Iguanainsularis (Iguanidae) from the southern Lesser Antilles: An endemic lineage endangered by hybridization.

The newly described horned iguanaIguanainsularis from the southern Lesser Antilles is separated in two easily recognized subspecies: I.insularissanctaluciae from St. Lucia and I.insularisinsularis from the Grenadines. Its former description is completed by the use of 38 new samples for genetic and morphological analysis. Seventeen microsatellites were used to estimate genetic diversity, population structure and the level of introgression with other Iguana species over nearly the whole range of the species. ND4 and PAC sequences were also used to better characterize hybridization and to complete the description of this lineage. The I.insularis population of St. Vincent shows a high level of introgression from I.iguana whereas in the Grenadines, most islands present pure insularis populations but several show evidence of introgressions. Of the two remaining populations of I.insularissanctaluciae, only one is still purebred. The recent identification of this and other distinct insular species and subspecies in the eastern Caribbean, and evaluation of where hybridization has occurred, are timely and important because the native iguanas are in urgent need of conservation action. Among the greatest threats is the ongoing human-mediated spread of invasive iguanas from Central and South America, which are destroying the endemic insular lineages through multiple diachronic introgression events.

An inter-laboratory study of DNA-based identity, parentage and species testing in animal forensic genetics.

The probative value of animal forensic genetic evidence relies on laboratory accuracy and reliability. Inter-laboratory comparisons allow laboratories to evaluate their performance on specific tests and analyses and to continue to monitor their output. The International Society for Animal Genetics (ISAG) administered animal forensic comparison tests (AFCTs) in 2016 and 2018 to assess the limitations and capabilities of laboratories offering forensic identification, parentage and species determination services. The AFCTs revealed that analyses of low DNA template concentrations (≤300 pg/µL) constitute a significant challenge that has prevented many laboratories from reporting correct identification and parentage results. Moreover, a lack of familiarity with species testing protocols, interpretation guidelines and representative databases prevented over a quarter of the participating laboratories from submitting correct species determination results. Several laboratories showed improvement in their genotyping accuracy over time. However, the use of forensically validated standards, such as a standard forensic short tandem repeat (STR) kit, preferably with an allelic ladder, and stricter guidelines for STR typing, may have prevented some common issues from occurring, such as genotyping inaccuracies, missing data, elevated stutter products and loading errors. The AFCTs underscore the importance of conducting routine forensic comparison tests to allow laboratories to compare results from each other. Laboratories should keep improving their scientific and technical capabilities and continuously evaluate their personnel's proficiency in critical techniques such as low copy number (LCN) analysis and species testing. Although this is the first time that the ISAG has conducted comparison tests for forensic testing, findings from these AFCTs may serve as the foundation for continuous improvements of the overall quality of animal forensic genetic testing.

Inter-individual differences in foveal shape in a scavenging raptor, the black kite Milvus migrans.

Birds, and especially raptors, are believed to forage mainly using visual cues. Indeed, raptors (scavengers and predators) have the highest visual acuity known to date. However, scavengers and predators differ in their visual systems such as in their foveal configuration. While the function of the foveal shape remains unknown, individual variation has never been quantified in birds. In this study, we examined whether foveal shape differs among individuals in relation to eye size, sex, age, eye (left or right) and genetic proximity in a scavenging raptor, the black kite Milvus migrans. We assessed foveal shape in 47 individuals using spectral domain optical coherence tomography (OCT) and geometric morphometric analysis. We found that foveal depth was significantly related to eye size. While foveal width also increased with eye size, it was strongly related to age; younger individuals had a wider fovea with a more pronounced rim. We found no relationship between foveal shape and genetic proximity, suggesting that foveal shape is not a hereditary trait. Our study revealed that the shape of the fovea is directly linked to eye size and that the physical structure of the fovea may develop during the entire life of black kites.

Painted black: Iguana melanoderma (Reptilia, Squamata, Iguanidae) a new melanistic endemic species from Saba and Montserrat islands (Lesser Antilles).

