Rahma Attia

El Hili

Mohamed Sghaier Achouri

Olivier Verneau
email: verneau@univ-perp.fr

Cytochrome c oxydase I phylogenetic analysis of Haemogregarina parasites (Apicomplexa, Coccidia, Eucoccidiorida, Haemogregarinidae) confirms the presence of three distinct species within the freshwater turtles of Tunisia

Keywords:

Species of Haemogregarina are apicomplexan blood parasites that use vertebrates as intermediate hosts. Due to limited interspecific morphological characters within the genus during the last decade, 18S rRNA gene sequences were widely used for species identification.

As coinfection patterns were recently reported from nuclear molecular data for two sympatric freshwater turtles Mauremys leprosa and Emys orbicularis from Tunisia, our objectives were to design COI specific primers to confirm the presence of three distinct species in both host species. Blood samples were collected from 22 turtles, from which DNAs were extracted and used as templates for amplification. Following different rounds of PCR and nested PCR, we designed specific Haemogregarina COI primers that allowed the sequencing of nine distinct haplotypes. Phylogenetic Bayesian analysis revealed the occurrence of three welldifferentiated sublineages that clustered together into a single clade. Based on pairwise genetic distances (p-distance), we confirmed the occurrence of three distinct but phylogenetically closely related species coinfecting M. leprosa and E. orbicularis in the same aquatic environments. Our results demonstrate that the use of fast evolving genes within Haemogregarina will help to investigate the parasite diversity within both intermediate vertebrate and definitive invertebrate hosts, and to assess the evolution, historical biogeography and specificity of haemogregarines.

Introduction

The phylum Apicomplexa is a major group of protistan parasites that infect a wide variety of animals. While human pathogens, such as Toxoplasma gondii, Babesia spp., Cryptosporidium spp. and Plasmodium spp., have been extensively studied for their biological and genetic characteristics [START_REF] Janouškovec | Apicomplexan-like parasites are polyphyletic and widely but selectively dependent on cryptic plastid organelles[END_REF], the majority of apicomplexan parasites without medical importance are still poorly studied [START_REF] Karadjian | Highly rearranged mitochondrial genome in Nycteria parasites (Haemosporidia) from bats[END_REF], yet they play a critical role in ecosystem functioning [START_REF] Vargas | Eukaryotic plankton diversity in the sunlit ocean[END_REF][START_REF] Mahé | Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests[END_REF]. The identification and the description for all of these parasites, irrespective their degree of pathogenicity, remains a crucial step to encourage further studies in basic and applied research.

It is well known that morphological approaches alone can in some cases be misleading or unreliable in species identification due to the existence of cryptic taxa, differences in life stages or gender, genetic variability and/or phenotypic plasticity [START_REF] Hebert | Biological identification through DNA barcodes[END_REF][START_REF] Bourlat | Genomics in marine monitoring: New opportunities for assessing marine health status[END_REF][START_REF] Aylagas | Environmental status assessment using DNA metabarcoding: Towards a genetics based marine biotic index (gAMBI)[END_REF]. The DNA barcoding initiative that emerged following the development of molecular approaches in the 1990s now represents a central approach to circumvent taxonomic uncertainties [START_REF] Hebert | Biological identification through DNA barcodes[END_REF]. Today, the cytochrome c oxidase I (COI) is still the most widely used marker for molecular identification at species level within taxa as diverse as arthropods, fish and birds [START_REF] Hebert | Identification of birds through DNA Barcodes[END_REF][START_REF] Hogg | Biological identification of springtails (Hexapoda: Collembola) from the Canadian Arctic, using mitochondrial DNA barcodes[END_REF][START_REF] Hebert | Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator[END_REF][START_REF] Ward | DNA barcoding Australia's fish species[END_REF][START_REF] Costa | The barcode of life initiative: synopsis and prospective societal impacts of DNA barcoding of fish[END_REF]. Its maternal inheritance, limited recombination, robustness of mtDNA against degradation and rapid evolution make it the ideal marker for resolving recent phylogenetic events [START_REF] Avise | Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics[END_REF]. In contrast, the slow evolving nuclear 18S rRNA gene is one of the most valuable markers for resolving deep phylogenetic relationships [START_REF] Collins | Evaluating multiple alternative hypotheses for the origin of Bilateria: An analysis of 18S rRNA molecular evidence[END_REF][START_REF] Carranza | Are the platyhelminthes a monophyletic primitive group? An assessment using 18S rDNA sequences[END_REF][START_REF] Soltis | The phylogeny of land plants inferred from 18S rDNA sequences: pushing the limits of rDNA signal?[END_REF][START_REF] Peterson | Animal phylogeny and the ancestry of bilaterians: inferences from morphology and 18S rDNA gene sequences[END_REF][START_REF] Tsagkogeorga | An updated 18S rRNA phylogeny of tunicates based on mixture and secondary structure models[END_REF][START_REF] Meyer | Fast evolving 18S rRNA sequences from Solenogastres (Mollusca) resist standard PCR amplification and give new insights into mollusk substitution rate heterogeneity[END_REF].

