Biological invasions in international seaports: a case study of exotic rodents in Cotonou

Human activities contribute strongly to the displacement of species outside their original distribution area, thus leading to an increased number of biological invasions (Wilson et al. 2009; Blackburn et al. 2011; Gallardo and Aldridge 2013; Padayachee et al. 2017; Seebens et al. 2017). In particular, maritime trade plays a major role in the global spread of exotic plant and animal species around the world (Veale et al. 2021). Among them, the transport and involuntary introduction of commensal rodents and their associated ectoparasites and pathogens are particularly worrying (Aplin et al. 2011; Song et al. 2003, 2014; Bona 2020). The black rat Rattus rattus, the Norway rat Rattus norvegicus and the domestic mouse Mus musculus fall within the 100 most impactful invasive alien species (Lowe et al. 2000; www.​iucnredlist.​org), ranking within the top 15 rodent pest taxa in the world (Capizzi et al. 2014). These species have invaded all the continents except Antarctica (Long 2003). The intensification of trade has accelerated their spread to new territories with invasion modalities that remain largely uncontrolled (e.g., Dalecky et al. 2015; Berthier et al. 2016). Consequently, they have now reached a quasi-global distribution (St Clair 2011). All three species impact local biodiversity, leading to the decrease or extinction of endemic species (Wyatt et al. 2009; Harper and Bunbury 2015; Doherty et al. 2016). They cause damage to infrastructure and human activities (Garba et al. 2014b; Panti-May et al. 2017) and huge economic losses, essentially related to the destruction of food stocks (Dossou et al. 2020; Diagne et al. 2020a, b, 2021). They are also involved in the transport, maintenance, and transmission of many zoonotic agents (review in Meerburg et al. 2009; Colombe et al. 2019). Their tigh interaction with humans has made them privileged sources of infectious diseases, thus inducing the loss of hundreds thousands of lives annually (Colombe et al. 2019). In order to avoid rodent-mediated dissemination of zoonotic agents showing epidemic/pandemic potential, International Health Regulation (World Health Organization 2008) imposes signatory states to monitor and control invasive rodents at their entry points, especially within seaport areas. However, scientific data to guide rodent management actions in such habitats are very scarce.

Thanks to its international seaport in the city of Cotonou (officially “Autonomous Port of Cotonou”, hereafter designed as to APC), the Republic of Benin is at the heart of intercontinental trade between West African coastal as well as hinterland countries and the rest of the world. As a consequence, the APC constitutes a particularly favourable site for exotic rodents introduction and subsequent spread towards landlocked countries such as Niger, Burkina-Faso, Mali and Chad. For instance, trade flows from Benin to Niger are thought to have facilitated the introduction of the black rat and the house mouse in large cities such as Niamey, which is more than 1,000 km away from the coast (Berthier et al. 2016; Hima et al. 2019). This latter species was probably brought in by ships, settled in the seaport and then transported to Niamey with the goods loaded on the trucks (Hima et al. 2019).

Rattus rattus, Rattus norvegicus and Mus musculus are abundant within the APC, representing more than 80% of the small mammals caught (Hima et al. 2019; Dossou et al. 2020; Badou et al. submitted). In the industrial storehouses, Norway rats and house mice damage an average of 3,450 tons of imported rice, for an estimated loss of 58 k€ per warehouse and per year (Dossou et al. 2020). They may also be responsible for the destruction of the electricity networks, including those of some very expensive unloading machines (APC, pers. comm.). Additionally, they may be involved in the introduction of allochthonous zoonotic pathogens, such as Seoul Orthohantavirus that are responsible for hemorrhagic fevers (Castel et al. 2021). However, whether introduction events are frequent or not remains unknown, thus greatly limiting our ability to evaluate future risks.

By allowing indirect estimation of rodent origin and dispersal at the spatial and temporal scales that are relevant for surveillance and control, population genetics may help to address these issues. In this study, we describe the population genetic structures of R. rattus, R. norvegicus and M. musculus within the APC using species-specific sets of microsatellite markers, and we evaluate gene flows at a very fine spatial scale. Using longitudinal sampling, we investigate the effect of rodent control campaigns on the population genetic estimates from the three pest species. Finally, we rely on samples from foreign countries to mimic putative introduction events and examine whether our microsatellite panels allow us to identify newly disembarked individuals.

