Assessing changes in genomic divergence following a century of human‐mediated secondary contact among wild and captive‐bred ducks

Along with manipulating habitat, the direct release of domesticated individuals into the wild is a practice used worldwide to augment wildlife populations. We test between possible outcomes of human‐mediated secondary contact using genomic techniques at both historical and contemporary timescales for two iconic duck species. First, we sequence several thousand ddRAD‐seq loci for contemporary mallards (Anas platyrhynchos) throughout North America and two domestic mallard types (i.e., known game‐farm mallards and feral Khaki Campbell's). We show that North American mallards may well be becoming a hybrid swarm due to interbreeding with domesticated game‐farm mallards released for hunting. Next, to attain a historical perspective, we applied a bait‐capture array targeting thousands of loci in century‐old (1842–1915) and contemporary (2009–2010) mallard and American black duck (Anas rubripes) specimens. We conclude that American black ducks and mallards have always been closely related, with a divergence time of ~600,000 years before present, and likely evolved through prolonged isolation followed by limited bouts of gene flow (i.e., secondary contact). They continue to maintain genetic separation, a finding that overturns decades of prior research and speculation suggesting the genetic extinction of the American black duck due to contemporary interbreeding with mallards. Thus, despite having high rates of hybridization, actual gene flow is limited between mallards and American black ducks. Conversely, our historical and contemporary data confirm that the intensive stocking of game‐farm mallards during the last ~100 years has fundamentally changed the genetic integrity of North America's wild mallard population, especially in the east. It thus becomes of great interest to ask whether the iconic North American mallard is declining in the wild due to introgression of maladaptive traits from domesticated forms. Moreover, we hypothesize that differential gene flow from domestic game‐farm mallards into the wild mallard population may explain the overall temporal increase in differentiation between wild black ducks and mallards, as well as the uncoupling of genetic diversity and effective population size estimates across time in our results. Finally, our findings highlight how genomic methods can recover complex population histories by capturing DNA preserved in traditional museum specimens.     

Meller's duck: A threatened species receives recognition at last

Wetland areas of Madagascar, increasingly threatened by extensive habitat modification, contain many little known endemic species. One of these species, Meller's duck (Anas melleri), is restricted to the eastern slope of the central highlands but for many years was just considered a recent island isolate of the widespread common mallard (A. platyrhynchos) and, therefore, not worthy of protection. Phylogenetic analysis, based on mitochondrial d-loop DNA sequences, indicates that Meller's duck is distinct from the mallard and from its nearest geographical congener, the yellow-billed duck (A. undulata) and may have evolved from a non-dimorphic ancestor, the African black duck (A. sparsa). A decline in numbers of the Near-Threatened Meller's duck highlights the need for adequate protection for Madagascar's wetlands.     

Inheritance patterns of enzymes and serum proteins of mallard-black duck hybrids

From 1974 to 1976, a breeding program was used to produce hybrids of black ducks and mallards for the evaluation of inheritance patterns of serum proteins and serum, liver and muscle enzymes. In addition to the crosses designed to produce hybrids, a series of matings in 1975 and 1976 were designed to evaluate inheritance patterns of a hybrid with either a black duck or mallard. At the F₁ level, hybrids were easily distinguished using serum proteins. However, once a hybrid was crossed back to either a mallard or black duck, only 12–23% of the progeny were distinguishable from black ducks or mallards using serum proteins and 23–39% using esterases. Muscle, serum and liver enzymes were similar between the two species.     