Within the phylum of Apicomplexa, the most widely used mitochondrial markers for taxonomy, phylogeny and molecular idenification involved COI [START_REF] Ogedengbe | DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Apicomplexa, Alveolata)[END_REF][START_REF] Gjerde | Phylogenetic relationships among Sarcocystis species in cervids, cattle and sheep inferred from the mitochondrial cytochrome c oxidase subunit I gene[END_REF][START_REF] Rubiola | Molecular identification of Sarcocystis spp. in cattle: partial sequencing of cytochrome C oxidase subunit 1 (COI)[END_REF] and cytochrome b [START_REF] Karadjian | Highly rearranged mitochondrial genome in Nycteria parasites (Haemosporidia) from bats[END_REF][START_REF] Perkins | A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences[END_REF][START_REF] Hellgren | Diversity and phylogeny of mitochondrial cytochrome B lineages from six morphospecies of avian Haemoproteus (haemosporida: haemoproteidae)[END_REF] sequences. Ogedengbe et al. [START_REF] Ogedengbe | DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Apicomplexa, Alveolata)[END_REF] revealed that partial COI sequences provided many more variable characters than longer 18S rRNA gene sequences to distinguish closely related coccidian parasite species. On 07 January 2021, 308,627 COI sequences for vertebrates were publically available from the National Center for Biotechnology Information (NCBI) compared to 10,307 18S sequences for the same group of species (keywords used: Vertebrata and COI versus Vertebrata and 18S). Conversely, only 1,303 COI sequences were found within the Apicomplexa from the NCBI compared to 20,903 18S sequences within the same phylum (keywords used: Apicomplexa and COI versus Apicomplexa and 18S), which showed that the 18S rRNA gene was still the most investigated molecular marker for phylogenetic studies of apicomplexan parasites. Such differences in the proportion of COI versus 18S sequences in these groups of taxa (5,8% of COI sequences for the Apicomplexa compared to 96,7% for the Vertebrata) could be partially explained by the difficulties associated with the design of universal and specific amplification primers in the COI for groups that are still poorly studied. Until now, there have only been few PCR primer sets used to amplify COI sequences within apicomplexan parasites. The universal primers LCO1490F and HCO2198R, which were proven to amplify a 658bp fragment of the COI gene in a wide range of invertebrates [START_REF] Folmer | DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates[END_REF][START_REF] Hanner | Data standards for BARCODE records in INSDC (BRIs)[END_REF], were inadequate in the amplification process of the COI locus from protists and more specifically from apicomplexan parasites [START_REF] Ogedengbe | DNA barcoding of Apicomplexa: Mitochondrial evolution across the phylum[END_REF]. On the other hand, the high number of 18S copies and conserved terminal regions in the 18S rRNA locus made it easier to design such primers [START_REF] Morrison | Effects of nucleotide sequence alignment study on phylogeny estimation : A case study of 18S rDNA of Apicomplexa[END_REF][START_REF] Mugridge | Effects of sequence alignment and structural domains of ribosomal DNA on phylogeny reconstruction for the Protozoan family Sarcocystidae[END_REF][START_REF] Ouvrard | 18S rRNA secondary structure and phylogenetic position of Peloridiidae (Insecta, Hemiptera)[END_REF].

Haemogregarina is a genus which infects vertebrates as fish and freshwater turtles. It is classified in the family Haemogregarinidae of the order Eucoccidiorida. Until the 1990s, identification and recognition of Haemogregarina spp. were primarily based on morphological features. Due to limited interspecific morphological differences, traditional approaches were subsequently combined with molecular methods using 18S rRNA gene sequences for species identification [START_REF] Dvořáková | Haemogregarines of freshwater turtles from Southeast Asia with a description of Haemogregarina sacaliae sp. n. and a redescription of Haemogregarina pellegrini Laveran and Pettit[END_REF]. Genetic diversity of blood parasites within the sympatric freshwater turtles Mauremys leprosa and Emys orbicularis of Tunisia, based on 18S sequences, revealed the occurrence of three distinct Haemogregarina variants including H. stepanowi [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF]. As coinfection patterns were clearly reported from molecular data for both turtle species, morphological identification of these variants was challenging, if not impossible [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF]. Thus, our objectives in the present study were to design COI specific primers to investigate the COI Haemogregarina variations from blood samples in both turtle species and to then validate the presence of three distinct species of Haemogregarina.

Material and methods

Biological samples

Two freshwater turtle species, namely M. leprosa, the Mediterranean pond turtle, and E. orbicularis, the European pond turtle, were trapped using hoop nets baited with fish from three localities in Northern Tunisia (see Table 1 for GPS coordinates and also ref [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF]).

Mauremys leprosa was detected at all sampling sites corresponding to El Amaria, El Hania and Large Hammam Bourguiba pond, while E. orbicularis was found only at El Hania.

Leeches were present on their vertebrate hosts in all localities. Blood samples were collected from the dorsal coccygeal sinus at the base of the turtle tail and preserved at -80°C in 98% ethanol until DNA extraction. A drop from each blood sample was also smeared on a glass slide, air-dried, fixed in absolute methanol for 5 min and stained with 10% Giemsa for 20 min in the laboratory for the presence of hemoparasites. Results from microscopic observations were reported in [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF].

Primer design, DNA extractions, amplifications and sequencing

The first step involved the design of degenerate primers from an alignment comprising ten COI sequences of apicomplexan parasite species followed by the evaluation of their efficiency in amplifying Haemogregarina COI. These sequences, which were extracted from GenBank (see Table 2), corresponded to species belonging to Eucoccidiorida, Haemosporida and Piroplasmida orders. They were aligned using Clustal [START_REF] Thompson | improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF] implemented in MEGA X software [START_REF] Kumar | MEGA X : Molecular evolutionary genetics analysis across computing platforms[END_REF]. The most conserved regions in the 5' and 3' ends were selected to design three forward (HemFor1, HemFor2 and HemFor3) and three reverse (HemRev1, HemRev2 and HemRev3) degenerated primers (Table 3). DNA was extracted from 22 blood samples of M. leprosa and E. orbicularis with the E.Z.N.A Tissue DNA Kit (Omega bio-tek, USA), according to manufacturer recommendations. Six DNA samples, four from M. leprosa (Ml36AM, Ml34AM, Ml44HS and Ml10HB) and two from E. orbicularis (Eo18HS and Eo19HS) were selected to test the PCR efficiency of the newly designed degenerated primers used two by two. These DNA samples were also studied earlier in [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF] to investigate the genetic diversity of Haemogregarina based on 18S rRNA gene sequences. All PCRs were performed in a final volume of 25 µL under the following conditions: 5 min at 95°C for initial denaturation; 35 cycles of 30 sec each at 94°C for denaturation, 30 sec at 48°C for annealing, 1 min at 72°C for elongation; 10 min at 72°C for final extension. PCR products were run on a 1% agarose gel and stained with ethidium bromide for visualization. Since all of the nine primer combinations failed to amplify the expected PCR fragments (Figure 1a), nested PCRs were subsequently performed from PCR products resulting from the amplification of Ml30AM and Eo19HS DNA samples with primers HemoFor1 and HemoRev1. These two new templates allowed for four combinations of inner primers to be tested, namely HemoFor2/HemoRev2, HemoFor2/HemoRev3, HemoFor3/HemoRev2 and HemoFor3/HemoRev3. Nested PCRs were conducted in a final volume of 25 µL following the same thermal conditions as described above. Only the HemoFor2/HemoRev2 primer combination allowed for the amplification of a PCR product of the expected size from the preamplified Ml30AM DNA sample (results not shown).

Following sequencing by the Genoscreen Company (Lille, France), the chromatogram was imported and checked with Chromas 2.2.6 (Technelysium Pty Ltd). Blast search of this fragment at 436bp revealed about 70% identity with COI sequences of apicomplexan parasites. As the occurrence of Haemogregarina parasites was evidenced from the same blood sample using morphological and molecular (18S rRNA gene sequences) approaches [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF], the COI sequence we obtained was assigned to a species of Haemogregarina.

