Gabriele Gentile

Attempts to molecularly distinguish cryptic taxa in Anopheles gambiae s.s.

Analyses of inversions in polytene chromosomes indicate that, in West Africa, Anopheles gambiae (sensu stricto) may be a complex of more than a single taxonomic unit, and these units have been called chromosomal forms. In order to determine whether this genetic discontinuity extends to the rest of the genome, as would be expected if reproductive isolation exists, we have sequenced several regions of both the nuclear and mitochondrial genomes. With one exception, we were unable to identify any nucleotide sites that differentiate the chromosomal forms. The exception was the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). Three sites in this region distinguish Mopti chromosomal form from Savanna and Bamako in Mali and Burkina Faso. However, outside these two countries, the association between chromosomal form and rDNA type does not always hold. Together with the variants in the rDNA intergenic spacer (IGS) described in the accompanying papers ( della Torre et al., 2001 ; Favia et al., 2001 ), we can recognize two major types of rDNA, Type I and Type II (corresponding to molecular forms S and M in della Torre et al., 2001 ). Type I is widespread in West Africa and is the only type found outside of West Africa (i.e. Tanzania and Madagascar). Type II is confined to West Africa. We were unable to detect any heterozygosity for the ITS types even in five collections containing both types. A sample from the island of São Tomé could not be classified into either Type I or Type II as the rDNA had characteristics of both. In general, our results confirm that An. gambiae is not a single pan‐mictic unit, but exactly how to define any new taxa remains problematic. Finally, we have found minor variants of the major rDNA types fixed in local populations; contrary to most previous studies, this suggests restricted gene flow among populations of this species.

PHYLOGEOGRAPHY AND HISTORY OF GIANT GALAPAGOS TORTOISES

Abstract.— We examined the phylogeography and history of giant Galàpagos tortoise populations based on mito‐chondrial DNA sequence data from 161 individuals from 21 sampling sites representing the 11 currently recognized extant taxa. Molecular clock and geological considerations indicate a founding of the monophyletic Galàpagos lineage around 2–3 million years ago, which would allow for all the diversification to have occurred on extant islands. Founding events generally occurred from geologically older to younger islands with some islands colonized more than once. Six of the 11 named taxa can be associated with monophyletic maternal lineages. One, Geochelone porteri on Santa Cruz Island, consists of two distinct populations connected by the deepest node in the archipelago‐wide phylogeny, whereas tortoises in northwest Santa Cruz are closely related to those on adjacent Pinzón Island. Volcan Wolf, the northernmost volcano of Isabela Island, consists of both a unique set of maternal lineages and recent migrants from other islands, indicating multiple colonizations possibly due to human transport or multiple colonization and partial elimination through competition. These genetic findings are consistent with the mixed morphology of tortoises on this volcano. No clear genetic differentiation between two taxa on the two southernmost volcanoes of Isabela was evident. Extinction of crucial populations by human activities confounds whether domed versus saddleback carapaces of different populations are mono‐ or polyphyletic. Our findings revealed a complex phylogeography and history for this tortoise radiation within an insular environment and have implications for efforts to conserve these endangered biological treasures.

Multiple origins of knockdown resistance mutations in the Afrotropical mosquito vector Anopheles gambiae.

How often insecticide resistance mutations arise in natural insect populations is a fundamental question for understanding the evolution of resistance and also for modeling its spread. Moreover, the development of resistance is regarded as a favored model to study the molecular evolution of adaptive traits. In the malaria vector Anopheles gambiae two point mutations (L1014F and L1014S) in the voltage-gated sodium channel gene, that confer knockdown resistance (kdr) to DDT and pyrethroid insecticides, have been described. In order to determine whether resistance alleles result from single or multiple mutation events, genotyping of the kdr locus and partial sequencing of the upstream intron-1 was performed on a total of 288 A. gambiae S-form collected from 28 localities in 15 countries. Knockdown resistance alleles were found to be widespread in West Africa with co-occurrence of both 1014S and 1014F in West-Central localities. Differences in intron-1 haplotype composition suggest that kdr alleles may have arisen from at least four independent mutation events. Neutrality tests provided evidence for a selective sweep acting on this genomic region, particularly in West Africa. The frequency and distribution of these kdr haplotypes varied geographically, being influenced by an interplay between different mutational occurrences, gene flow and local selection. This has important practical implications for the management and sustainability of malaria vector control programs.