Population Genetics of a Translocated Population of Mottled Ducks and Allies

Translocating species is an important management tool to establish or expand the range of species. Success of translocations requires an understanding of potential consequences, including whether a sufficient number of individuals were used to minimize founder effects and if interspecific hybridization poses a threat. We provide an updated and comprehensive genetic assessment of a 1970s–1980s translocation and now established mottled duck (Anas fulvigula) population in South Carolina, USA. In addition to examining the population genetics of these mottled ducks, we simulated expected genetic assignments for generational hybrids (F1–F10), permitting formal purity assignment across samples to identify true hybrids and establish hybridization rates. In addition to wild mallards (A. platyrhynchos), we tested for presence of hybrids with migrant American black ducks (A. rubripes) and released domestic game-farm mallards (A. p. domesticus). We used wild reference populations of North American mallard-like ducks and sampled game-farm mallards from 2 sites in South Carolina that could potentially interbreed with mottled ducks. Despite 2 different subspecies of mottled duck (Florida [A. f. fulvigula] and the Western Gulf Coast [A. f. maculatlus]) used in original translocations, we determined the gene pool of the Western Gulf Coast mottled duck was overwhelmingly represented in South Carolina's current population. We found no evidence of founder effects or inbreeding and concluded the original translocation of 1,285 mottled ducks was sufficient to maintain current genetic diversity. We identified 7 hybrids, including an F1 and 3 late-staged (i.e., F2–F3 backcrosses) mottled duck × black duck hybrids, 1 F2-mottled duck backcrossed with a wild mallard, and 2 F3-mottled ducks introgressed with game-farm mallard. We estimated a 15% hybridization rate in our mottled duck dataset; however, the general lack of F1 and intermediate hybrids were inconsistent with scenarios of high hybridization rates or presence of a hybrid swarm. Instead, our results suggested a scenario of infrequent interspecific hybridization between South Carolina's mottled ducks and congeners. We concluded that South Carolina's mottled duck population is sufficiently large now to absorb current hybridization rates because 85% of sampled mottled ducks were pure. These results demonstrate the importance in managing and maintaining large parental populations to counter hybridization. As such, future population management of mottled ducks in South Carolina will benefit from increased geographical and continued sampling to monitor hybridization rates with closely related congeners. We also suggest that any future translocations of mottled ducks to coastal South Carolina should originate from the Western Gulf Coast. © 2021 The Wildlife Society.     

Microsatellite markers for two stifftail ducks: the white‐headed duck,Oxyura leucocephala,and the ruddy duck,O. jamaicensis

Hybridization with a close relative, the North American ruddy duck (Oxyura jamaicensis), is a major problem for the conservation of the endangered white‐headed duck (Oxyura leucocephala). We report the development of 11 microsatellite markers that can facilitate the identification of hybrids as well as the study of the population structure of both species across their distributions. These markers were tested in 63 white‐headed ducks and 50 ruddy ducks and show a larger diversity in the latter species.     

FISH mapping of 57 BAC clones reveals strong conservation of synteny between Galliformes and Anseriformes

Karyotypes of chicken (Gallus gallus domesticus; 2n = 78) and mallard duck (Anas platyrhynchos; 2n = 80) share the typical organization of avian karyotypes including a few macrochromosome pairs, numerous indistinguishable microchromosomes, and Z and W sex chromosomes. Previous banding studies revealed great similarities between chickens and ducks, but it was not possible to use comparative banding for the microchromosomes. In order to establish precise chromosome correspondences between these two species, particularly for microchromosomes, we hybridized 57 BAC clones previously assigned to the chicken genome to duck metaphase spreads. Although most of the clones showed similar localizations, we found a few intrachromosomal rearrangements of the macrochromosomes and an additional microchromosome pair in ducks. BAC clones specific for chicken microchromosomes were localized to separate duck microchromosomes and clones mapping to the same chicken microchromosome hybridized to the same duck microchromosome, demonstrating a high conservation of synteny. These results demonstrate that the evolution of karyotypes in avian species is the result of fusion and/or fission processes and not translocations.     

Occupancy Patterns of Breeding American Black Ducks

Occupancy patterns can assist with the determination of habitat limitation during breeding or wintering periods and can help guide population and habitat management efforts. American black ducks (Anas rubripes; black ducks) are thought to be limited by habitat and food availability during the winter, but breeding sites may also limit the size or growth potential of the population. The Canadian Wildlife Service conducts an annual breeding waterfowl survey that we used to explore the hypothesis that black duck carrying capacity is limited by wetlands available for breeding in Québec, Canada. We applied single-visit, multi-species occupancy models to the 1990–2015 population survey data to determine if there was evidence the black duck population was limited by breeding habitat. Using a dynamic (multi-season) occupancy modeling approach, we estimated latent occupancy (occupancy accounting for imperfect detection) of black ducks and then used latent occupancy estimates to derive occupancy, colonization, and extirpation rates. We jointly modeled the occupancy dynamics of black ducks and other duck species in wetlands where both species were present. Throughout the duration of the survey, 44% of wetlands were never observed to be occupied by black ducks. Occupancy models showed wetland size was positively associated with occupancy at the first time step (initial occupancy) and colonization. All 2-species models indicated initial black duck occupancy, persistence (continued occupancy), and colonization were positively associated with the presence of a second species. Colonization rate over the 26-year period ranged from 7% to 27% across all models. Extirpation rates were similar and were constant through time within each model. Low occupancy rates, combined with approximately equal colonization and extirpation rates, suggest there are available wetlands for breeding black ducks in their core breeding area. If breeding habitats are not saturated, this suggests migration or wintering areas may be more limiting to black duck population abundance. © 2019 The Wildlife Society.     

