A molecular tool described here allows in one step for specific discrimination among three cryptic freshwater snail species (genus Galba) involved in fasciolosis transmission, a worldwide infectious disease of humans and livestock. The multiplex PCR approach taken targets for each species a distinctive, known microsatellite locus which is amplified using specific primers designed to generate an amplicon of a distinctive size that can be readily separated from the amplicons of the other two species on an agarose gel. In this way, the three Galba species (G. cubensis, G. schirazensis, and G. truncatula) can be differentiated from one another, including even if DNA from all three were present in the same reaction. The accuracy of this new molecular tool was tested and validated by comparing multiplex PCR results with species identification based on sequences at mitochondrial and nuclear markers. This new method is accurate, inexpensive, simple, rapid, and can be adapted to handle large sample sizes. It will be helpful for monitoring invasion of Galba species and for developing strategies to limit the snail species involved in the emergence or re-emergence of fasciolosis.

Cryptic species can present a significant challenge to the application of systematic and biogeographic principles, especially if they are invasive or transmit parasites or pathogens. Detecting cryptic species requires a pluralistic approach in which molecular markers facilitate the detection of coherent taxonomic units that can then be analyzed using various traits (e.g., internal morphology) and crosses. In asexual or self-fertilizing species, the latter criteria are of limited use. We studied a group of cryptic freshwater snails (genus Galba) from the family Lymnaeidae that have invaded almost all continents, reproducing mainly by self-fertilization and transmitting liver flukes to humans and livestock. We aim to clarify the systematics, distribution, and phylogeny of these species with an integrative approach that includes morphology, molecular markers, wide-scale sampling across America, and data retrieved from GenBank (to include Old World samples). Our phylogenetic analysis suggests that the genus Galba originated ca. 22 Myr ago and today comprises six species or species complexes. Four of them show an elongated-shell cryptic phenotype and exhibit wide variation in their genetic diversity, geographic distribution, and invasiveness. The remaining two species have more geographically restricted distributions and exhibit a globose-shell cryptic phenotype, most likely phylogenetically derived from the elongated one. We emphasize that no Galba species should be identified without molecular markers. We also discuss several hypotheses that can explain the origin of cryptic species in Galba, such as convergence and morphological stasis.

Abstract. Chagas disease is a parasitic disease caused by the protozoan parasite Trypanosoma cruzi and about 230,000 persons are estimated to be infected in Ecuador. However, limited studies have been performed in the Amazon region, on the eastern side of the country. We evaluated here the seroprevalence of Trypanosoma cruzi infection in 12 rural villages of the Loreto canton, Orellana Province in schoolchildren aged 5-15 years and in pregnant women. A total of 1,649 blood samples were tested for Trypanosoma cruzi antibodies by enzyme-linked immunosorbent assay and indirect hemaglutination, and discordant samples were tested by indirect immunofluorescence assay. We detected a seroprevalence of anti-Trypanosoma cruzi antibodies of 1.3% in schoolchildren aged 5-15 years, indicating the persistence of a constant and active vectorial transmission in the Loreto County and confirming the need of the implementation of nonconventional vector control. We also observed a seroprevalence of 3.8% in pregnant women, indicating a clear risk of congenital transmission. Further studies should help define this risk more precisely and implement current international guidelines for the diagnosis, treatment, and care of these cases.

Ecuador was an early COVID-19 hotspot with substantial COVID-19-mortality. In developed countries, low socioeconomic status is associated with COVID-19 infection and low compliance with non-pharmaceutical interventions (NPIs). However, if NPI were successful in resource-limited settings with high human mobility and informal labour is still unclear. We performed a retrospective observational molecular and serological study of Ecuador’s reference laboratory. We tested 1,950 respiratory samples from COVID-19 surveillance for SARS-CoV-2 and 12 respiratory viruses using RT-PCR, characterized 642 SARS-CoV-2 genomes, and examined SARS-CoV-2 seroprevalence in 1,967 samples from patients with fever in Ecuador’s reference laboratory during 2020-2021. Molecular and serological data were compared to NPI stringency in Bayesian, maximum-likelihood and modelling frameworks.SARS-CoV-2 (Pearson correlation test; r=-0.74; p=0.01) and other respiratory viruses (r=-0.68; p=0.02) detection correlated negatively with NPI stringency. SARS-CoV-2 seroprevalence increased from <1% during February-March 2020 to 50% within 6 weeks and plateaued after NPI implementation. Decrease of effective reproduction number <1 and antibody reactivity over time suggested intense SARS-CoV-2 transmission during pandemic onset, subsequently limited by NPIs. Phylogeographic analyses revealed that travel restrictions were implemented late not preventing 100 near-parallel SARS-CoV-2 introductions, and implementation of NPIs modified SARS-CoV-2 geographic spread by restricting recreational activity. NPIs stringency correlated negatively with the number of circulating SARS-CoV-2 lineages (r=-0.69; p=0.02). Virological evidence supports NPIs restricting human movement as an effective public health tool to control the spread of respiratory pathogens in resource-limited settings, providing a template for emerging SARS-CoV-2 variants and future epidemics.

Cryptic species can present a significant challenge to the application of systematic and biogeographic principles, especially if they are invasive or transmit parasites or pathogens. Detecting cryptic species requires a pluralistic approach in which molecular markers facilitate the detection of coherent taxonomic units that can then be analyzed using various traits (e.g., internal morphology) and crosses. In asexual or self-fertilizing species, the latter criteria are of limited use. We studied a group of cryptic freshwater snails (genus Galba) from the family Lymnaeidae that have invaded almost all continents, reproducing mainly by self-fertilization and transmitting liver flukes to humans and livestock. We aim to clarify the systematics, distribution and phylogenetic relationships of these species with an integrative approach that includes morphology (shell and reproductive anatomy), molecular markers, wide-scale sampling across America, and data retrieved from GenBank (to include Old World samples). Our phylogenetic analysis suggests that the genus Galba originated ca. 22 Myr ago and today comprises six clusters of species. Four of these clusters (G. truncatula, G. cubensis/viator, G. humilis and G. schirazensis) are morphologically cryptic and constitute species or species complexes with wide variation in their genetic diversity, geographic distribution and invasiveness. The other two clusters constitute a single species or a species complex (Galba cousini/meridensis) that demonstrate more geographically restricted distributions and exhibit an alternative morphology more phylogenetically derived than the cryptic one. Further genetic studies are required to clarify the status of both G. cousini/meridensis and G. cubensis/viator. We emphasize that no Galba species should be identified without molecular markers and that additional sampling is required, especially in North America, Eurasia and Africa to clarify remaining questions in systematics and biogeography. We also discuss several hypotheses that can explain crypsis in Galba, such as convergence and morphological stasis, and hypothesize a role for stabilizing selection in amphibious (rather than strictly freshwater) habitats.