Co-occurrence of East and West African kdr mutations suggests high levels of resistance to pyrethroid insecticides in Anopheles gambiae from Libreville, Gabon

Abstract.  Point mutations in the voltage‐gated sodium channel gene involved in knockdown resistance to DDT and pyrethroid insecticides have been described in several insect species. In the malaria vector Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) two mutations have been identified. The first, consisting of a leucine–phenylalanine substitution at amino acid position 1014, is widespread in West Africa. The second, a leucine–serine substitution at the same position, has to date only been detected in western Kenya. Analysis of the kdr polymorphism in a sample of 106 An. gambiae s.s. of the rDNA S‐form/Type I collected in Libreville (Gabon) surprisingly revealed the presence of both East and West African kdr mutations with frequencies of 63% and 37%, respectively. No wild‐type alleles were detected and there was an excess of heterozygous genotypes (P = 0.04). In addition, an inconsistency was found during the kdr genotyping procedures by polymerase chain reaction, which could have lead to an underestimation of resistance alleles. The implications of these findings are discussed.

Tracking acquired antibiotic resistance in commensal bacteria of Galápagos land iguanas: no man, no resistance.

Background Antibiotic resistance, evolving and spreading among bacterial pathogens, poses a serious threat to human health. Antibiotic use for clinical, veterinary and agricultural practices provides the major selective pressure for emergence and persistence of acquired resistance determinants. However, resistance has also been found in the absence of antibiotic exposure, such as in bacteria from wildlife, raising a question about the mechanisms of emergence and persistence of resistant strains under similar conditions, and the implications for resistance control strategies. Since previous studies yielded some contrasting results, possibly due to differences in the ecological landscapes of the studied wildlife, we further investigated this issue in wildlife from a remote setting of the Galapagos archipelago. Methodology/Principal Findings Screening for acquired antibiotic resistance was carried out in commensal enterobacteria from Conolophus pallidus, the terrestrial iguana of Isla Santa Fe, where: i) the abiotic conditions ensure to microbes good survival possibilities in the environment; ii) the animal density and their habits favour microbial circulation between individuals; and iii) there is no history of antibiotic exposure and the impact of humans and introduced animal species is minimal except for restricted areas. Results revealed that acquired antibiotic resistance traits were exceedingly rare among bacteria, occurring only as non-dominant strains from an area of minor human impact. Conclusions/Significance Where both the exposure to antibiotics and the anthropic pressure are minimal, acquired antibiotic resistance traits are not normally found in bacteria from wildlife, even if the ecological landscape is highly favourable to bacterial circulation among animals. Monitoring antibiotic resistance in wildlife from remote areas could also be a useful tool to evaluate the impact of anthropic pressure.

Population genetics of Galápagos land iguana (genus Conolophus) remnant populations

The Galápagos land iguanas (genus Conolophus) have faced significant anthropogenic disturbances since the 17th century, leading to severe reduction of some populations and the extinction of others. Conservation activities, including the repatriation of captive‐bred animals to depleted areas, have been ongoing since the late 1970s, but genetic information has not been extensively incorporated. Here we use nine species‐specific microsatellite loci of 703 land iguanas from the six islands where the species occur today to characterize the genetic diversity within, and the levels of genetic differentiation among, current populations as well as test previous hypotheses about accidental translocations associated with early conservation efforts. Our analyses indicate that (i) five populations of iguanas represent distinct conservation units (one of them being the recently discovered rosada form) and could warrant species status, (ii) some individuals from North Seymour previously assumed to be from the natural Baltra population appear related to both Isabela and Santa Cruz populations, and (iii) the five different management units exhibit considerably different levels of intrapopulation genetic diversity, with the Plaza Sur and Santa Fe populations particularly low. Although the initial captive breeding programmes, coupled with intensive efforts to eradicate introduced species, saved several land iguana populations from extinction, our molecular results provide objective data for improving continuing in situ species survival plans and population management for this spectacular and emblematic reptile.