The resultant COI sequence was aligned with the ten selected apicomplexan COI sequences (see above) and used to develop six new specific PCR primers in the most conserved regions of the 5' and 3' ends. Two forward primers, namely HemoFor4 and HemoFor5, and four reverse primers, namely HemoRev4, HemoRev5, HemoRev6 and HemoRev7, were designed (Table 3). Subsequently all primers used two by two were tested on the six DNA samples that were used during the first step of amplification. Following the same thermal PCR conditions, with the exception of the annealing temperature that was fixed to 60°C, only four combinations of primers (HemoFor4/HemoRev4, HemoFor5/HemoRev4, HemoFor5/HemoRev6 and HemoFor5/HemoRev7) gave PCR fragments of the expected size with low PCR background for all six DNA samples (Figure 1b). These primers were thus used two by two to amplify the 22 DNA samples extracted from blood of M. leprosa and E. orbicularis. When the amplification step was successful with a strong PCR signal, PCR products were sent to the Genoscreen Company (Lille, France) for sequencing.

Chromatograms were then imported and edited with Chromas 2.2.6 (Technelysium Pty Ltd) and resulting sequences were aligned using Clustal W implemented in MEGA X software.

Phylogenetic and distance analyses

Fifty-three COI sequences corresponding to species of distinct genera spanning Eucoccidiorida, Haemosporida and Piroplasmida orders were extracted from GenBank (Table 2) and aligned with the COI haplotypes confirmed within this study with Clustal W implemented in MEGA X. Another COI sequence corresponding to Scrippsiella sweeneyae (Dinoflagellata) was also revovered from GenBank for outgroup comparison. A JC + G model was selected following the Akaike Information Criterion (AIC) implemented in Modeltest 3.06 [START_REF] Posada | Modeltest: testing the model of DNA substitution[END_REF]. A single substitution model and gamma rates with four categories were applied to the COI partition that comprised 418 characters. The Bayesian analysis was run using MrBayes 3.04b [START_REF] Huelsenbeck | MRBAYES: Bayesian inference of phylogenetic trees[END_REF], with four chains running for five million generations and sampled every 100 cycles. The Bayesian consensus tree was drawn after removing the first 5,000 trees (10%) as the burn-in phase and opened under TreeView version 1.6 [START_REF] Page | Tree View: An application to display phylogenetic trees on personal computers[END_REF]. Pairwise distances (pdistance) were also estimated both within two closely related species of Eimeria (E.

lancasterensis and E. sciurorum) as well as between these two species, to assess levels of genetic divergences for species delimitation within the Eucoccidiorida.

Results

Following amplification of the 22 DNA samples using the four specific primer combinations, 21 were positive using HemoFor4/HemoRev4 primer set of which 11 were sequenced, 21 were positive using the HemoFor5/HemoRev4 primer set of which 14 were sequenced, 22 were positive using the HemoFor5/HemoRev6 primer set of which 11 were sequenced and 21 were positive using the HemoFor5/HemoRev7 primer set of which 18 were sequenced.

Between one and three distinct haplotypes were identified per blood sample depending on the combination of primers used for PCR, indicating that several genetic variants may occur in the same turtle specimen. A total of nine distinct COI haplotypes were identified (Table 4).

The Bayesian analysis of the COI sequences resulted in a tree which grouped all of the COI Hamogregarina haplotypes together in a single clade, which can be subdivided into three distinct sublineages: Haemogregarina sp_Hap1 includes haplotypes 1a and 1b, Haemogregarina sp_Hap2 includes a single haplotype 2a and Haemogregarina sp_Hap3 includes haplotypes 3a to 3f. Isolates of the species of Haemogregarina were most closely related to Hepatozoon as compared to any of the other apicomplexans (Figure 2). While pairwise distances within both Haemogregarina sublineages Hap1 and Hap 3 were less than 0,01 on average (Table 5), they were higher between the three distinct sublineages (Table 6).

Pairwise distances were 0.077 between Haemogregarina sp_Hap1 and Haemogregarina sp_Hap2, ranged from 0,066 to 0,074 with an average of 0,069 ± 0,003 between Haemogregarina sp_Hap1 and Haemogregarina sp_Hap3 and ranged from 0,086 to 0,094 with an average of 0,089 ± 0,003 between Haemogregarina sp_Hap2 and Haemogregarina sp_Hap3 (Table 6). For comparison, intra specific pairwise distances within Eimeria lancasterensis and E. sciurorum were less than 0,006 on average (Table 5), and interspecific pairwise distances between these two species ranged from 0,014 to 0,029 with an average of 0,021 ± 0,005 (Table 6).

Discussion

Although the mitochondrial COI is the most widely used marker for DNA barcoding across most animal taxa [START_REF] Hebert | Identification of birds through DNA Barcodes[END_REF][START_REF] Hogg | Biological identification of springtails (Hexapoda: Collembola) from the Canadian Arctic, using mitochondrial DNA barcodes[END_REF][START_REF] Hebert | Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator[END_REF][START_REF] Ward | DNA barcoding Australia's fish species[END_REF][START_REF] Costa | The barcode of life initiative: synopsis and prospective societal impacts of DNA barcoding of fish[END_REF], the use of COI sequences as DNA barcodes within Protozoa is still in its initial stage. While the usefulness of this genetic marker for differentiation of cryptic species has already been proven successful for several apicomplexans [START_REF] Gjerde | Sarcocystis species in red deer revisited: with a re-description of two known species as Sarcocystis elongata n. sp. and Sarcocystis truncata n. sp. based on mitochondrial cox1 sequences[END_REF][START_REF] Gjerde | The resurrection of a species: Sarcocystis bovifelis Heydorn et al., 1975 is distinct from the current Sarcocystis hirsuta in cattle and morphologically indistinguishable from Sarcocystis sinensis in water buffaloes[END_REF][START_REF] Hu | Sarcocystis tuagulusi n.sp. (Apicomplexa: Sarcocystidae) from Williamson's mouse deer (Tuagulus williamsoni) (Artiodactyla: Tragulidae)[END_REF][START_REF] Hu | Sarcocystis menglaensis n. sp. (Apicomplexa: Sarcocystidae) from Williamson's mouse deer Tuagulus williamsoni (Kloss) (Artiodactyla: Tragulidae)[END_REF], no species of the Haemogregarinidae was processed until now for COI barcodes [START_REF] Léveillé | Next generation sequencing from Hepatozoon canis (Apicomplexa: Coccidia: Adeleorina): Complete apicoplast genome and multiple mitochondrion-associated sequences[END_REF]. In addition, molecular species identification of Haemogregarina was solely based on 18S rRNA gene sequences, which may hidden the true species diversity. The molecular characterization of Haemogregarina using specific COI primers revealed the presence of nine distinct haplotypes within the two freshwater turtles E. orbicularis and M. leprosa in Tunisia. Based on Bayesian analyses, Haemogregarina COI haplotypes formed a monophyletic group that was subdivided into three sub-lineages, namely Haemogregarina sp_Hap1, Haemogregarina sp_Hap2 and Haemogregarina sp_Hap3 (Figure 2). Pairwise distances estimated between the three sublineages are greater than 6.6%, which is much higher than the interspecific average pairwise distance, i.e. 2.1%, estimated between two valid distinct species, namely E. lancasterensis and E. sciurorum (Table 6). Additionally, the pairwise distances within Haemogregarina sp_Hap1 and within Haemogregarina sp_Hap3 are less than 1.1% (Table 5). These outcomes suggest that each Haemogregarina sub-lineage may represent a distinct species, which was previously suggested by Attia El Hili et al. [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF] based on nuclear 18S rRNA gene sequences.

