Introduction
One of the most iconic species of the Galapagos Islands is the endemic and endangered Galapagos sea lion, Zalophus wollebaeki (Lorden et al., Reference Lorden, Sambrook and Mitchell2012; Trillmich, Reference Trillmich2015). As the smallest sea lion species in the world, this otariid exhibits remarkable adaptations to the unique tropical environment of the archipelago (Trillmich et al., Reference Trillmich, Jeglinski, Meise and Piedrahita2014; Riofrío-Lazo and Páez-Rosas, Reference Riofrío-Lazo and Páez-Rosas2023). With a population of around 24 000 individuals (Páez-Rosas et al., Reference Páez-Rosas, Torres, Espinoza, Marchetti, Seim and Riofrío-Lazo2021), this species has been evolutionarily separated from California sea lions, Zalophus californianus, for approximately 0.65 million years (Asadobay et al., Reference Asadobay, Urquía, Künzel, Espinoza-Ulloa, Vences and Páez-Rosas2023). The oceanographic dynamics of the archipelago were essential for the reproductive success and growth of its rookeries (Riofrío-Lazo and Páez-Rosas, Reference Riofrío-Lazo and Páez-Rosas2021), such that the quality of its feeding areas is an indicator of environmental degradation, making it a sentinel of the ecosystem’s health in the region (Páez-Rosas and Guevara, Reference Páez-Rosas and Guevara2017).
Their population has declined by about 50% over the past 4 decades, mainly due to climate variability effects, such as the El Niño-Southern Oscillation event (Kalberer et al., Reference Kalberer, Meise, Trillmich and Krüger2018; Páez-Rosas et al., Reference Páez-Rosas, Torres, Espinoza, Marchetti, Seim and Riofrío-Lazo2021), along with anthropogenic influences (i.e. habitat degradation and introduced species) (Moreira-Mendieta et al., Reference Moreira-Mendieta, Garcia-Garin, Muñoz-Pérez, Urquía, Drago, Borrell and Páez-Rosas2023; Ruiz-Saenz et al., Reference Ruiz-Saenz, Barragan, Grijalva-Rosero, Diaz and Páez-Rosas2023). The introduction of domestic animals, particularly dogs and cats, serves as an example of the impact of human activities on islands (Jimenez et al., Reference Jimenez, Mariño, Stapleton, Prieto and Bowman2020; Sarzosa et al., Reference Sarzosa, Duignan, DeRango, Field, Ríos, Sanchez, Espinoza, Loyola, Rueda and Páez-Rosas2021). Free-roaming domestic dogs are present on all inhabited islands of Galapagos (i.e. San Cristóbal, Santa Cruz, Isabela and Floreana islands) (Culda et al., Reference Culda, Dionnet, Barbu, Cârstolovean, Dan, Grijalva, Espin, Vinueza, Cruz, Páez-Rosas, Leon and Mihalca2022; Diaz et al., Reference Diaz, Mendez, Grijalva, Walden, Cruz, Aragon and Hernandez2016; Hernandez et al., Reference Hernandez, Yoak, Walden, Thompson, Zuniga, Criollo, Duque and Cruz2020) and they represent important reservoirs for invasive pathogens that create new challenges for Galapagos sea lion populations (Culda et al., Reference Culda, Rodriguez, Puleo, Sosa, Panait, Cazan, Deak, Leon, Vinueza, Páez-Rosas and Mihalca2024; Jimenez et al., Reference Jimenez, Vega-Mariño, Villacres and Houck2024; Vega-Mariño et al., Reference Vega-Mariño, Olson, Howitt, Criollo, Figueroa, Orlando, Cruz and Garcia-Bereguiain2023).