S100‐A9 protein in exosomes derived from follicular fluid promotes inflammation via activation of NF‐κB pathway in polycystic ovary syndrome

Exosomes have recently emerged as key mediators of different physiological and pathological processes. However, there has been few report about proteomic analysis of exosomes derived from human follicular fluid and their association with the occurrence of PCOS. Herein, we used TMT‐tagged quantitative proteomic approach to identify proteomic profiles in exosomes derived from follicular fluid of PCOS patients and healthy controls. We identified 662 proteins in exosomes derived from human ovarian follicular fluid. Eighty‐six differently expressed proteins (P < .05) were found between PCOS and healthy women. The alterations in the proteomic profile were related to the inflammation process, reactive oxygen species metabolic process, cell migration and proliferation. Importantly, we observed that follicular fluid exosomes contain S100 calcium‐binding protein A9 (S100‐A9) protein. Exosome‐enriched S100‐A9 significantly enhanced inflammation and disrupted steroidogenesis via activation of nuclear factor kappa B (NF‐κB) signalling pathway. These data demonstrate that exosomal proteins are differentially expressed in follicular fluid during disease process, and some proteins may play important roles in the regulation of granulosa cell function. These results highlight the importance of exosomes as extracellular communicators in ovarian follicular development.     

Mallard–Black Duck Hybridization and Population Genetic Structure in North Carolina

North Carolina, USA, represents the southern extent of the American black duck's (Anas rubripes) breeding range. Mallards (A. platyrhynchos) are present on the breeding grounds of the American black duck and hybridization is observed between these species; therefore, we assessed the genetic integrity, hybridization rates, and population structure of this local breeding population. We extracted genomic and mitochondrial DNA from chorioallantoic membranes and contour feathers from monitored black duck nests. We then prepared the extracted DNA for analysis using high-throughput DNA sequencing methods (ddRAD-seq). First, we assessed nuclear and mitochondrial population structure, genetic diversity, and differentiation across samples from North Carolina, and compared them against 199 genetically vetted mallards, black ducks, and mallard × black duck hybrids that served as genetic references. Next, we tested for parentage and sibling relationship and overall relatedness of black ducks in North Carolina. We recovered strong population structure and high co-ancestry across genetic markers due to interrelatedness among sampled nests in North Carolina and concluded that black ducks have been locally breeding in this area for a prolonged period of time. Despite a high level of interrelatedness among our samples, nucleotide diversity was similar to the reference continental black duck population, suggesting little effect of genetic drift, including inbreeding. Additionally, we conclude that molecular diversity of black ducks in North Carolina is maintained at reference population levels through the influx of genetic material from unrelated, migrating male black ducks. Finally, we report a hybridization level of 47.5%, covering 3 filial generations. Of identified hybrids, 54.7% and 53% were the direct result of interbreeding between black ducks and captive-reared or wild mallards, respectively. We conclude that because of high rates of interspecific hybridization and successive backcrossing events, introgression from wild and feral mallards is occurring into this population of breeding black ducks and requires careful consideration in future management efforts. © 2021 The Wildlife Society.     

Comparative Proteomic Analysis of the Hepatic Response to Heat Stress in Muscovy and Pekin Ducks: Insight into Thermal Tolerance Related to Energy Metabolism