Furthermore the current study showed that the COI gene is a more variable marker than the 18S gene indicating that the COI gene may be useful in both systematics as well as in phylogeography studies of species of Haemogregarina. For all these reasons, and because it is not necessary to sequence the total length of the COI to get variable and informative characters, focusing on COI instead of 18S marker may be more advantageous in terms of cost and time.

The occurrence of several COI variants within some turtle specimens is in agreement with the molecular results inferred from the analysis of 18S rRNA gene sequences obtained from the same blood samples [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF]. This confirms co-infection patterns of species of Haemogregarina within freshwater turtles in Tunisia, which was also observed in other apicomplexans such as species of Hepatozoon, Eimeria and Isospora of Eucoccidiorida [START_REF] Dolnik | Individual oocysts of Isospora (Apicomplexa: Coccidia) parasites from avian feces: from photo to sequence[END_REF][START_REF] Györke | Prevalence and distribution of Eimeria species in broiler chicken farms of different capacities[END_REF][START_REF] Marzal | Prevalence and genetic diversity of blood parasite mixed infections in Spanish terrapins, Mauremys leprosa[END_REF][START_REF] Hamid | Chicken coccidiosis in Central Java, Indonesia: A Recent update[END_REF], and species of Plasmodium and Haemoproteus of Haemosporida [START_REF] Bernotienė | Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections[END_REF]. Due to the presence of several Haemogregarina COI variants in the same turtle specimen, as is also the case with Haemogregarina 18S variants [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF], it remains difficult to associate a particular COI haplotype to one of the three identified 18S haplotypes. Besides H. stepanowi, for which it is still impossible to assign a particular COI haploclade, the morphological description of the two new Haemogregarina species will be possible once unique COI and 18S haplotypes are identified from the same turtle population.

Based on morphological and molecular analyses, a low diversity of Haemogregarina species, accounting for less than 20 species [START_REF] Dvořáková | Haemogregarines of freshwater turtles from Southeast Asia with a description of Haemogregarina sacaliae sp. n. and a redescription of Haemogregarina pellegrini Laveran and Pettit[END_REF][START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF][START_REF] Dvořáková | Haemogregarines from western Palaearctic freshwater turtles (genera Emys, Mauremys) are conspecific with Haemogregarina stepanowi Danilewsky[END_REF][START_REF] Özvegy | Cytological and molecular identification of Haemogregarina stepanowi in blood samples of the European pond turtle (Emys orbicularis) from quarantine at Belgrade zoo[END_REF][START_REF] Arizza | The good, the bad and the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa)[END_REF][START_REF] Rakhshandehroo | Morphological and molecular characterisation of Haemogregarina sp. (Apicomplexa: Adeleina: Haemogregarinidae) from the blood of the Caspian freshwater turtle Mauremys caspica (Gmelin) (Geoemydidae) in Iran[END_REF][START_REF] Alhaboubi | Molecular and morphological characterization of a haemogregarine in the alligator snapping turtle, Macrochelys temminckii (Testudines: Chelydridae)[END_REF][START_REF] De Oliveira | Molecular detection of hemogregarines and haemosporidians in Brazilian free-living testudines[END_REF][START_REF] Úngari | Haemogregarina podocnemis sp. nov.: description of a new species of Haemogregarina Danilewsky 1885 (Adeleina: Haemogregarinaidae) in free-living and captive yellow-spotted river turtles Podocnemis unifilis (Testudines: Podocnemididae) from Brazil[END_REF], successfully infect freshwater turtles. With the exception of Attia El Hili et al. [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF] who reported from 18S rRNA gene sequences the presence of three Haemogregarina species within the same individuals, all other reports that focused on both morphological and genetic data of Haemogregarina revealed the presence of single or low divergent 18S variants per individual, suggesting the occurrence of a single parasite species in sympatric hosts [START_REF] Dvořáková | Haemogregarines of freshwater turtles from Southeast Asia with a description of Haemogregarina sacaliae sp. n. and a redescription of Haemogregarina pellegrini Laveran and Pettit[END_REF][START_REF] Dvořáková | Haemogregarines from western Palaearctic freshwater turtles (genera Emys, Mauremys) are conspecific with Haemogregarina stepanowi Danilewsky[END_REF][START_REF] Özvegy | Cytological and molecular identification of Haemogregarina stepanowi in blood samples of the European pond turtle (Emys orbicularis) from quarantine at Belgrade zoo[END_REF][START_REF] Arizza | The good, the bad and the ugly: Emys trinacris, Placobdella costata and Haemogregarina stepanowi in Sicily (Testudines, Annelida and Apicomplexa)[END_REF][START_REF] Rakhshandehroo | Morphological and molecular characterisation of Haemogregarina sp. (Apicomplexa: Adeleina: Haemogregarinidae) from the blood of the Caspian freshwater turtle Mauremys caspica (Gmelin) (Geoemydidae) in Iran[END_REF][START_REF] Alhaboubi | Molecular and morphological characterization of a haemogregarine in the alligator snapping turtle, Macrochelys temminckii (Testudines: Chelydridae)[END_REF][START_REF] De Oliveira | Molecular detection of hemogregarines and haemosporidians in Brazilian free-living testudines[END_REF][START_REF] Úngari | Haemogregarina podocnemis sp. nov.: description of a new species of Haemogregarina Danilewsky 1885 (Adeleina: Haemogregarinaidae) in free-living and captive yellow-spotted river turtles Podocnemis unifilis (Testudines: Podocnemididae) from Brazil[END_REF]. Following PCR assays that were conducted on haemosporidian parasites, Bernotienė et al. [START_REF] Bernotienė | Avian haemosporidian parasites (Haemosporida): A comparative analysis of different polymerase chain reaction assays in detection of mixed infections[END_REF] showed several cases of mixed infections indicating that single PCR assays could underestimate the true parasitic diversity. This can explain why, until recently [START_REF] Hili | The genetic diversity of blood parasites within the freshwater turtles Mauremys leprosa and Emys orbicularis in Tunisia reveals coinfection with Haemogregarina spp[END_REF], so few Haemogregarina species were documented within wild freshwater turtles from 18S rRNA gene sequences. Because of the low specificity of these parasites towards their chelonian hosts, the use of fast evolving mitochondrial markers should help to assess the true diversity of Haemogregarina infections within turtles. Similarly, investigating this parasite diversity within leeches may provide new insights into the specificity of species of Haemogregarina and their definitive hosts. Finally, even if M. leprosa is found predominantly in the Southern parts of the Mediterranean basin, and E. orbicularis in the Northern parts [START_REF] Van Dijk | Turtles of the world, 7th edition: annotated checklist of taxonomy, synonymy, distribution with maps and conservation status[END_REF], they can also be found in sympatry in some areas of Spain and Portugal, as well as in restricted areas of Southern France, Morocco, Algeria and Tunisia. Therefore, it may be interesting to investigate the genetic diversity of Haemogregarina spp. in both host species along their range in order to focus on the overall parasite diversity and specificity and to further explore additional aspects of their origin, evolution and historical biogeography.