Variation in an intron sequence of the voltage-gated sodium channel gene correlates with genetic differentiation between Anopheles gambiae s.s. molecular forms

We present the results of a geographical survey of genetic variation in Anopheles gambiae M and S molecular forms from ten African countries at Intron I of the voltage‐gated sodium channel gene. We found two major haplotypes separated by a single mutational step, which cosegregate almost completely with the rDNA sites that identify M and S, consistent with previous estimates of strong reductions of gene flow between the two forms. We also report ten additional haplotypes stemming from the two major haplotypes, mostly present in single localities. The low levels of genetic variation found in this intronic region are discussed in light of a possible selective sweep. These findings offer additional elements to the ongoing debate on the amount of genetic differentiation and isolation between the two molecular forms and on their taxonomic status.

Temporal and Spatial Covariation of Gender and Oxidative Stress in the Galápagos Land Iguana Conolophus subcristatus

Physiological responses to organismal stress can have direct impacts on individual fitness. While responses to stressors mediated by glucocorticoid hormones are well studied, the regulation of the redox system via pro‐oxidant and antioxidant balance as well as the natural causes of oxidative stress in nature remain poorly known, especially for reptiles. In this study, we investigate the interpopulation and intersex variation in oxidative damage and plasma antioxidant capacity in the Galápagos land iguana, Conolophus subcristatus, over a 3‐yr study to evaluate what factors (e.g., season, food availability, reproductive activity) can explain levels and patterns of oxidative damage and of plasma antioxidant capacity. Our results indicate that (1) males showed lower levels of oxidative damage, higher levels of plasma antioxidant capacity, and better body condition than females and (2) significant interactions exist among patterns of oxidative damage across sexes, sampling localities, body condition, and season. These results suggest that reproductive activity and food abundance might act as determinants shaping levels and patterns of oxidative stress of land iguanas.

An overlooked pink species of land iguana in the Galápagos

Despite the attention given to them, the Galápagos have not yet finished offering evolutionary novelties. When Darwin visited the Galápagos, he observed both marine (Amblyrhynchus) and land (Conolophus) iguanas but did not encounter a rare pink black-striped land iguana (herein referred to as “rosada,” meaning “pink” in Spanish), which, surprisingly, remained unseen until 1986. Here, we show that substantial genetic isolation exists between the rosada and syntopic yellow forms and that the rosada is basal to extant taxonomically recognized Galápagos land iguanas. The rosada, whose present distribution is a conundrum, is a relict lineage whose origin dates back to a period when at least some of the present-day islands had not yet formed. So far, this species is the only evidence of ancient diversification along the Galápagos land iguana lineage and documents one of the oldest events of divergence ever recorded in the Galápagos. Conservation efforts are needed to prevent this form, identified by us as a good species, from extinction.

Characterization of Salmonella Occurring at High Prevalence in a Population of the Land Iguana Conolophus subcristatus in Galápagos Islands, Ecuador

The aim of the study was to elucidate the association between the zoonotic pathogen Salmonella and a population of land iguana, Colonophus subcristatus, endemic to Galápagos Islands in Ecuador. We assessed the presence of Salmonella subspecies and serovars and estimated the prevalence of the pathogen in that population. Additionally, we investigated the genetic relatedness among isolates and serovars utilising pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA and determined the antimicrobial susceptibility to a panel of antimicrobials. The study was carried out by sampling cloacal swabs from animals (n = 63) in their natural environment on in the island of Santa Cruz. A high prevalence (62/63, 98.4%) was observed with heterogeneity of Salmonella subspecies and serovars, all known to be associated with reptiles and with reptile-associated salomonellosis in humans. Serotyping revealed 14 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 48), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 1), and S. enterica subsp. houtenae (n = 7). Four serovars were predominant: S. Poona (n = 18), S. Pomona (n = 10), S. Abaetetuba (n = 8), and S.Newport (n = 5). The S. Poona isolates revealed nine unique XbaI PFGE patterns, with 15 isolates showing a similarity of 70%. Nine S. Pomona isolates had a similarity of 84%. One main cluster with seven (88%) indistinguishable isolates of S. Abaetetuba was observed. All the Salmonella isolates were pan-susceptible to antimicrobials representative of the most relevant therapeutic classes. The high prevalence and absence of clinical signs suggest a natural interaction of the different Salmonella serovars with the host species. The interaction may have been established before any possible exposure of the iguanas and the biocenosis to direct or indirect environmental factors influenced by the use of antimicrobials in agriculture, in human medicine or in veterinary medicine.