Interactions between Galapagos sea lions and domestic dogs pose health risks due to the potential transmission of pathogens (Denkinger et al., Reference Denkinger, Guevara, Ayala, Murillo, Hirschfeld, Montero-Serra, Fietz, Goldstein, Ackermann, Barragán, Cabrera, Chavez, Dubovi, Martinez and Trueba2017; Sarzosa et al., Reference Sarzosa, Duignan, DeRango, Field, Ríos, Sanchez, Espinoza, Loyola, Rueda and Páez-Rosas2021; Vega-Mariño et al., Reference Vega-Mariño, Olson, Howitt, Criollo, Figueroa, Orlando, Cruz and Garcia-Bereguiain2023; Walden et al., Reference Walden, Grijalva, Páez-Rosas and Hernandez2018). These include the canine distemper virus, parvoviruses, herpesviruses, caliciviruses, Leptospira or Brucella (Denkinger et al., Reference Denkinger, Guevara, Ayala, Murillo, Hirschfeld, Montero-Serra, Fietz, Goldstein, Ackermann, Barragán, Cabrera, Chavez, Dubovi, Martinez and Trueba2017; Ruiz-Saenz et al., Reference Ruiz-Saenz, Barragan, Grijalva-Rosero, Diaz and Páez-Rosas2023). However, among the pathogens identified in dogs on the archipelago, Dirofilaria immitis has a potential impact on the pinniped populations of the region (Culda et al., Reference Culda, Dionnet, Barbu, Cârstolovean, Dan, Grijalva, Espin, Vinueza, Cruz, Páez-Rosas, Leon and Mihalca2022). The Galapagos sea lion rookeries on San Cristóbal Island are the largest population in the archipelago (Riofrío-Lazo et al., Reference Riofrío-Lazo, Arreguín-Sánchez and Páez-Rosas2017). In El Malecón, a rookery close to the town of San Cristóbal Island, sea lions rest overlap with mosquito breeding sites, and dogs roam freely (Alagona, Reference Alagona2022; Culda et al., Reference Culda, Panait, Cazan, Vinueza, Páez‐Rosas, Guerrero Vásquez, Leon and Mihalca2025), which increases the possibility of infections.
Barnett (Reference Barnett1985) demonstrated the presence of microfilariae in the blood of Galapagos sea lions on Floreana, but without molecular confirmation and with no detailed on the larval morphology to allow clear identification as D. immitis. Recent studies have detected the presence of D. immitis in Galapagos sea lions through necropsy, antigen testing and DNA analysis (Gregory et al., Reference Gregory, Livingston, Hawkins, Loyola, Cave, Vaden, Deresienski, Breen, Riofrío-Lazo, Lewbart and Páez-Rosas2023; Livingston et al., Reference Livingston, Gregory, Hawkins, Cave, Loyola, Vaden, Deresienski, Riofrío-Lazo, Lewbart, Páez-Rosas and Breen2024). An action plan for eradicating the canine heartworm in the Galapagos was recently developed (Culda et al., internal report), which revolves around the question of whether domestic dogs represent the sole source of D. immitis infection for mosquitoes or Galapagos sea lions may also contribute to the spread of the parasite. In this context and with the recent findings of endemic foci of D. immitis on San Cristóbal Island (Gregory et al., Reference Gregory, Livingston, Hawkins, Loyola, Cave, Vaden, Deresienski, Breen, Riofrío-Lazo, Lewbart and Páez-Rosas2023; Livingston et al., Reference Livingston, Gregory, Hawkins, Cave, Loyola, Vaden, Deresienski, Riofrío-Lazo, Lewbart, Páez-Rosas and Breen2024), the current research aimed to evaluate the potential role of sea lions as hosts, which can develop a patent infection.
Materials and methods
Sample collection and examination
The fieldwork and sample collection were carried out following the protocols of ethics and animal handling approved by the Galapagos National Park Directorate (GNPD) and the Universidad San Francisco de Quito (USFQ) under research permit PC-19-23. The sampling was conducted in August 2023 at 2 different rookeries on San Cristóbal Island. One site was located in the urban area of Puerto Baquerizo Moreno (El Malecón rookery, 0°54ʹ05.7″S and 89°36ʹ43.1″W, while the other site was in the protected natural area on the opposite side of the island (Punta Pitt rookery, −0°42ʹ59.4″S and 89°14ʹ47.2″W). A total of 50 blood samples were collected from both adult and juvenile Galapagos sea lions at these 2 rookeries (Figure 1; Supplementary File 1).
