, if highly competent sterile insects can be intensively released at sites with high densities of wild populations, even small numbers of sterile insects can effectively reduce the number of wild individuals¹⁶. As salmonids congregate at specific spawning grounds during the reproductive season, the methodology developed in this study should make it relatively easy to meet this requirement. In this case, eradication could be achieved with relatively low overflooding ratios in salmonids compared with those needed for insects, which can mate anywhere in their habitat. However, further precise studies are still needed.

Regarding salmonids, several studies have reported successful population reductions to as few as dozens of individuals^17,18. Given that a single researcher can produce more than 100 surrogate recipients in a day, it is feasible to generate an excess number of surrogates for release against a target population of dozens of remaining invasive fish through a multiday germ-cell-transplantation experiment.

Triploid males of salmonid species are known to exhibit courtship and oviposition behavior toward diploid females¹⁹. Since these triploid males produce only a small amount of sperm lacking fertilizing ability, eggs from females that mate with them remain unfertilized²⁰. However, as shown in Supplementary Fig. 4, sneaker males frequently participate in natural mating. Since triploid males release only a very limited quantity of sperm, most eggs are expected to be fertilized by sneaker sperm when such males are present. Thus, the reproductive suppression achieved by releasing triploid males without germ cell transplantation is limited. In contrast, releasing surrogate males, as developed in this study, is expected to provide an extremely effective strategy, as it substantially reduces the opportunities for fertilization by sneaker males.

Theoretically, whether the methodology developed in this study can be applied to a particular fish species depends on (1) the existence of a closely related species, the interspecies mating with which will cause hybrid lethality and (2) the feasibility of producing recipients that produce gametes of the closely related species mentioned in (1). The phenomenon of hybrid lethality has been observed in crosses of many fish species^21,22. However, if the genetic distance between two species is too close, the resulting hybrid is often viable, so it is important to find a combination with an appropriate genetic distance. With respect to the second abovementioned requirement, surrogate broodstock technology has been applied to a wide range of taxa, including Salmonidae^23,24,25, Cyprinidae^26,27,28, Cobitidae²⁶, Ictaluridae²⁹, Adrianichthyidae³⁰, Atherinopsidae³¹, Sciaenidae³², Scombridae^33,34, Carangidae³⁵, Paralichthyidae³⁶, Tetraodontidae³⁷ and Cichlidae³⁸, and it is also being developed for other taxa. When germ cells from a donor species are transplanted into the exotic species targeted for elimination, the recipient must be capable of producing sperm derived from the donor species. In general, sperm production is relatively efficient and successful when the species is of the same genus, and even when it is of a different genus, it is more likely to be successful when the species is of the same family. Therefore, it is crucial to identify a partner species within the same family, preferably within the same genus, that can cause hybrid lethality for each target exotic species. In addition, germ cell transplantation is not limited to fish. Although the methodologies differ slightly, germ cell transplantation has been successful in some species of birds³⁹ and mammals⁴⁰. Therefore, the strategy proposed in this study is expected to be applicable to the control of invasive species in nonfish vertebrates.

In insects, the sterile insect technique (SIT) has been developed as a pest control technique^11,41. This methodology uses irradiation of pupae or infection of embryos with Wolbachia, a type of intracellular symbiotic bacteria, to produce a sterile target species population, which is then released in large excess to mate with the wild population. The next, resulting generation that mates with sterile insects is lethal because of developmental abnormalities, greatly reducing the population of wild individuals. By repeating this cycle, it is possible to completely eradicate the target species. This methodology has been applied with positive results to eradicate mosquitoes that carry contagious diseases⁴². Although the mechanism underlying the lethality of the next generation is different, our methodology is similar to SIT regarding the concept of eradication by inhibiting the reproduction of the target species. Therefore, this method of inhibiting the reproduction of target invasive species through surrogate recipients may, at least theoretically, be applicable to the eradication of invasive species in natural environments.

In certain regions, chemical control methods, such as the use of rotenone and antimycin, are employed to eradicate invasive alien salmonids ⁶. When this eradication method is implemented, native species are temporarily relocated; however, the majority of aquatic organisms that are not relocated are eliminated. This raises concerns about the significance of the impact on the ecosystem. In contrast, the methodology developed in this study minimises the impact on the ecosystem and specifically suppresses the reproduction of the target species. In recent years, new pest control methods have been developed using gene drive technology⁴³. However, this technology comes with a significant risk of releasing large amounts of genetically modified organisms into the environment⁴⁴. Conversely, the method developed in this study uses surrogate males to inhibit the reproduction of nonnative fish in the absence of any genetic modification and is therefore considered more acceptable by the general public.