Figure legends Figure 1a .

 legends1a Figure legends

Figure 1b .

 1b Figure 1b. Schematized alignment of apicomplexan COI sequences, from top to bottom

Acknowledgements 13 The research was funded by The Fonds Départemental de Coopération Extérieure 2018, Département des Pyrénées-Orientales as part of the project « Parasites, émergence des maladies à transmission vectorielle et changement climatique: Conséquences écologiques et évolutives» and by the PHC Maghreb project 19MAG50/41431WD. Rahma Attia El Hili was funded by a grant from the mobility Erasmus+ program in 2018-2019 (Erasmus+ KA107).

Authors are grateful to two anonymous reviewers for their constructive remarks on an earlier draft of this manuscrip. English corrections were made by Jeanine Almany (N° SIRET 8283968040027).

Columba livia domestica Australia KT305929 [61] Haemogregarina sp_Hap1a M. leprosa / E. orbicularis Tunisia MW052245 This study Haemogregarina sp_Hap1b M. leprosa / E. orbicularis Tunisia MW052246 This study Haemogregarina sp_Hap2a M. leprosa / E. orbicularis Tunisia MW052247 This study Haemogregarina sp_Hap3a M. leprosa / E. orbicularis Tunisia MW052248 This study Haemogregarina sp_Hap3b M. leprosa / E. orbicularis Tunisia MW052249 This study Haemogregarina sp_Hap3c M. leprosa / E. orbicularis Tunisia MW052250 This study Haemogregarina sp_Hap3d M. leprosa / E. orbicularis Tunisia MW052251 This study Haemogregarina sp_Hap3e M. leprosa / E. orbicularis Tunisia MW052252 This study Haemogregarina sp_Hap3f M. leprosa / E. orbicularis Tunisia MW052253 This study Hepatozoon canis Canis lupus familiaris Israel

Photographs J to M show PCR products resulting from amplification of DNAs Ml36AM (well n°1), Ml34AM (well n°2), Ml44HS (well n°3), Ml10HB (well n°4), Eo18HS (well n°5) and Eo19HS (well n°6) with HemoFor4 -HemoRev4 in J (expected size ≃ 465bp), HemoFor5 -HemoRev4 in K (expected size ≃ 410bp), HemoFor5 -HemoRev6 in L (expected size ≃ 400bp) and HemoFor5 -HemoRev7 in M (expected size ≃ 405bp).


domain: chim phys sdv

text:
Florence Charnay-Pouget

Matthieu Le Liepvre

Hendrik Eijsberg

Régis Guillot

Jean Ollivier

Francesco Secci

Angelo Frongia

David J Aitken
email: david.aitken@universite-paris-saclay.fr

A short synthesis of both enantiomers of 2-aminobicyclo[3.2.0]heptane-2,7-dicarboxylic acid

Keywords: Glutamic acid analog Synthetic photochemistry Bicyclo[3, 2, 0]heptane derivatives Resolution procedure

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Glutamic acid is the most abundant mammalian excitatory amino acid and is involved in many aspects of brain function and in the regulation of numerous neuropathological conditions. 1 It interacts with both fast-acting ionotropic (iGlu) receptors and slow-acting G protein-coupled metabotropic (mGlu) receptors. Eight mGlu receptor subtypes have been identified and classified into three groups, and have been targeted over several decades in the search for treatment of anxiety and stress disorders, including schizophrenia, Alzheimer's disease and Parkinson's disease. [START_REF] Muguruza | [END_REF] The search for analogues of glutamic acid that behave as selective ligands for the group II receptors, mGlu2 and mGlu3, led to the discovery of the potent agonist LY354740 (Eglumegad) 3 (Fig. 1), which entered clinical trials as a potential anti-psychotic agent 4 and has recently been shown to alleviate dyskinesia 5 and to counter retinal cell apoptosis in glaucoma. 6 While the bicyclo [3.1.0]hexane core of LY354740 has featured in other group II mGlu receptor ligands, 7 numerous cyclic glutamic acid derivatives have been investigated over the years. [START_REF] Chernykh | For examples of cyclic conformationally constrained analogs of glutamic acid[END_REF] One such compound is (1S,2S,5S,7S)-2aminobicyclo[3.2.0]heptane-2,7-dicarboxylic acid (1) whose molecular structure is very similar to that of LY354740, having just one extra carbon atom in the smaller of the two rings (Fig. 1). Pharmacological evaluation of 1 revealed a weak agonist activity for mGlu1, mGlu2, mGlu4 and mGlu6 receptor subtypes, although the activity towards the mGlu3 receptor subtype was not reported. 9 There is still a need for glutamate analogs that differentiate between mGlu2 and mGlu3, 10 so we felt that the mGlu3 activity should also be assessed. Furthermore, in a more general context there is considerable current interest in polyfunctional compounds with a bicyclo[3.2.0]heptane core for drug discovery 11 and as rigid molecular scaffolds for multiple functional group displays. The published synthesis 9 of 1 adopted a "chiral pool" strategy, starting from L-serine. It involved a lengthy sequence (10 steps; undisclosed yield) featuring a late-stage intramolecular photochemical [2+2]-cycloaddition reaction to construct the bicyclic core, which took place with low diastereoselectivity. We report here our own investigation focusing on an alternative synthesis of 1, which we reasoned should be more expedient. The pivot of this simplified strategy is to conduct an early-stage intermolecular photochemical

[2+2]-cycloaddition reaction, A R T I C L E I N F O A B S T R A C T
which is followed by a resolution protocol. As we will show, it has the advantage of providing both (1S,2S,5S,7S)-2aminobicyclo[3.2.0]heptane-2,7-dicarboxylic acid (1) and its enantiomer (ent-1).