The Pekin duck, bred from the mallard (Anas platyrhynchos) in china, is one of the most famous meat duck species in the world. However, it is more sensitive to heat stress than Muscovy duck, which is believed to have originated in South America. With temperature raising, mortality, laying performance, and meat quality of the Pekin duck are severely affected. This study aims to uncover the temperature-dependent proteins of two duck species using comparative proteomic approach. Duck was cultured under 39°C ± 0.5°C for 1 h, and then immediately returned to 20°C for a 3 h recovery period, the liver proteins were extracted and electrophoresed in two-dimensional mode. After analysis of gel images, 61 differentially expressed proteins were detected, 54 were clearly identified by MALDI TOF/TOF MS. Of the 54 differentially expressed protein spots identified, 7 were found in both species, whereas 47 were species specific (25 in Muscovy duck and 22 in Pekin duck). As is well known, chaperone proteins, such as heat shock protein (HSP) 70 and HSP10, were abundantly up-regulated in both species in response to heat stress. However, we also found that several proteins, such as α-enolase, and S-adenosylmethionine synthetase, showed different expression patterns in the 2 duck species. The enriched biological processes were grouped into 3 main categories according to gene ontology analysis: cell death and apoptosis (20.93%), amino acid metabolism (13.95%) and oxidation reduction (20.93%). The mRNA levels of several differentially expressed protein were investigated by real-time RT-PCR. To our knowledge, this study is the first to provide insights into the differential expression of proteins following heat stress in ducks and enables better understanding of possible heat stress response mechanisms in animals.     

Origin and domestication history of Peking ducks deltermined through microsatellite and mitochondrial marker analysis

In order to elucidate the domestication history of Peking ducks, 190 blood samples from six Chinese indigenous duck breeds were collected with186 individualsgenotyped by 15 microsatellite markers. Both the F_ST and Nei’s standard genetic distances (D_s) from the microsatellite data indicated high genetic differentiation between Peking duck and other Chinese indigenous breeds. The haplotype network with mtDNA data showed that most of the Peking duck haplotypes were distinctly different from those of other domestic breeds. Although the H01 haplotype was shared by all domesticated duck breeds, Peking ducks displayed 12 specific domestic duck haplotypes, including four similar haplotypes H02, H04, H08 and H22, that formed a single haplogroup (A). Both H02 and H22 haplotypes were also shared by mallard and Peking ducks, indicating that Peking ducks originated from wild mallard ducks.     

Divergent Proteome Patterns of Egg Albumen from Domestic Chicken,Duck, Goose,Turkey, Quail and Pigeon

To uncover a diversity of genetic and biological unknowns, a comprehensive and comparative proteomic analysis is performed on egg albumen of domestic chicken, duck, goose, turkey, quail, and pigeon with tandem mass tags quantification technology. In this study, a total of 148, 138, 150, 162, 183, and 179 proteins are identified in egg albumen of the above six species, respectively. Venn plots, PCA, and cluster analysis all reveal the highest similarity of protein composition between duck and goose (≈75%). Additionally, the six species have 52 proteins detected in common in the egg albumen. As revealed by GO and pathway analyses, the plausible functions of these highly conserved proteins are to provide a secure environment and prevent the early death of embryonic cells. Species‐specific proteins such as haptoglobin in pigeon, serpin‐like protein HMSD in duck, and ovodefensin in chicken are also screened and are likely associated with species‐dependent biological processes. Furthermore, Enzyme Code analysis indicated egg albumen have abundant enzyme activity, with hydrolases accounting for more than half of the total enzymes. This study is the first to provide the proteome profiles of egg albumen for the major poultry species, which will be instructive for the understanding of species‐specific biological problems with egg albumen.     

Comparisons of nine heavy metals in salt gland and liver of greater scaup (Aythya marila), black duck (Anas rubripes) and mallard (A. platyrhynchos)

Levels of nine heavy metals were measured in the livers and salt glands of greater scaup (Aythya marila), black duck (Anas rubripes) and mallard (A. platyrhynchos) from Raritan Bay, New Jersey to determine if the functioning avian salt gland concentrates heavy metals. Heavy metals examined were cadmium, cobalt, chromium, copper, lead, mercury, manganese, nickel and zinc. Heavy metal levels varied significantly by species and tissue for chromium, copper, lead, and manganese, and by tissue for cobalt, mercury, nickel and zinc. In comparing tissues cobalt was higher in the salt glands than in livers of all three species; chromium and nickel were higher in the salt gland than liver for mallard and black duck; and lead, manganese and zinc were higher in the liver than the salt gland in greater scaup. Generally metal levels were higher in the salt gland for mallard and black duck, and in the liver for greater scaup.     