Sampling sites of Galapagos sea lions on San Cristóbal Island in August 2023.
These 2 rookeries were selected to evaluate the level of exposure to pathogens and contact with domestic animals (dogs and cats). The distance between these 2 rookeries is approximately 50 km.
All animals captured in this study were monitored by a veterinarian and a GNPD ranger. The sea lions were weighed using an electronic scale, then removed from the net and restrained by experts handling this species. A physical examination was performed, and routine morphometric measurements were taken. Blood was collected from the caudal gluteal vein and aliquoted into citrate tubes. The samples were stored in a cooler and processed within 12 h. A total volume of 5 mL of blood was collected from each animal. Subsequently in the laboratory, the whole blood was divided into 2 equal aliquots: 0.5 mL in citrate tubes, for performing Knott’s test to evaluate the presence of D. immitis larvae (L1 – microfilariae) (Knott and Earle, Reference Knott and Earle1939; Newton and Wright, Reference Newton and Wright1956), the remaining 0.5 mL of blood was mixed with ethanol and kept at −20°C for further molecular analysis. The morphological differentiation of microfilariae was done according to Magnis et al. (Reference Magnis, Lorentz, Guardone, Grimm, Magi, Naucke and Deplazes2013) and Saari et al. (Reference Saari, Näreaho and Nikander2019).
DNA extraction and polymerase chain reaction amplification
Genomic DNA was extracted from blood samples using the DNeasy Blood & Tissue Kit (Qiagen, Germany), following the manufacturer’s protocol. A positive control was included in the DNA extraction process, which was sourced from the blood of a canine infected with D. immitis. The concentration and purity of the extracted DNA were assessed in duplicate using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA).
Following the extraction, polymerase chain reaction (PCR) reactions were conducted to target different genes associated with various filarial species (Table 1). Each PCR reaction set included a positive control from an infected dog with D. immitis and a negative control that contained purified water instead of DNA. The positive samples were prepared to be sequenced by Macrogen Europe (Amsterdam, the Netherlands) and analysed using Geneious® 4.85 software and BLASTn to identify the closest matching sequences stored in GenBank®.
Primer sequences used to identify filarial species and genes in Galapagos sea lions
Statistical analysis
The statistical analysis was performed using the EpiTools software. Age groups were established according to the potential risk of infection related to the duration of exposure to possible vectors for each sea lion. Odds ratios (ORs), 95% confidence intervals (CIs) and P-values were determined using univariate logistic regression to assess statistically significant prevalence differences. A P-value of ≤0.05 was considered statistically significant. Additionally, binomial proportions with 95% CIs were calculated for positive Galapagos sea lions.
Results
From the 50 sea lion blood samples, one (prevalence 2.0%, 95% CI = 0–0.1) was microscopically positive for one microfilaria of D. immitis. The positive sample was collected from a juvenile female at the El Malecón rookery (Table 2; Figure 2). The anterior edge of circulating microfilariae was conical, with the nuclei situated at a distance from the cuticle. All blood samples were molecularly analysed using conventional PCR to identify possible filarial species, targeting 2 different genes: cox1 and ITS2. The sample that tested positive in Knott’s test was successfully confirmed as D. immitis using PCR protocols. The resulting DNA sequences showed a similarity of 99–100% with available DNA sequences of D. immitis in the NCBI GenBank database (Table 3). All other samples were negative. The sequences obtained from the positive sample can be found in Supplementary File 2.
Microfilaria of D. immitis in circulating Galapagos sea lion blood from San Cristóbal Island.
Prevalence of microfilariae of D. immitis in Galapagos sea lion from San Cristóbal
OR, odds ratio; 95% CI, 95% confidence interval; NA. not applicable.
BLAST comparisons between the obtained sequences and the GenBank sequences (November 2024)
* The percentage of identical nucleotides between the two sequences.