In the present study, some of the released surrogate males did not participate in spawning behaviour. Although the reason could not be determined in the present study, previous studies have shown considerable variation in the number of spermatozoa produced by rainbow trout surrogate males, ranging from 2.4 × 10⁹–1.9 × 10¹¹²⁵. In addition, the gonadal size of the recipients of germ cell-transplanted triploids and dnd-KOs is highly variable^45,46. Taking these facts into account, one possible interpretation is that the variation in surrogate male behaviour may be due to differences in the number of spermatozoa produced by the recipients and the variation in sex steroid levels caused by the difference in testicular size. The amount of sperm produced by surrogate males is likely related to the number of germline stem cells incorporated into the recipient gonadal anlagen immediately after transplantation^45,47. In the future, it may be possible to improve the production efficiency of donor-derived spermatozoa by surrogate males via the transplantation of larger numbers of germline stem cells⁴⁸ or the amplification⁴⁹ or concentration¹⁰ of stem cells before their use for transplantation.

While considerable research has been conducted on pheromonal stimulation of males by female salmonids, the available literature on stimulation of females by males is quite limited. In Arctic char (Salvelinus alpinus), prostaglandin F-series (PGFs) released by mature males have been demonstrated to function as sex pheromones, attracting mature females and inducing digging behaviour, a female reproductive behaviour occurring immediately before spawning⁵⁰. Since PGFs are known to be produced in various tissues of the fish body, it is not clear whether germ cells are involved in their production pathway. However, the confirmation of communication between rainbow trout wild-type females and surrogate males during mating allows the inference that the germline does not play a significant role in the production of species-specific sex pheromones in male rainbow trout. Moreover, visual stimuli play an important role in salmonid mate choice⁵¹. As the external morphology of surrogate recipients is identical to that of normal rainbow trout, this may also be an important cause of the high frequency of mating between surrogate males and wild-type females.

For surrogate males to successfully, naturally mate with wild females, they must win intermale competition with wild males. In salmonids, larger and heavier males generally have an advantage in intermale competition^{52,53,54,55,56}. In the future, optimising the conditions necessary for surrogate males to outcompete other males is expected to enable efficient mating between surrogate males and females of invasive alien species. It is also known that fighting behaviour is controlled by some neuropeptides⁵⁷ and hormones⁵⁸. Thus, it may be possible to increase aggressiveness by administering substances that induce fighting behaviour to surrogate males prior to release.

Fish

The fish used in this study were reared in spring water at 10.5 oC at Oizumi Station (Yamanashi, Japan) of Tokyo University of Marine Science and Technology. All experimental procedures were followed in accordance with protocols approved by Institutional Animal Care and Use Committee (IACUC) of Tokyo University of Marine Science and Technology (TUMSAT), and all methods are reported in accordance with ARRIVE guidelines. Triploid rainbow trout offspring were produced via the suppression of second polar-body extrusion using heat shock treatment (28 oC, 15 min) starting 10 min after fertilisation (Okutsu et al., 2007) and were used as recipients for ASG transplantation. The donor brown trout used for transplantation had a mean body length of 108.6 ± 4.3 mm, a mean body weight of 24.0 ± 1.6 g, and a gonadal weight of 12.5 ± 1.0 mg (n = 8).

Interspecies hybridisation

The donor species used in this experiment was required to meet two requirements: the hybrid with rainbow trout must be lethal, and the genetic distance should be close enough to the rainbow trout so that the rainbow trout recipients can produce gametes derived from the donor-derived cells by germ cell transplantation. Therefore, we conducted artificial fertilisation between brown trout and rainbow trout and confirmed that all the hybrids were lethal. The rainbow trout eggs were artificially inseminated with brown trout sperm via the dry method, the resulting eggs were reared in 10.5 °C running water, and the fertilisation rate, eyed rate, hatched rate, and swim-up rate were measured (Supplementary Table 1).