Molecular population genetics, phylogeography, and conservation biology of the mottled duck (Anas fulvigula)

The mottled duck (Anas fulvigula) is a year-round endemicresident of the Gulf Coast and one of two non-migratory dabbling ducksthat inhabit North America. To investigate population genetic structureof allopatric mottled duck populations, we collected 5' control regionsequences (bp 78–774) from the mitochondria of 219 mottled duckssampled at 11 widely spaced geographic localities in Texas, Louisiana,and Florida and compared them to each other and to homologous sequencesfrom 4 Mexican ducks (A. diazi), 13 American black ducks(A. rubripes), and 10 mallards (A. platyrhynchos). Weidentified 57 unique haplotypes composed of 665 or 666 nucleotides inthe 246 control region sequences. Of the 665 homologous positions,8.3% (n = 55) vary among haplotypes, and98.2% (n = 54) of these occur within the first351 nucleotides from the 5' end of the outgroup sequence.Neighbor-joining analysis shows a large distal clade (52.5% ofmottled ducks sampled in our study) composed of two reciprocallymonophyletic clades of mottled duck haplotypes, one of which is endemicto Texas and Louisiana and the other endemic to Florida. No mottledducks sampled in Florida occur in the clade composed of mottled ducksfrom Texas and Louisiana or vice versa, suggesting that (1) an enduringgeographic split has existed for many years between east and west, and(2) gene flow currently is non-existent (or at least undetectable)across the central Gulf Coast. The remaining 47.5% of mottledducks sampled in our study branch basally from this derived clade, showsubstantially less hierarchical structure, and fall into various lineagegroups of mixed species composition with no geographic orspecies-specific pattern. Pairwise F _ST valuescorroborate the pattern of strong differentiation observed betweenTexas/Louisiana and Florida. Our findings are consistent with apattern of partial lineage sorting from a polymorphic ancestral genepool reshuffled by hybridizing mallards. Control region data andpatterns of divergence in mallard-like species worldwide, furthermore,suggest that mottled ducks are close relatives of Mexican ducks, and inturn nested within black ducks. Genetic similarities to nominatemallards are less likely to be the product of common ancestry, but theresult of past hybridization with a dichromatic mallard ancestor thatinvaded North America from Asia many generations ago. Our findings haveseveral important consequences for the conservation biology of mottledducks across the Gulf Coast and our understanding of the phylogeographyof mallard-like species worldwide.     

In this issue: Proteomics 11/2008

In this issue of Proteomics you will find the following highlighted articles: Pancreatic cancer signs autograph on micro antibody array Pancreatic cancer has been one of the nastier members of the “Discovered‐too‐late‐to‐do‐anything‐about‐it” disease club. Its 5‐year survival rate is 3–5 % because of late diagnosis and no effective therapy for advanced disease cases. This paper by Ingvarsson et al. reports their encouraging findings on the use of recombinant antibody microarrays to survey serum for diagnostic and prognostic proteins. In these “proof‐of‐concept” experiments they found a signature of 19 unique scFv antibodies, specific for immunoregulatory proteins, that could distinguish pancreatic cancer from normal and from Helicobacter pylori (an indicator of inflammation, 3 out of 14 overlap). The test panel distinguished long and short survivors (with only one long survivor misclassified). Data was classified using a Support Vector Machine. The classifier was validated by multiple splits of the data and leave‐one‐out tests. Ingvarsson, J. et al., Proteomics 2008, 8, 2211–2219. Of cadmium and zinc: Brothers or not? Cadmium and zinc occupy the same column in the periodic table so you might expect some biological similarities. Not much luck – mercury is also in that column. Zinc, under tight control, is an essential mineral; cadmium is toxic and induces a variety of defensive responses. A highly zinc‐resistant cell line (HZR) has been derived from the human HeLa line. Rousselet et al. have compared the proteomes of HZR and HeLa cultured in Cd and Zn using a variety of proteomic and genomic tools. MALDI‐TOF MS after 2‐DE revealed examples of a co‐chaperone, a heat‐shock organizing protein (Hop), ubiquitin and a number of reactive oxygen species control proteins elevated in HZR. Of special interest was 4‐hydroxyphenyl‐pyruvate dioxygenase (HPPD), catalyst of one of the first breakdown steps of tyrosine. The complex relationships revealed will require a lot more than one paragraph for explanation. Rousselet, E. et al., Proteomics 2008, 8, 2244–2255. Grey box proteomics of salty species In the classic black box experiment you know nothing about the contents of the box. I propose a grey box for experiments directed by homologous knowledge – like these. Pandhal et al. have developed a protocol for proteomic analysis of an unsequenced species by homology. The organism of interest is a halotolerant cyanobacterium, Euhalothece sp. which can grow in NaCl concentrations ranging from 0% to >9% NaCl. The nearest sequenced relative is a Synechocystis sp. By metabolic labeling with ¹⁵N/­¹⁴N, the researchers were able to use MS to match proteins from the two species and also quantitate changes in levels of proteins in response to salt levels. Three labelling experiments ([% NaCl], 0% +3%, 3% +6%, and 3% +9%) yielded 229, 212, and 96 proteins, respectively, by MASCOT search of proteins with two peptides of each isotope. MS BLAST found 32, 30, and 7 more proteins, respectively. Pandhal, J. et al., Proteomics 2008, 8, 2266–2284.     