Discussion
This study highlights new epidemiological data for D. immitis in Galapagos otariids. Worldwide, there are only few cases of heartworm infection reported in pinnipeds, all diagnosed by various methods from necropsy, antigen test, smear, modified Knott’s test to PCR and qPCR tests (Alho et al., Reference Alho, Marcelino, Colella, Flanagan, Silva, Correia, Latrofa, Otranto and Madeira de Carvalho2017; Barnett, Reference Barnett1985; Farriols et al., Reference Farriols, Arellano-Carbajal, Elorriaga-Verplancken, Adame-Fernández, Garrido, Álvarez-martínez, Bárcenas, Flores-Morán and Acevedo-Whitehouse2020; Gregory et al., Reference Gregory, Livingston, Hawkins, Loyola, Cave, Vaden, Deresienski, Breen, Riofrío-Lazo, Lewbart and Páez-Rosas2023; Jung et al., Reference Jung, Kim, Lee, Choi, Kim, Lee, Kim, So, Kang and Choi2019; Kang et al., Reference Kang, Kim, Kwon and Park2002; King, Reference King1964; Livingston et al., Reference Livingston, Gregory, Hawkins, Cave, Loyola, Vaden, Deresienski, Riofrío-Lazo, Lewbart, Páez-Rosas and Breen2024; Sato et al., Reference Sato, Higuchi, Shibuya, Ohba, Nogami, Shirai, Watanabe and Honda2002; White, Reference White1975). Our findings, together with preliminary data by Barnett (Reference Barnett1985), strongly suggest that the Galapagos sea lion can act as a suitable definitive host and reservoir for D. immitis. However, the duration of microfilariemia in sea lions is not known.
As shown in other non-canid hosts such as cats, the duration of microfilariemia and its intensity are significantly lower than in the preferred hosts, which are canids (American Heartworm Society, 2014; Simón et al., Reference Simón, Siles-Lucas, Morchón, González-Miguel, Mellado, Carretón and Montoya-Alonso2012). Recent findings revealed that Culex quinquefasciatus mosquitoes, known vectors for D. immitis, are feeding on Galapagos sea lions in the same area where circulating microfilariae were present in the sea lions’ blood (Culda et al., Reference Culda, Panait, Cazan, Vinueza, Páez‐Rosas, Guerrero Vásquez, Leon and Mihalca2025). Additionally, D. immitis was identified by performing PCR tests on engorged mosquitoes near the Galapagos sea lion rookery (Culda et al., Reference Culda, Panait, Cazan, Vinueza, Páez‐Rosas, Guerrero Vásquez, Leon and Mihalca2025). Another microfilariemic case was reported in pinnipeds, specifically in Cape fur seal, Arctocephalus pusillus pusillus, in an area highly endemic for canine dirofilariasis (Alho et al., Reference Alho, Marcelino, Colella, Flanagan, Silva, Correia, Latrofa, Otranto and Madeira de Carvalho2017).
Our PCR successfully detected the presence of D. immitis in the blood of sea lions. Indeed, molecular analysis detected one sample as being positive for D. immitis, which was also positive for circulating microfilariae by Knott’s test. Recent studies have revealed that Galapagos sea lions’ blood has tested positive for D. immitis using antigen tests and PCR techniques (Gregory et al., Reference Gregory, Livingston, Hawkins, Loyola, Cave, Vaden, Deresienski, Breen, Riofrío-Lazo, Lewbart and Páez-Rosas2023; Livingston et al., Reference Livingston, Gregory, Hawkins, Cave, Loyola, Vaden, Deresienski, Riofrío-Lazo, Lewbart, Páez-Rosas and Breen2024). Both studies identified positive cases in the same rookeries of the current study, located at El Malecón. This study found no evidence in Punta Pitt, a remote area far from the port with minimal human interaction. Furthermore, Gregory et al. (Reference Gregory, Livingston, Hawkins, Loyola, Cave, Vaden, Deresienski, Breen, Riofrío-Lazo, Lewbart and Páez-Rosas2023) performed both morphological and molecular identification on 20 adult D. immitis worms recovered from the right ventricle of an adult Galapagos sea lion carcass found on Santa Cruz Island.