Spermatogonial transplantation

After the donor individuals were anaesthetised, the abdomens were incised, and the testes were removed. The testicular mesentery and accompanying blood vessels were removed from the testes using tweezers under a stereomicroscope. The testes were then cut into small pieces using Weckel scissors (MB-41, NAPOX, Natsume Seisakusho Co., Ltd., Tokyo, Japan) to obtain testis fragments. The resulting testis fragments were transferred into 1 mL trypsin (150 U/mg P, 88% protein; Worthington Biochemical Corp., New Jersey, USA)/phosphate-buffered saline ( +) solution and subjected to enzymatic digestion for 2 h at 20 °C. During the enzymatic treatment, gentle pipetting was applied every 30 min to promote the dissociation of the remaining testicular fragments. The resulting testicular cell suspension was filtered through a 42-µm mesh nylon screen to remove cell clumps resulting from incomplete dissociation. The resulting donor testicular cells were stored at 4 °C until transplantation. To visualise type A spermatogonia, immunocytochemistry was conducted with Alexa Fluor 488-conjugated antibody No. 95. Approximately 2 × 10⁶ dissociated testicular cells were incubated with 1000 µL Alexa Fluor 488-conjugated antibody diluted tenfold in L-15 medium for 8 h at 4 °C. After the immunoreaction, the testicular cells were washed three times with L-15 medium, and the labelled cells were subsequently observed under a fluorescence microscope (BX53, Olympus Corporation, Tokyo, Japan).

Glass micropipettes were prepared for germ cell transplantation by using a glass capillary (G-1, Narishige Scientific Instruments Laboratory, Tokyo, Japan) with a puller (PW-6, Narishige Scientific Instruments Laboratory). The inner diameter of the tip of the glass micropipette was adjusted to 70–90 µm using a micro grinder (EG-3, Narishige Scientific Instruments Laboratory). A micromanipulator (BP-1, Narishige Scientific Instruments Laboratory) and microinjector (IM-9A, Narishige Scientific Instruments Laboratory) set up under a stereomicroscope were used to transplant 80 to 100 nL of the donor cell suspension containing 80,000 to 100,000 testicular cells into the abdominal cavity of each newly hatched recipient larvae^59,60. The resulting recipients were reared at 10.5 °C. Fourteen days after transplantation, six recipients were dissected, and the incorporation of donor spermatogonia into the gonads of the recipients was analysed using a fluorescence microscope (BX-53 equipped with a U-MWIB, Olympus).

Ploidy determination

The ploidy of the putative triploid fish used for the transplantation experiments was assessed. Blood samples from mature recipients were used for flow cytometry to test the ploidy status of each recipient. The relative DNA content of each blood sample was measured via a Guava easyCyte instrument equipped with a 695/50 nm filter (Cytek Biosciences, California, USA). Blood samples were collected from mature recipients with a heparinised syringe, fixed in 70% ethanol for at least 30 min and centrifuged at 300 × g for 10 min at 4 °C; approximately 1 × 10⁶ of these fixed cells were resuspended in propidium iodide (PI) staining solution containing 60 µg/mL PI (Dojindo, Kumamoto, Japan) and 30 µg/mL RNase A (Sigma‒Aldrich, St. Louis, MO) in PBS (-) for 1 h at 20 °C. Blood samples from normal diploids were used to represent the standard diploid DNA content value.

Analyses of gametes obtained from recipients

Gamete production was determined by applying gentle pressure to the abdomen of each recipient. This operation was performed once a week during the spawning season. If sperm were obtained, then a small amount of semen was squeezed. PCR-amplified fragment length polymorphism analysis using sperm DNA was performed to confirm whether the sperm obtained from the male recipients were of donor origin. Total DNA was extracted from 1 µL milt with a Gentra Puregene Tissue Kit (QIAGEN, Venlo, The Netherlands) according to the attached protocol. Primers conserved among rainbow trout and brown trout were used to amplify partial fragments of the vasa, nos2, and stm genes (Supplementary Table 2). PCR products were electrophoresed on 0.7% (vasa) and 2.0% (nos2 and stm) agarose gel together with 2-log DNA Ladder (New England Biolabs, Ipswich, MA, USA) as a molecular weight marker. The lengths of the amplified fragments were compared with those of fragments from rainbow trout and brown trout to confirm that the sperm produced by the recipient were of donor origin.