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi‐functional platelets and cross‐species function evolution

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high‐throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin‐stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across‐species evolutionary link that the orthologous proteins representing “core proteome”, and the “evolutionary proteome” is actually a relatively static proteome.     

Frequency of mallard-black duck hybrids along the atlantic coast determined by electrophoresis and plumage analysis

The frequency of mallard and black duck hybrids along the Atlantic flyway was estimated based on serum protein and serum esterase electrophoresis and plumage analysis. Hybrid frequencies for black ducks were low (below 10%) in Nova Scotia - Maine, Ontario and New Jersey and high (above 10%) in Maryland and Massachusetts. Plumage estimates tended to be higher for black ducks in Massachusetts and Maryland than estimates based on the electrophoretic work.     

Cover Picture: Proteomics 11/2008

In this issue of Proteomics you will find the following highlighted articles: Pancreatic cancer signs autograph on micro antibody array Pancreatic cancer has been one of the nastier members of the “Discovered‐too‐late‐to‐do‐anything‐about‐it” disease club. Its 5‐year survival rate is 3–5 % because of late diagnosis and no effective therapy for advanced disease cases. This paper by Ingvarsson et al. reports their encouraging findings on the use of recombinant antibody microarrays to survey serum for diagnostic and prognostic proteins. In these “proof‐of‐concept” experiments they found a signature of 19 unique scFv antibodies, specific for immunoregulatory proteins, that could distinguish pancreatic cancer from normal and from Helicobacter pylori (an indicator of inflammation, 3 out of 14 overlap). The test panel distinguished long and short survivors (with only one long survivor misclassified). Data was classified using a Support Vector Machine. The classifier was validated by multiple splits of the data and leave‐one‐out tests. Ingvarsson, J. et al., Proteomics 2008, 8, 2211–2219. Of cadmium and zinc: Brothers or not? Cadmium and zinc occupy the same column in the periodic table so you might expect some biological similarities. Not much luck – mercury is also in that column. Zinc, under tight control, is an essential mineral; cadmium is toxic and induces a variety of defensive responses. A highly zinc‐resistant cell line (HZR) has been derived from the human HeLa line. Rousselet et al. have compared the proteomes of HZR and HeLa cultured in Cd and Zn using a variety of proteomic and genomic tools. MALDI‐TOF MS after 2‐DE revealed examples of a co‐chaperone, a heat‐shock organizing protein (Hop), ubiquitin and a number of reactive oxygen species control proteins elevated in HZR. Of special interest was 4‐hydroxyphenyl‐pyruvate dioxygenase (HPPD), catalyst of one of the first breakdown steps of tyrosine. The complex relationships revealed will require a lot more than one paragraph for explanation. Rousselet, E. et al., Proteomics 2008, 8, 2244–2255. Grey box proteomics of salty species In the classic black box experiment you know nothing about the contents of the box. I propose a grey box for experiments directed by homologous knowledge – like these. Pandhal et al. have developed a protocol for proteomic analysis of an unsequenced species by homology. The organism of interest is a halotolerant cyanobacterium, Euhalothece sp. which can grow in NaCl concentrations ranging from 0% to >9% NaCl. The nearest sequenced relative is a Synechocystis sp. By metabolic labeling with ¹⁵N/­¹⁴N, the researchers were able to use MS to match proteins from the two species and also quantitate changes in levels of proteins in response to salt levels. Three labelling experiments ([% NaCl], 0% +3%, 3% +6%, and 3% +9%) yielded 229, 212, and 96 proteins, respectively, by MASCOT search of proteins with two peptides of each isotope. MS BLAST found 32, 30, and 7 more proteins, respectively. Pandhal, J. et al., Proteomics 2008, 8, 2266–2284.