The Lesser Antilles, in the Eastern Caribbean, is inhabited by three Iguana species: the Lesser Antillean iguanaIguana delicatissima, which is endemic to the northernmost islands of the Lesser Antilles, the introduced common iguana from South America, Iguana iguana iguana, represented also by the two newly described endemic subspecies Iguana iguana sanctaluciae from Saint Lucia and Iguana iguana insularis from Saint Vincent and the Grenadines, and Grenada, and the introduced Iguana rhinolopha from Central America. Drawing on both morphological and genetic data, this paper describes the Iguana populations from Saba and Montserrat as a new species, Iguana melanoderma. This species is recognized on the basis of the following combination of characteristics: private microsatellite alleles, unique mitochondrial ND4 haplotypes, a distinctive black spot between the eye and tympanum, a dorsal carpet pattern on juveniles and young adults, a darkening of body coloration with aging (except for the anterior part of the snout), a black dewlap, pink on the jowl, the high number of large tubercular nape scales, fewer than ten medium sized-triangular dewlap spikes, high dorsal spikes, and lack of horns on the snout. This new melanistic taxon is threatened by unsustainable harvesting (including for the pet trade) and both competition and hybridization from escaped or released invasive alien iguanas (I. iguana iguana and I. rhinolopha) from South and Central America, respectively. The authors call for action to conserve Iguana melanoderma in Saba and Montserrat and for further research to investigate its relationship to other melanistic iguanas from the Virgin Islands and coastal islands of Venezuela.

A story of nasal horns: two new subspecies of Iguana Laurenti, 1768 (Squamata, Iguanidae) in Saint Lucia, St Vincent amp; the Grenadines, and Grenada (southern Lesser Antilles).

The Lesser Antilles, in the Eastern Caribbean, were long considered to have only two species in the genus Iguana Laurenti 1768: the Lesser Antillean iguana Iguana delicatissima, which is endemic to parts of the Lesser Antilles, and the Common green iguana Iguana iguana, which also occurs throughout Central and South America. No subspecies are currently recognised. However, herpetologists and reptile collectors have pointed out strong physical differences between some of the island populations of Iguana iguana and those from the continent. Drawing on both morphological and genetic data, this paper describes two subspecies of the Common green iguana Iguana iguana from the southern Lesser Antilles, specifically the countries of Saint Lucia Iguana iguana sanctaluciae and Iguana iguana insularis from St Vincent the Grenadines, and Grenada. The form on the island of Saint Vincent has not been identified. The new subspecies are described based on the following unique combination of characters: Presence of high median and medium to small lateral horns on the snout; Small subtympanic plate not exceeding 20% of the eardrum size; Two or three scales of decreasing size anterior to the subtympanic plate; Fewer than ten small to medium triangular gular spikes; Medium sized dewlap; Low number of small to medium dispersed nuchal tubercles; Dark brown iris, with the white of the eye visible; Oval, prominent nostril; Short and relatively flat head; High dorsal spines; No swelling of the jowls in reproductively active males.                Iguana iguana sanctaluciae has in adults vertical black stripes on body and tail and a black dewlap whereas Iguana iguana insularis is pale grey or creamy white in adults.                Both subspecies are globally threatened by unsustainable hunting (including the pet trade) and by invasive alien species,     including hybridization from invasive iguanas from South and Central America (I. iguana iguana and I. rhinolopha, considered here as full species) that have become established in all three countries. The authors call for stronger measures to conserve the remaining purebred Iguana i. insularis and Iguana i. sanctaluciae ssp. nov. throughout their ranges and for further research to identify other cryptic species and subspecies of Iguana in the Lesser Antilles.

Strong genetic isolation of the black-lipped pearl oyster (Pinctada margaritifera) in the Marquesas archipelago (French Polynesia).