Artificial spawning ground

Two artificial spawning grounds with optimal environmental conditions for rainbow trout spawning (water depth of 20 cm, bottom velocity of 10–20 cm/s, surface velocity of 20–35 cm/s, and gravel diameter of 10–100 mm) were constructed in an experimental stream 3.2 m wide at the Oizumi Station of Tokyo University of Marine Science and Technology⁶¹. Because there was a reservoir downstream of the experimental stream, any released fish that were swept downstream could be recovered. Bird nets were placed around the artificial spawning grounds to prevent herons from entering. Before the mating experiment, as many wild-type rainbow trout as possible were removed via an electric shocker.

Flow acclimation of surrogate recipients

To acclimate the recipients to the fast water flow of the stream, the recipients were reared in a 1.5 t FRP tank installed with a 150 L/min submersible pump (TSURUMI MANUFACTURING, Osaka, Japan) to generate an average flow velocity of 10 cm/s. The recipients were kept in this tank for 7 days or 125 days. The acclimated recipients were then released into the experimental river where an artificial spawning ground was created, and the number of days until each fish was swept downstream from the artificial spawning ground was measured.

Surrogate recipient mating study in the experimental stream

Female wild-type rainbow trout and male surrogate recipients were released into the artificial spawning grounds, and their reproductive behaviour was observed. Six recipient males within one month of spermiation and 17 wild-type females one day after ovulation were used in the experiment. The released fish were tagged with dart tags and anchor tags (Hallprint, Hindmarsh Valley, Australia). The mature males had a mean standard length of 374.7 ± 28.2 mm and a mean body weight of 2063.5 ± 211.4 g. Four underwater cameras were used to observe and video record reproductive behaviour for 7 days. Data collection and battery replacement were performed at night to avoid disturbing reproductive behaviour. The collected video data were immediately reviewed on the same day, and if spawning behaviour was confirmed, their spawning positions were recorded. The eggs were collected by excavating the predicted spawning redds. The collected eggs were kept overnight in rainbow trout isotonic solution and then reared in running water at 10.5 °C. The number, fertilisation rate, eyed rate, hatched rate, and swim-up rate of the retrieved eggs were measured. Eggs that died during development were immediately collected, and the embryos were isolated and frozen. After the eggs were collected, the spawning redds were reconstructed to their prespawning condition and prepared for the next day’s spawning. Observations were made from the day of release until 7 days after release. At the end of the observation period, all the recipient fish used in the experiment were collected.

Identification of parental species of embryos

The fertilised eggs produced from mating between recipient males and wild-type females were subjected to species identification analysis. The same methods employed in the species identification analysis of the sperm produced by the recipients were utilised to confirm that the next generation produced by this mating was a rainbow trout × brown trout hybrid. If proven to be a hybrid, the maternal species was identified by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis of the D-loop region of the mitochondrial DNA (Supplementary Table 2). The amplified D-loop fragments were digested with Mbo I (Takara Bio, Inc.) to distinguish between rainbow trout-derived sequences and brown trout. PCR products were electrophoresed on 0.7% (vasa) and 2.0% (nos2, stm and D-loop) agarose gel together with 2-log DNA Ladder (New England Biolabs, Ipswich, MA, USA) as a molecular weight marker.

Statistical analysis

All the data are presented as the means ± standard errors of the means (SEMs). Statistical significance was determined for comparisons of three or more groups by one-way analysis of variance with Tukey’s multiple comparison test at a statistical significance level of P < 0.05. All analyses were performed using R.

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Authors and Affiliations

1. Department of Marine Biosciences, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato, Tokyo, 108-8477, Japan

   Yuichi Amano, Haruto Baba & Goro Yoshizaki

2. Gifu Prefectural Research Institute for Fisheries and Aquatic Environments, Gifu, 501-6021, Japan

   Daisuke Kishi & Yushi Shimomura

Contributions

Y.A., H.B., D.K. and Y.S. conducted experiments and analysed the data. Y.A. and G.Y. designed the research. Y.A. and G.Y. conceived the study and wrote the manuscript. All authors critically revised the report, commented on drafts of the manuscript, and approved the final report.

Corresponding author

Correspondence to Goro Yoshizaki.

Competing interests

The authors declare no competing interests.

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