Short-term stability of wastewater samples for storage and shipment in the context of the EU Sewage Sentinel System for SARS-CoV-2.

In the context of an EU-wide surveillance system for SARS-CoV-2 in wastewater, recommended by the European Commission, this study aims to provide scientific support to the adequacy of transport and storage conditions of samples both in terms of duration and samples temperature. Three laboratories in Slovenia, Cyprus and Estonia investigated the short-term, one-week, isochronous stability of wastewater samples by RT-qPCR based detection of SARS-CoV-2 genes. The results were tested for statistical significance to determine uncertainty of quantification and shelf-life, at testing temperatures of + 20 °C and - 20 °C, relative to reference at + 4 °C. Samples were collected from three urban wastewater treatment plant influents and analysed respectively for SARS-CoV-2 genes N1, N2 (Laboratory 1), N2, E (Laboratory 2) and N3 (Laboratory 3), with various analytical methods. For a period of 7/8 days at + 20 °C, decreasing trends of measured concentrations were observed for all genes resulting in instability according to the statistical analysis, while at - 20 °C the trend of variation was stable only for N1, N2 (Laboratory 1) and N3 (Laboratory 3). Trends for gene E concentrations at - 20 °C (Laboratory 2) could not be tested statistically for stability because of lack of data. Over a period of just 3 days at + 20 °C, the variation was statistically non-significant indicating stability for genes N1, E and N3 for laboratories 1, 2 and 3, respectively. Nonetheless, the outcome of the study presents evidence to support the choice of the selected temperature at which samples shall be preserved during storage before analysis or transport to the laboratory. The conditions (+4 °C, ∼ few days) chosen for EU wastewater surveillance are in accordance with these results, highlighting the importance of stability testing of environmental samples to determine the short-term analytical uncertainty.

Haploid females in the parasitic wasp Nasonia vitripennis.

The insect order of Hymenoptera (ants, bees, sawflies, and wasps) consists almost entirely of haplodiploid species. Under haplodiploidy, males develop from unfertilized eggs and are haploid, whereas females develop from fertilized eggs and are diploid. Although diploid males commonly occur, haploid females have never been reported. In analyzing the phenomenon of gynandromorphism in the parasitoid wasp Nasonia vitripennis, we found a line that generates complete phenotypic females from unfertilized eggs. These females have ovaries, can lay eggs, and are haploid, as shown by cytological and flow cytometric analyses. The data show that diploidy is not necessary for female development.

Mosaic haploid-diploid embryos and polyspermy in the tellinid bivalve Macoma balthica.

We investigated meiosis, fertilization, and early development in eggs of the tellinid bivalve Macoma balthica (L.), which has external fertilization. Meiosis is standard but polyspermy is found to be very common. In all eight crosses examined, mosaic embryos consisting of a mixture of diploid (2n = 38) and haploid cells occur at a frequency ranging from 2.7 to 29.1%. The earliest mosaic found is in the two-cell stage. We propose that an androgenic haploid cell lineage can originate from one supernumerary sperm that decondenses into a functional haploid nucleus, starts mitosis, and is incorporated in the developing embryo.

Cytology of Wolbachia-induced parthenogenesis in Leptopilina clavipes (Hymenoptera: Figitidae).

Parthenogenesis induced by cytoplasmatically inherited Wolbachia bacteria has been found in a number of arthropod species, mainly Hymenoptera. Previously, two different forms of diploidy restoration have been reported to underlie parthenogenesis induction in Hymenoptera by Wolbachia. Both are a form of gamete duplication, but each differs in their timing. We investigated the cytology of the early embryonic development of a Wolbachia-infected strain of the parasitoid wasp Leptopilina clavipes and compared it with that of an uninfected sexual strain. Both strains have a similar meiosis. In the infected parthenogenetic strain, diploidy is restored by anaphase restitution during the first somatic mitosis, similar to Trichogramma, but not to Muscidifurax. Our results confirm the occurrence of different cytological mechanisms of diploidy restoration associated with parthenogenesis-inducing Wolbachia in the order Hymenoptera.