Human movements and migrations have played a leading role in the global spread of domestic rodents (Roberts 1991; Kovács 2012; Puckett et al. 2020). In particular, commercial exchanges (imports and/or exports) through maritime trade largely contribute to the long-distance transport and introduction of invasive rodent species in seaports around the world (Veale et al. 2021), from which they colonise entire continents. Among these species, R. rattus, R. norvegicus and M. musculus are currently spreading in Africa (Konečný et al. 2013; Dalecky et al. 2015; Berthier et al. 2016; Hima et al. 2019). Here, we investigated and compared for the first time the population genetic structures of these three species that coexist in an international seaport.

Rodent sampling in 9 different sites of the port first indicated spatial segregation of the species: R. rattus was found in much higher numbers in the artisanal seaport, where the two other species did not occur, or occur only on very rare occasions. Rattus norvegicus and M. musculus co-occurred in the industrial seaport and dominated largely the trapping results in the warehouses located along the unloading docks (APC4-7). The spatial segregation observed between these three species in APC could be due to habitat preferences, interspecific competition and/or historical factors that are beyond the scope of the present paper, but discussed in Badou et al. (submitted).

Population genetic studies using microsatellite markers of commensal rodents in urban contexts were often conducted at larger spatial scales than that considered in our study. For instance, the rare studies conducted on R. rattus all focused on West African cities with sampling covering several different districts (Niamey, Niger: Berthier et al. 2016; Franceville, Gabon: Mangombi et al. 2016; Cotonou, Benin: Badou et al. 2021). Urban R. norvegicus genetics was investigated in a few cities in North and South America (i.e., Baltimore, USA: Gardner-Santana et al. 2009; Salvador, Brazil: Kajdacsi et al. 2013; New York City, USA: Combs et al. 2018a; Salvador, Brazil; New Orleans, USA; Vancouver, Canada; and New York City, USA: Combs et al. 2018b). The only study on urban M. musculus we are aware of was conducted in Dakar, Senegal (Stragier et al. 2022), and again was conducted at the scale of the city. To our knowledge, the only genetic study available on rodent populations from a seaport area and its surroundings was conducted in Paranagua, Brazil on 71 R. norvegicus using 11 microsatellite loci (Gatto-Almeida et al. 2022). Differences in spatial scales and genetic markers used in all these studies make meaningful comparisons difficult. However, it is noteworthy that genetic diversity estimates observed for all three species within the APC were of the same order of magnitude as those obtained in other studies. This suggests similar introduction histories throughout the world (Gardner-Santana et al. 2009; Desvars-Larrive et al. 2019; Gatto-Almeida et al. 2022).

Our results suggest that new introductions of rodents are rare in the APC. Indeed, no outlier individual was detected by the FCA analysis in rodents sampled within the APC during our 3 year-long monitoring study. Also, allele numbers and allelic richness in R. rattus from Cotonou (a_r = 4.41; Badou et al. 2021) and in the APC (a_r = 4.62; this study) are quite similar, whereas one would expect these estimates to be higher in the seaport if individuals had been regularly (and, especially, recently) disembarked off moored ships and then persisted within the local black rat population. We were surprised by such patterns since the absence of signal reflecting new introduction events suggest that rats and mice do not descend frequently from the boats. Yet, considering the quasi-global distribution of these three species in the world, it is unlikely that this does not occur on a rather frequent basis. Moreover, we observed that anti-rat discs along mooring cables, though mandatory by IHR, were poorly and/or badly used by ships docking at APC: out of 13 vessels and 119 cables inspected, only 79 discs (69%) were indeed installed, many of which being badly installed; in total, no ship was perfectly protected with all the cables with well-laid discs (personal observations). Finally, the recent detection of Seoul virus in R. norvegicus captured in the APC in 2018 (Castel et al. 2021) suggested that the introduction of some migrant rats sometimes occurs. Those individuals could however have just transmitted their pathogens before dying, being unable to establish themselves and reproduce with resident rodents. Indeed, the non-detection of new introductions in genetic data may be explained by the presence of small mammal communities already on-site, which could render the settlement of newly immigrating individuals or species difficult. This hypothesis is supported by eco-ethological studies on commensal rodents, showing that introduced individuals may be rapidly identified as intruders by the residents, and subsequently rejected (e.g., in R. rattus: Ewer 1971; Granjon and Cheylan 1989; Barnett 1958; in R. norvegicus and M. musculus Berdoy and Drickamer 2007). If this is indeed the case, it is important to note that deratisation campaigns leading to the temporarily extirpation of the local rodent populations may open the gate to the successful settlement of newly introduced rodents (and associated pathogens). Although data are lacking to conclude definitely, it may be an additional argument in favour of a rigorous prevention strategy against rodent introduction through ships, rather than rodent control only.

