New view of population genetics of zooplankton: RAD-seq analysis reveals population structure of the North Atlantic planktonic copepod Centropages typicus.

Detection of population genetic structure of zooplankton at medium-to-small spatial scales in the absence of physical barriers has remained challenging and controversial. The large population sizes and high rates of gene flow characteristic of zooplankton have made resolution of geographical differentiation very difficult, especially when using few genetic markers and assuming equilibrium conditions. Next-generation sequencing now allows simultaneous sampling of hundreds to thousands of genetic markers; new analytical approaches allow studies under nonequilibrium conditions and directional migration. Samples of the North Atlantic Ocean planktonic copepod, Centropages typicus, were analysed using restriction site-associated DNA (RAD) sequencing on a PROTON platform. Although prior studies revealed no genetic differentiation of populations across the geographical range of the species, analysis of RAD tags showed significant structure across the North Atlantic Ocean. We also compared the likelihood for models of connectivity among NW Atlantic populations under various directional flow scenarios that replicate oceanographic conditions of the sampled domain. High-density marker sampling with RAD sequencing markedly outperformed other technical and analytical approaches in detection of population genetic structure and characterization of connectivity of this high geneflow zooplankton species.

MtDNA sequencing from zooplankton after long-term preservation in buffered formalin.

Molecular genetic analysis of zooplankton has been slowed by the usual practice of preservation and storage of samples in dilute formalin solutions, which are not always adequately buffered for pH. We report here the determination of DNA sequences for Meganyctiphanes norvegica (Crustacea, Euphausiacea) preserved and stored in buffered formalin for up to 25 years. Specifically designed molecular protocols for DNA extraction and PCR amplification yielded valid sequence data for short (approximately 100-200 bp) regions of the mitochondrial cytochrome b (mtCYB) gene for individual euphausiids. Critical aspects of our approach include: extended extraction and proteinase-K digestion to maximize DNA yield; use of protocols requiring short DNA fragments; design of species-specific PCR primers to minimize risks of contamination by exogenous DNA; and comparison with published DNA sequences for the same gene and species. We conclude that the yield of DNA and the success of subsequent molecular analyses depend primarily on the length of time the tissue has been exposed to formalin and the pH of the solution. Zooplankton samples intended for molecular analysis should preferably be preserved and maintained in ethanol or deep-frozen, but long-term storage in buffered formalin does not preclude some types of molecular genetic analysis.

Low mitochondrial diversity and small effective population sizes of the copepods Calanus finmarchicus and Nannocalanus minor: possible impact of climatic variation during recent glaciation.

Molecular population genetic diversity of two planktonic copepods of the North Atlantic, Calanus finmarchicus and Nannocalanus minor (Crustacea, Copepoda, Calanoida), was characterized using the sequence variation in a 350 bp region of the mitochondrial 16S rRNA gene. The subarctic species, C. finmarchicus, shows lower population genetic diversity (haplotype diversity, h = 0.368, SD = 0.043; nucleotide diversity, pi = 0.00370, SD = 0.0026) than the temperate/subtropical species, N. minor (h = 0.824, SD = 0.024; pi = 0.00502, SD = 0.0032). Effective population sizes (N(e), estimated from numbers of haplotypes) and effective female population sizes (Nf(e), estimated from nucleotide diversities) for the two species are 10(7) to 10(10) smaller than census female population sizes (Nf) estimated from observed densities and areal distributions. For both C. finmarchicus and N. minor, Nf approximately 10(15), N(e) approximately 10(8), and Nf(e) approximately 10(5). We hypothesize that the cause of both low levels of molecular diversity and small effective population sizes of the two species is the impact of glaciation during the past 20,000 years. C. finmarchicus may have experienced 75% range reduction and latitudinal displacement during the last glacial maximum at 18,000 years BP, giving rise to a genetic bottleneck; this may explain low diversity and an L-shaped distribution of pairwise haplotype differences. In contrast, N. minor may have experienced range reduction of only 30% and less change in latitudinal extent, with less impact of levels of molecular diversity and the shape of the pairwise difference distribution. Although marine zooplankton species are highly abundant, conservation biologists should note that their numbers may vary significantly on climatic to evolutionary time scales, generating low levels of molecular genetic diversity.

Endotoxin neutralization with rabbit antisera to Escherichia coli J5 and other gram-negative bacteria.

To study the mechanisms of protection against endotoxin challenge offered by antisera to smooth and rough gram-negative organisms, we have developed an assay to quantitate endotoxin neutralization based on inhibition of the Limulus amoebocyte lysate test. Dilutions of different bacterial lipopolysaccharides (LPSs) were incubated with hyperimmune rabbit sera against Escherichia coli O113, E. coli O18, and rough mutants E. coli J5 and Salmonella minnesota Re595 and were then combined with limulus lysate. The gelation reaction induced by LPS in the lysate was monitored spectrophotometrically, and the concentration of LPS resulting in a 50% lysate response was determined and correlated with antibody titers measured by enzyme-linked immunosorbent assay. Antisera to smooth organisms neutralized homologous LPS markedly and heterologous LPSs only minimally relative to neutralization by preimmune serum. Neutralization of homologous LPS occurred immediately without preincubation of serum and LPS. Antisera to rough mutants neutralized more heterologous LPS than did antisera to smooth organisms. However, this heterologous neutralization required preincubation of serum and LPS and did not appear to be correlated with antibody concentrations. We conclude that antisera to LPS rapidly neutralize the biological activity of the homologous LPS, as detected by limulus lysate, and that neutralization is at least in part antibody mediated. Antisera to rough-mutant organisms slowly neutralized the activity of heterologous LPSs, but this effect appeared not to be correlated with concentrations of antibody to the LPS of the rough mutant, as measured by enzyme-linked immunosorbent assay.