The photochemical [2+2]-cycloaddition reaction between cyclopent-2-enone and methyl acrylate was previously reported to give a roughly 1:1 mixture of head-to-head and head-to-tail regioisomers, in which the former was a 86:14 mixture of exo:endo stereoisomers, respectively. 13 We reexamined this photochemical reaction using ethyl acrylate as the alkene component (Scheme 1). When the gram scale reaction was carried out in acetone (25 °C, 4 h, 10 equiv. ethyl acrylate, 400 W Hg vapor lamp) we obtained a 2:1 mixture of regioisomers which were separated by simple column chromatography in good overall yield. The major compound was the head-to-head regioisomer (±)-2, isolated in 49% yield as a single diastereomer. By analogy with the literature, 13 the exo configuration was expected and was subsequently confirmed by transformation into compound 5 (vide infra). The minor head-to-tail regioisomer 3 was isolated in 24% yield as a 2:1 mixture of exo and endo diastereomeric forms. To confirm the identity of 3 we took advantage of compound 4, [START_REF]The sample of compound 4 used here was obtained from the work described in ref. 12b and was non racemic, with the 1S,5R[END_REF] available from previous studies. 12b A four-step procedure involving hydrogenation, ester hydrolysis, oxidation then esterification gave an authentic sample of endo-3 in 65% overall yield (Scheme 2). Comparison of the spectroscopic data indicated that this was the minor diastereomer present in the endo/exo-3 mixture obtained in Scheme 1.

Scheme 1. Synthesis of (±)-1.

Scheme 2. Independent preparation of endo-3.

To validate the photochemical strategy for accessing the target amino acid, (±)-2 was transformed into a hydantoin using a Bucherer-Bergs reaction to provide derivative (±)-5 as a single diastereomer, albeit in modest yield, after crystallization from water. 15 The molecular structure and the relative stereochemistry of (±)-5 were confirmed by X-ray diffraction analysis (Fig. 2); [START_REF]Crystallographic data for compounds 5 and 7b have been deposited at the CCDC with[END_REF] the requisite endo orientation of the ring-bound nitrogen is in agreement with the attack of cyanide on the least hindered face of the bicyclic core during the Bucherer-Bergs reaction. Rapid transformation of (±)-5 into the target compound now seemed clearly feasible. Treatment with moderately strong aqueous base resulted in the efficient selective hydrolysis of the ester to provide (±)-6, while slightly stronger basic conditions achieved the desired complete hydrolysis to give (±)-1 in 46% isolated yield. In this way, a very short and simple synthesis (3 steps) of (1S*,2S*,5S*,7S*)-2-aminobicyclo[3.2.0]heptane-2,7dicarboxylic acid (±)-1 was validated (Scheme 1). In order to adapt this strategy to allow access to enantiomerically pure samples, our initial plan was to incorporate a resolution step implicating fractional crystallization of a diastereomeric salt mixture obtained by treatment of (±)-6 with readily available (S)-α-methylbenzylamine. Indeed, this approach had proven successful for the resolution of the corresponding hydantoin-acid precursor of LY354740. 3 Despite considerable efforts, however, we were unable to crystallize salt samples with any significant diastereoselectivity.

We then turned our attention to a resolution strategy based on diastereomeric derivatization by covalent attachment of a chiral resolving agent. Previously, our group has employed nonracemic oxazolidinones in this way to resolve a number of cyclic amino acids. [START_REF] Chang | [END_REF] Derivatization of (±)-6 seemed likely to be problematic due to the reactivity of the hydantoin so we focused on the simpler keto derivative. Saponification of (±)-2 in aqueous base gave the corresponding carboxylic acid which was activated as a mixed anhydride by reaction with pivaloyl chloride, then treated with the lithium salt of (R)-4-phenyloxazolidine-2-one (Scheme 3). The two diastereomers 7a and 7b obtained from this three-step procedure were separated easily by column chromatography and were isolated as single compounds in gratifying overall yields of 33% and 24% respectively from (±)-2. Single crystal X-ray diffraction analysis of 7b provided the absolute configuration assignment (Fig. 3). [START_REF]Crystallographic data for compounds 5 and 7b have been deposited at the CCDC with[END_REF] Scheme 3. Resolution procedure and synthesis of 1 and ent-1. The expedient synthesis of both enantiomers of the title compound was then completed in three steps, requiring minimal work-up of intermediates. A Bucherer-Bergs reaction was carried out on compounds 7a and 7b to provide the corresponding hydantoin as a single stereoisomer with roughly the same efficiency as had been observed in the racemic route. Each hydantoin was treated, in a one-pot protocol, with lithium hydroperoxide to cleavage the oxazolidine-2-one then with stronger base to hydrolyze the hydantoin, to furnish the free amino acids 1 and ent-1 in overall yields of 20% and 33%, respectively, for the three steps (Scheme 3). Spectroscopic data of these compounds were in full agreement with the proposed structures and the specific rotation values, which had not been previously reported, were [α]D 23 = -26.3 (c = 0.99, H2O) for 1 and [α]D 23 = +26.4 (c = 1.00, H2O) for ent-1.

Compounds 1 and ent-1 were tested as ligands for the mGlu2 and mGlu3 receptors using a HTRF (Homogeneous Time-Resolved FRET) immuno-assay with LY354740 as the control ligand. Response curves indicated little or no affinity of the test compounds for either the mGlu2 or the mGlu3 receptor. These observations confirm and complete the previous findings regarding 1. 9

Conclusion

In summary, we have established a short and simple synthesis of the title compound in single enantiomer form. The key initial photochemical step allowed facile access to the core bicyclic skeleton on near-gram-scale and although the Bucherer-Bergs reactions gave moderate yields they had the advantage of being entirely stereoselective. The resolution protocol confirms the synthetic value of oxazolidinones as chiral resolving agents.