Within APC, population genetic analyses showed very marked spatial patterns for the three species. This of course has important consequences in terms of rodent control since the genetic clusters observed should serve as a basis to guide deratization campaigns through the adequate definition of eradication units (Richardson et al. 2019). In R. norvegicus and M. musculus, two and three genetic clusters were observed within the industrial seaport, respectively. In R. rattus, two different groups were observed within the artisanal seaport, but only one within the industrial seaport. This pattern for the last species strongly contrasts with that observed using the same microsatellite markers at the whole Cotonou city scale, where only two genetic clusters associated with lower mean F_ST (0.107) were detected in black rats (Badou et al. 2021).

Genetic structure may reflect genetic isolation by distance, which is significant in all species. Indeed, invasive pests that occupy a broad range of urban conditions (primarily constrained by access to water, food/rubbish and nesting/burrowing sites) might be expected to exhibit spatial genetic patterns driven only by isolation by distance, thus reflecting spatial limitation of dispersal (Combs et al. 2018b). As such, the gradual pattern of differentiation observed between both sides of the industrial seaport in R. norvegicus (Fig. 2B) could suggest IBD at the scale of the port. IBD may also result from social behaviour of rodent species. Indeed, R. norvegicus (Combs et al. 2018a, b; Gardner-Santana et al. 2009) and M. musculus (Lippens et al. 2017) are known for strong social structure and very limited active dispersal. In M. musculus in particular, home ranges of a few tens of metres in commensal habitats (Pocock et al. 2005), and effective dispersal of only few hundred meters (Lippens et al. 2017) may explain the high F_ST values observed in this species (within the industrial seaport: mean F_ST = 0.09 for M. musculus; 0.06 for R. norvegicus; 0.02 for R. rattus). High genetic structure levels such as those observed within the APC in R. rattus (F_ST > 0.16) and M. musculus (F_ST > 0.13) between genetic groups, may also be explained by other factors. Indeed, they may reflect the presence of physical barriers to individual dispersal. Such barriers within the urban landscape have already been evidenced for Norway rats, such as major waterways in Baltimore (Gardner-Santana et al. 2009), Salvador (Kajdacsi et al. 2013) and New Orleans (Combs et al. 2018b), roadways in Salvador and Vancouver, or resource deserts in New York City (Combs et al. 2018b). Within the APC, however, we could not identify obvious physical barriers that could explain the observed marked genetic partitions. Finally, genetic structure may result from variations in effective population size, i.e. demographic barrier (Piry et al. 2004; Berthier et al. 2016; Richardson et al. 2017; Stragier et al. 2019). Such a demographic process may be especially relevant for commensal rodents whose natural dispersal distances (i.e. not human-mediated) are generally short ranged (< 1 km) and mainly prompted by the lack of feeding and harborage sites (Byers et al. 2019; Pocock et al. 2005). Control measures could also be another factor generating important gaps in densities and thus genetic discontinuities, such as for instance between the deratised industrial port (which may have been recolonized from the city) and the non-deratised artisanal port in R. rattus.