The El Malecón rookery where the positive sea lion was found is on one of the most populated islands in the archipelago, characterized by the presence of free-roaming dogs; ships and ferryboats; a high number of tourists attracted by local restaurants; and specific shops (Culda et al., Reference Culda, Dionnet, Barbu, Cârstolovean, Dan, Grijalva, Espin, Vinueza, Cruz, Páez-Rosas, Leon and Mihalca2022; Páez-Rosas and Guevara, Reference Páez-Rosas and Guevara2017). Additionally, the presence of mangroves creates favourable conditions for mosquito breeding, all of which contribute to the transmission of D. immitis (Asigau and Parker, Reference Asigau and Parker2018; Asigau et al., Reference Asigau, Salah and Parker2019; Barnett, Reference Barnett1985; Culda et al., Reference Culda, Panait, Cazan, Vinueza, Páez‐Rosas, Guerrero Vásquez, Leon and Mihalca2025). Previous studies in this island found a 1.7% prevalence of microfilariemia caused by D. immitis in dogs, based on a sample size of 587 animals (Culda et al., Reference Culda, Dionnet, Barbu, Cârstolovean, Dan, Grijalva, Espin, Vinueza, Cruz, Páez-Rosas, Leon and Mihalca2022).
The transmission cycle of D. immitis poses a significant threat to the endangered Galapagos sea lions. This was first noted in 1980 on Floreana Island, where D. immitis was detected in dogs, mosquitoes, Galapagos sea lions and even humans (Barnett, Reference Barnett1985). Floreana Island was the first to be colonized in the entire archipelago, allowing for the observation of how the delicate balance of island ecology can be disrupted by the changing life cycle of D. immitis. Gradually, other islands such as Isabela, San Cristóbal and Santa Cruz were also colonized, accompanied by the introduction of dogs and other invasive species. Currently, according to the International Union for Conservation of Nature, the settlements on San Cristóbal, Santa Cruz and Isabela Islands pose a significant risk of disease transmission from domestic carnivores to Galapagos fauna (Jimenez et al., Reference Jimenez, Vega-Mariño, Villacres and Houck2024). This situation can be attributed to several factors, including the increase in the human population, the impact of tourism and administrative management by environmental authorities.
The evidence suggests that the Galapagos sea lion can act as a definitive host for D. immitis. Their high mobility raises the risk of the parasite spreading across the island, across various locations and potentially throughout the entire archipelago. Addressing these factors is crucial for creating a programme aimed at preventing this disease.
Conclusion
This study reveals a new potential definitive host for D. immitis on San Cristóbal Island. Both the current study and previous research indicate that the dynamics of this multi-host parasite can pose a significant threat to Galapagos sea lions. Protecting this endemic and endangered species requires enhanced monitoring and conservation efforts. This knowledge is crucial for developing an effective eradication plan for canine heartworm and ensuring the long-term health of the region’s wildlife.
Supplementary material
The supplementary material for this paper can be found at https://doi.org/10.1017/S0031182025100425.
Data availability statement
All data generated or analysed during this study are included in this publication.
Acknowledgements
The authors express their gratitude to the Galápagos National Park staff for their assistance with sample collection and the entire fieldwork process.
Author contributions
C.A.C.: study design, lab work, molecular analyses, data curation and manuscript preparation; R.L.V.: manuscript preparation; M.R.-L.: field work, study design, resources and manuscript preparation; R.L.: manuscript preparation; DPR: field work, study design, resources and manuscript preparation; and A.D.M.: conceptualization, funding, supervision, study design and manuscript preparation.
Financial support
A part of the study was funded by the Department of Parasitology and Parasitic Disease, Faculty of Veterinary Medicine, Cluj-Napoca, Romania.
Competing interests
The authors declare they have no actual or potential conflicts of interest.
Ethical standards
This study followed the protocols of ethics and animal handling approved by the GNPD and the USFQ under research permit PC-19-23.
Open access