12

 12

Figure 1 .

 1 Figure 1. Structures of glutamic acid and two restricted analogs.

Figure 2 .

 2 Figure 2. X-ray structure of compound (±)-5.

Figure 3 .

 3 Figure 3. X-ray structure of compound 7b.

Acknowledgments

M. L. L. and H. E. were beneficiaries of PhD grants from the Ministère de l'Enseignement Supérieur et de la Recherche.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Olivier Garraud
email: olivier.garraud@univ-st-etienne.fr

Karine Lacombe

Pierre Tiberghien

The Clinical Trial Section

. The question, in other words, remains as to how to protect against what: from the infection itself or from the severity of the infection? Convalescent plasma treatment, i.e. passive polyclonal antibody administration to provide immediate immunity, has been used to improve the survival rate of patients with severe acute respiratory syndromes of viral etiology, as reviewed by Mair-Jenkins J et al. in their 2015 metanalysis [5]. Indeed, a number of convalescent plasma studies, unfortunately none

adequately controlled for bias, have reported positive outcomes, including decreased mortality in the so-called Spanish Influenza A (H1N1) infections in 1915-1917, the more recent Influenza A (H1N1)pdm09 infections in 2009/2010 as well SARS-CoV infections in 2003. On the contrary, convalescent plasma was found to provide no benefit for the treatment of Ebola disease [START_REF] Van Griensven | Evaluation of Convalescent Plasma for Ebola Virus Disease in Guinea[END_REF] while being beneficial in the treatment of another hemorrhagic fever i.e. the Argentinian hemorrhagic fever [START_REF] Ruggiero | Traitement de la fièvre hémorragique argentine par le plasma de convalescent : 4,433 cas [Treatment of Argentine hemorrhagic fever with convalescent's plasma. 4433 cases[END_REF].

Very early after the SARS-CoV-2 infection appeared to be potentially severe and lethal in a number of patients, most presenting with frailties, proposals were made to assess convalescent plasma therapy, either in compassionate situations or in randomized controlled trials (RCTs) [START_REF] Shen | Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma[END_REF][START_REF] Bloch | Deployment of convalescent plasma for the prevention and treatment of COVID-19[END_REF][START_REF] Duan | Effectiveness of convalescent plasma therapy in severe COVID-19 patients[END_REF]. Two paramount questions were to be addressed prior to promoting such a treatment: firstl, to assess whether the recovering person was capable to raise sustainable amounts of antibodies to be collected by plasmapheresis; and secondly, whether antibodies were indeed neutralizing and not facilitating (such as in certain viral infections, the most exemplary being Dengue viruses, against the other three serotypes) [START_REF] Casadevall | The convalescent sera option for containing COVID-19[END_REF][START_REF] Lee | DeKovsky BJ Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies[END_REF].

At a speed never seen so far, robust data accumulated regarding the complexity of antibodies made against SARS-CoV-2 moieties (the virus signature track) or by COVID-19 suffering patients, not necessarily overlapping. Indeed, while COVID-19 healers were proven to raise significant amounts of antibodies that do not vanish immediately and last for weeks and likely months [START_REF] Chen | Quick COVID-19 Healers Sustain Anti-SARS-CoV-2 Antibody Production[END_REF][START_REF] Atyeo | Distinct Early Serological Signatures Track with SARS-CoV-2 Survival[END_REF], pathogenic antibodies were also made in parallel. These can bind to macrophages and cause acute lung injury [14], damage vascular endothelium leading to endotheliopathy [START_REF] Rauch | Members of the LICORNE Scientific Committee. Endotheliopathy Is Induced by Plasma From Critically Ill Patients and Associated With Organ Failure in Severe COVID-19[END_REF] and possibly coagulopathy [START_REF] Zuo | Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19[END_REF], and dampen antiviral natural defense when directed to type I interferons [START_REF] Bastard | Milieu Intérieur Consortium; CoV-Contact Cohort; Amsterdam UMC Covid-19 Biobank; COVID Human Genetic Effort[END_REF]. It has been shown that-not unlikely as several other infections, such as HIV and others-the SARS-CoV-2 virus alters the germinal center and forces extrafollicular antibody making [START_REF] Kaneko | Massachusetts Consortium on Pathogen Readiness Specimen Working Group. Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19[END_REF][START_REF] Woodruff | Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19[END_REF]. In spite of this, recent evidence indicates that robust antibody protection persists for months [START_REF] Wajnberg | Robust neutralizing antibodies to SARS-CoV-2 infection persist for months[END_REF][START_REF] Iyer | Persistence and decay of human antibody responses to the receptor binding domain of SARS-CoV-2 spike protein in COVID-19 patients[END_REF], and, if any, enhancing or facilitating antibodies were of moderate concentration.

Collection of convalescent plasma preceded accumulation of scientific evidence that antibodies were neutralizing and not enhancing, and in a quantity large enough to mediate protection, demonstrating that an unprecedented race was engaged against the pandemic, barely allowing time to discuss ethical issues [START_REF] Garraud | COVID-19, transfusion, and publishing ethics[END_REF][START_REF] Barroga | Fundamental Shifts in Research, Ethics and Peer Review in the Era of the COVID-19 Pandemic[END_REF], with the objective to learn as much as possible to justify the strongly wished vaccine approach.

Less than a single year after the initial reports of the severity of the disease, knowledge has accumulated regarding the potential of convalescent plasma: encouraging data were presented after 3 months of inclusion in a large series of recipients by Joyner et al. [START_REF] Joyner | Effect of Convalescent Plasma on Mortality among Hospitalized Patients with COVID-19: Initial Three-Month Experience[END_REF].