The French Polynesian islands are internationally known for their black pearls, produced by culture of the black lipped pearl oyster Pinctada margaritifera. The ongoing development of hatcheries for P. margaritifera in French Polynesia poses new challenges for the industry, particularly regarding the maintenance of genetic diversity in the hatchery stocks. This emphasizes the necessity to characterize the genetic diversity and differentiation within natural and exploited populations, to carefully select putative parental populations. The present study aimed at validating the phylogenetic status and investigating genetic attributes of populations from the only two non-exploited archipelagos of French Polynesia, the Marquesas archipelago, and the Australes archipelago, never analysed before. We found that individuals from both archipelagos belonged to P. margaritifera species. However, while the Australes population was genetically similar to non-exploited populations of the Tuamotu, the Marquesas populations were highly differentiated from the rest of the populations. This differentiation cannot not be only attributed to geographic distance and aquaculture status, but likely to hydrodynamic barriers allowing vicariant events to take place. Our results add up to other studies describing the Marquesas archipelago as a hotspot for biodiversity and differentiation, with some of the highest levels of endemism and vicariance found among marine species worldwide and provide precious information on available genetic resources for the implementation of P. margaritifera selective breeding and its genetic conservation in French Polynesia.

Experimental infection of Pacific oyster Crassostrea gigas spat by ostreid herpesvirus 1: demonstration of oyster spat susceptibility.

In 2008 and 2009, acute mortalities occurred in France among Pacific cupped oyster, Crassostrea gigas, spat. Different hypothesis including the implication of environmental factors, toxic algae and/or pathogens have been explored. Diagnostic tests indicated that OsHV-1 including a particular genotype, termed OsHV-1 µVar, was detected in most of samples and especially in moribund oysters with the highlighting of virus particles looking like herpes viruses by TEM examination. In this study, an experimental protocol to reproduce OsHV-1 infection in laboratory conditions was developed. This protocol was based on the intramuscular injection of filtered (0.22 µm) tissue homogenates prepared from naturally OsHV-1 infected spat collected on French coasts during mortality outbreaks in 2008. Results of the experimental trials showed that mortalities were induced after injection. Moreover, filtered tissue homogenates induced mortalities whereas the same tissue homogenates exposed to an ultraviolet (UV) treatment did not induce any mortality suggesting that oyster spat mortalities require the presence of a UV sensitive agent. Furthermore, analysis of injected oyster spat revealed the detection of high amounts of OsHV-1 DNA by real-time quantitative PCR. Finally, TEM analysis demonstrated the presence of herpes virus particles. The developed protocol allowed to maintain sources of infective virus which can be useful for the development of further studies concerning the transmission and the development of OsHV-1 infection.

Assessing the effects of FBC ash treatments of metal-contaminated soils using life history traits and metal bioaccumulation analysis of the earthworm Eisenia andrei.

Earthworms (Eisenia andrei) were exposed, in controlled conditions, to metal-contaminated soils previously treated in situ with two types of fluidized bed combustion ashes. Effects on this species were determined by life history traits analysis. Metal immobilizing efficiency of ashes was indicated by metal bioaccumulation. Ashes-treated soils reduced worm mortality compared to the untreated soil. However, these ashes reduced both cocoon hatching success and hatchlings numbers compared to the untreated soil. In addition, sulfo-calcical ashes reduced or delayed worm maturity and lowered cocoon production compared to silico-alumineous ones. Metal immobilizing efficiency of ashes was demonstrated for Zn, Cu and to a lesser extent Pb. Only silico-alumineous ashes reduced Cd bioaccumulation, although Cd was still bioconcentrated. Thus, although ash additions to metal-contaminated soils may help in immobilizing metals, their use might result, depending on the chemical nature of ashes, to severe detrimental effects on earthworm reproduction with possible long term consequences to populations.

Deciphering the immune function and regulation by a TLR of the cytokine EMAPII in the lesioned central nervous system using a leech model.

A highly conserved ortholog of the human complex p43/endothelial monocyte-activating polypeptide II (EMAPII) was characterized in the CNS of the leech Hirudo medicinalis. As observed in mammals, the leech complex is processed to release the cytokine HmEMAPII. Taking advantages of these similarities, we have attempted to elucidate the role of EMAPII in the CNS using the leech model. Although EMAPII is considered a modulator of inflammatory reactions within the peripheral innate immune response in humans, its function in CNS immunity has yet to be described. Chemotaxis assays were conducted, revealing the ability of EMAPII to exert a chemoattractant effect on both leech and human microglial cells, indicating a novel function of this cytokine in the human brain. Quantitative RT-PCR analysis together with in situ hybridization and immunohistochemistry approaches showed that bacterial challenge induced the expression of HmEMAPII at the lesion site where microglial cells accumulated. Moreover, gene silencing experiments have demonstrated that the gene expression of HmEMAPII is under the control of a signaling pathway associated with the TLR HmTLR1, newly characterized in the CNS of our model. To the best of our knowledge, this is the first report showing evidence for (1) the chemoattractant properties of EMAPII on leech and human microglial cells, (2) the regulation by a TLR of the expression of a gene encoding a cytokine in the CNS of an invertebrate, and (3) an immune function of a TLR in a lophotrochozoan model.