Some genetic signatures may suggest an effect of control measures on population structures. For instance, high LD values and significant tests for bottleneck signals were observed in both R. rattus and M. musculus. Increased linkage disequilibrium can indeed reflect bottleneck events (Slatkin 2008). However, similar results (i.e., LD disequilibrium and significant tests for bottleneck) were also observed for R. rattus in the non-deratized APC1 site, thus suggesting that other processes are at work. Also, high LD values may reflect the recent mixing of individuals from several subpopulations that have different allele frequencies (Slatkin 2008), which may also result from rodent control events. Rodent reinfestation after control operations may occur either by reproduction of non-poisoned or poison-resistant individuals, or by immigration of new individuals from neighbouring sites (Richardson et al. 2019).

Whatever the invasive rodent species considered here, our FCA analyses demonstrated that microsatellite markers makes it possible to detect recently introduced, hence expectedly genetically distant individuals. Indeed, for all three species, all individuals collected within the Autonomous Port of Cotonou over the three years of our study appeared much more similar to each other than to “allochthonous” ones. The only exception was APC and some Niamey black rat, which cluster together; but this supports the hypothesis that Niamey black rat originated from Benin seaport (Hima et al. 2019). These results thus highlight the interest of our panel of microsatellite loci as a valuable tool to trace back the geographic origin of house mice potentially introduced at APC. This should be useful to help seaport authorities to survey new rodent introductions in their facilities, thus allowing them to operationalize the IHR in terms of surveillance of maritime trade-associated rodent reservoirs introduction at entry points (World Health Organization, 2006). In the particular case of APC, this seems particularly realistic because of the recent implementation of a lab following an academic / seaport partnership initiative, and essentially dedicated to invasive terrestrial and marine species monitoring and management within Cotonou seaport (“Portuary Platform for Environmental Surveillance”; see https://​view.​genial.​ly/​6059bb3f64e78f0d​9cb32a7f).

With the high increasing rate of invasions (Pimentel et al. 2005; Seebens et al. 2017), there is a serious need for the development of methods for invasive species control (Brockerhoff et al. 2010; Glen et al. 2013; Thresher et al. 2014). Our trapping data suggested that populations of all three species rapidly recovered in terms of number of individuals (i.e., six and twelve months after control). In R. rattus, population genetic analyses integrating fifteen individuals from the core city showed that they grouped within the same genetic cluster than those from the industrial seaport. This pattern confirms the limited effect of the control measures deployed in the industrial seaport, at least on black rats which, otherwise, should result in higher genetic differentiation of the local populations (Gatto-Almeida et al. 2022). This adds to our previous results here above that poisoning campaigns alone (and as they stand) are insufficient to reach long-term decrease of invasive rodent abundances within the seaport area, and that accompanying measures are required. The latter could consist of environmental modifications towards less rodent-favoring habitats (e.g., rearrangement and physical protection of food stocks inside warehouses, rat-proofing of buildings, fueling of cracks and holes, setup of rat-proof grids along sewer networks, favoring of cats within the seaport, etc.). This would also reduce the dependency on toxicons such as anticoagulant rodenticides that may be dangerous for non-target species as well as to humans (Salas et al. 2000; Hendges et al. 2019; Kasiotis et al. 2021), and favor resistance evolution among target rodents (Hodroge et al. 2011; Buckle 2013). Such more suitable and long-term adapted measures have already been suggested in other settings (e.g., sewer networks: Channon et al. 2006; United States: Witmer and Shiels 2017; Brazilian slum: Hacker et al. 2016; Richardson et al. 2019). Unfortunately, we are not aware of such environmentally-based rodent management strategies in seaport habitats while their implementation in this type of close areas sounds quite feasible. Our study highlights the importance of monitoring of what descends from the ships to detect the eventual introduction of new invasive species (i.e. R. tanezumi; Bastos et al. 2011; Kosoy et al. 2015; Ringani et al. 2022) and the pathogens they could host. On the other hand, to monitor those that are carried on trucks to avoid propagations beyond the APC or even in the hinterland countries.