Next, Li et al [START_REF] Li | Effect of Convalescent Plasma Therapy on Time to Clinical Improvement in Patients With Severe and Life-threatening COVID-19: A Randomized Clinical Trial[END_REF] concluded that: "Among patients with severe or life-threatening COVID-19, convalescent plasma therapy added to standard treatment did not significantly improve the time to clinical improvement within 28 days, although the trial was terminated early and may have been underpowered to detect a clinically important difference". In contrast, Liu et al found that their retrospective, propensity score-matched case-controlled study assessed the effectiveness of convalescent plasma therapy with less oxygen requirements on day 14 after transfusion and less worsened cases in plasma recipients compared to propensity score-matched controls [START_REF] Liu | Firpo-BConvalescent plasma treatment of severe COVID-19: a propensity score-matched control study[END_REF]. Salazar et al reported a significant reduction (P = 0.047) in mortality within 28 days, specifically in patients transfused within 72 hours of admission with plasma with an anti-spike protein receptor binding domain titer of ≥1:1350, and suggested that that treatment of COVID-19 with high anti-receptor binding domain IgG titer convalescent plasma is efficacious in early-disease patients [START_REF] Salazar | Significantly Decreased Mortality in a Large Cohort of Coronavirus Disease 2019 (COVID-19) Patients Transfused Early with Convalescent Plasma Containing High-Titer Anti-Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike Protein IgG[END_REF]. Agarwal et al, however, found that convalescent plasma was not associated with a reduction of progression to disease or all cause of mortality [START_REF] Agarwal | Convalescent plasma in the management of moderate covid-19 in adults in India: open label phase II multicentre randomised controlled trial (PLACID Trial)[END_REF]. In aggregate, Joyner et al, having compiled (nearly) all submitted data-not necessarily peer-reviewed as many are loaded on the MedRXiv siteconcluded in favor of a protective effect of convalescent plasma [START_REF] Joyner | Early safety indicators of COVID-19 convalescent plasma in 5000 patients[END_REF]. More recently, convalescent plasma was found to be not superior to placebo in a randomized trial involving hospitalized patients with COVID-19 pneumonia [START_REF] Simonovich | for the PlasmaR study group. A ranndomized trial of convalescent plasma in Covid-19 penumonia[END_REF]. Apparent discrepancies in conclusions may well be the result of certain inconsistency in defining patient groups (severity, timecourse of infection, and outcomes), as was observed in other main trials addressing controversies in blood transfusion; the same concerns were for e.g. raised in this Journal à propos the age of blood [START_REF] Garraud | Clinical trials in Transfusion Medicine and hemotherapy: Worth moving forward in evaluating 'fresh' versus 'old' blood cell components?[END_REF]. These types of inconsistencies are, in general, pondered in metanalyses. It remained to confirm that transfusion of convalescent plasma was safe, which Joyner reviewed in 5,000 transfused COVID-19 patients [START_REF] Joyner | Early safety indicators of COVID-19 convalescent plasma in 5000 patients[END_REF]. Nguyen et al reviewed, in detail, the adverse reactions in 427 patients having received convalescent plasma and did not report increased risks compared to anticipated side effects [START_REF] Joyner | Early safety indicators of COVID-19 convalescent plasma in 5000 patients[END_REF]. Of note, the observed reaction rate was 12.9%, among which 82% (mainly fever, hypoxia or both) were attributed to the underlying disease.

Interestingly, convalescent plasma therapy has proven to be beneficial in B-cell depleted patients having protracted COVID-19 [START_REF] Nguyen | Transfusion reactions associated with COVID-19 convalescent plasma therapy for SARS-CoV-2[END_REF], with an acknowledged limit inherent to uncontrolled observations [START_REF] Hueso | Convalescent plasma therapy for B-cell-depleted patients with protracted COVID-19[END_REF]. Compassionate plasma therapy was applied early in the disease course [START_REF] Murphy | COVID-19, plasma, and hypogammaglobulinemia[END_REF][START_REF] Vélez | A practical approach for the compassionate use of convalescent plasma in patients with severe COVID-19 in developing countries[END_REF][START_REF] Maor | Compassionate use of convalescent plasma for treatment of moderate and severe pneumonia in COVID-19 patients and association with IgG antibody levels in donated plasma[END_REF]. As many more RCTs or compassionate programs are run in major healthcare facilities or within international consortiums, regulatory bodies-such as the FDA-also made more precise recommendations [START_REF] Vlachogianni | A case of COVID-19 Convalescent Plasma Donation in Greece: Directed donation for compassionate use in the donor's critically ill father[END_REF].

Very recently, investigators have reported (in a not-yet peer-reviewed format) positive results regarding the very early (within 72 hours of mild COVID-19 symptoms) transfusion of convalescent plasma in patients over 75 years old [39]. In this randomized study, convalescent plasma transfusion was associated with significant reduction in the occurrence of severe respiratory disease. These findings, reminiscent of recently reported findings with an anti-SARS-Cov-2 monoclonal treatment [START_REF] Libster | Prevention of severe COVID-19 in the elderly by early high titer plasma[END_REF] as well as much older findings with convalescent plasma treatment of the Argentinian hemorrhagic fever [START_REF] Enria | Antiviral treatment of Argentine hemorrhagic fever[END_REF], highlight the importance of early intervention when considering passive immunotherapy to treat infectious diseases. As we now move into the COVID-19 vaccination era, passive immunotherapy may become an important early therapeutic intervention for those poorly responding to vaccination (such as patients with acquired immunodeficiency, and possibly the elderly).

In conclusion, convalescent plasma therapy remains a solid option to treat COVID patients, though this option falls into a portfolio of many other therapeutic approaches and does not provide complete cure. The treatment schedule is likely to be refined to sort out the target populations and the most appropriate time frame, and also when to collect plasma from convalescent donors [START_REF] Chen | SARS-CoV-2 Neutralizing Antibody LY-CoV555 in Outpatients with Covid-19[END_REF]. With a broader perspective, it is interesting to note that once more (after e.g. the Ebola virus saga) plasma therapy competes with vaccine development, the former being not completely disregarded when the latter comes into reality as antibodyenriched plasma may well be obtained from vaccinated persons, to prepare purified, neutralizing antibodies as also recommended by the FDA [START_REF] Tiberghien | Collecting and evaluating convalescent plasma for COVID-19 treatment: why and how?[END_REF]. Two sets of conclusions can be temporarily drawn at this time. First, this pandemic has revealed how fragile the combination of healthcare and economy, forcing to state decisions that altered autonomy of populations and individual freedom. The swiftness of incremental health care measures to handle actual patients and to protect the rest of the population is barely compatible with the necessary in-depth ethical analysis of all situations and consequences. Secondly, there is an urgent need to set up-in forthcoming preparedness plans for the next pandemic (as this seems to be inevitable)-clearer outcomes to measure, in order to allow for the best-fitted clinical trials. Methods and ethics are more than ever paramount to accompany the vortex of medical and scientific achievements under pressure such as seen in this SARS-CoV-2 crisis.

Acknowledgements: Authors express their gratitude to Prof. Gail Rock for her help in editing this manuscript and critical reading.

Disclosures: None of the authors disclose any conflict of interest with respect to the present study.