Microbial challenge promotes the regenerative process of the injured central nervous system of the medicinal leech by inducing the synthesis of antimicrobial peptides in neurons and microglia.

Following trauma, the CNS of the medicinal leech, unlike the mammalian CNS, has a strong capacity to regenerate neurites and synaptic connections that restore normal function. In this study, we show that this regenerative process is enhanced by a controlled bacterial infection, suggesting that induction of regeneration of normal CNS function may depend critically upon the coinitiation of an immune response. We explore the interaction between the activation of a neuroimmune response and the process of regeneration by assaying the potential roles of two newly characterized antimicrobial peptides. Our data provide evidence that microbial components differentially induce the transcription, by microglial cells, of both antimicrobial peptide genes, the products of which accumulate rapidly at sites in the CNS undergoing regeneration following axotomy. Using a preparation of leech CNS depleted of microglial cells, we also demonstrate the production of antimicrobial peptides by neurons. Interestingly, in addition to exerting antibacterial properties, both peptides act as promoters of the regenerative process of axotomized leech CNS. These data are the first to report the neuronal synthesis of antimicrobial peptides and their participation in the immune response and the regeneration of the CNS. Thus, the leech CNS appears as an excellent model for studying the implication of immune molecules in neural repair.

Hedistin: A novel antimicrobial peptide containing bromotryptophan constitutively expressed in the NK cells-like of the marine annelid, Nereis diversicolor.

A novel antimicrobial peptide, named hedistin was identified from the coelomocytes of Nereis diversicolor. Hedistin shows no obvious similarities with other known peptides and constitutes the first antimicrobial peptide containing bromotryptophans demonstrated in annelids. cDNA and mass spectrometry analysis revealed that, upon bacteria challenge, this peptide is secreted following processing of a precursor containing a signal peptide and prosequences. Hedistin was shown to possess an activity against a large spectrum of bacteria including the methicillin resistant Staphylococcus aureus and Vibrio alginolyticus. The gene was demonstrated to be constitutively and exclusively expressed in circulating NK cells like known to play an important role in the immunity of the sand worm. These data contrast with those observed in another annelid, the leech, in which genes coding for antimicrobial peptides are upregulated in a specific tissue and peptides are rapidly released into the hemolymph after septic injury.

Metallothionein response following cadmium exposure in the oligochaete Eisenia fetida.

We studied the metallothionein (MT) response in cadmium-exposed worms (Eisenia fetida) both at the protein level by Dot Immunobinding Assay (DIA) with a polyclonal antibody raised against the most immunogenic part of this protein and at the expression level by Northern blotting using a specific probe. MT appeared as two close isoforms. DIA results clearly demonstrated significant differences in MT level of whole worm heat-treated supernatants between E. fetida exposed to Cd concentrations as low as 8 mg Cd kg(-1) of dry soil compared to controls. Northern blotting analysis performed on whole bodies of worms revealed that a single exposure to 8 mg Cd kg(-1) of dry soil for 1 day resulted in the production of MT mRNA. This response was maintained for exposure of at least 1 month. Clear differences of MT gene expression were also observed between worms exposed to different Cd concentrations (8, 80 or 800 mg Cd kg(-1) of dry soil). Immunocytochemistry demonstrated that MT was located in the chloragogenous tissue surrounding the gut where metals are known to be accumulated. This work revealed that E. fetida MT is a sensitive and relevant biomarker of Cd exposure and especially when considering gene expression response. Further experiments have now to prove its usefulness in natural metal-contaminated soil toxicity assessments.