Throughout the animal kingdom, adaptive colouration serves critical functions ranging from inconspicuous camouflage to ostentatious sexual display, and can provide important information about the environment and biology of a particular organism. The most ubiquitous and abundant pigment, melanin, also has a diverse range of non-visual roles, including thermoregulation in ectotherms. However, little is known about the functional evolution of this important biochrome through deep time, owing to our limited ability to unambiguously identify traces of it in the fossil record. Here we present direct chemical evidence of pigmentation in fossilized skin, from three distantly related marine reptiles: a leatherback turtle, a mosasaur and an ichthyosaur. We demonstrate that dark traces of soft tissue in these fossils are dominated by molecularly preserved eumelanin, in intimate association with fossilized melanosomes. In addition, we suggest that contrary to the countershading of many pelagic animals, at least some ichthyosaurs were uniformly dark-coloured in life. Our analyses expand current knowledge of pigmentation in fossil integument beyond that of feathers, allowing for the reconstruction of colour over much greater ranges of extinct taxa and anatomy. In turn, our results provide evidence of convergent melanism in three disparate lineages of secondarily aquatic tetrapods. Based on extant marine analogues, we propose that the benefits of thermoregulation and/or crypsis are likely to have contributed to this melanisation, with the former having implications for the ability of each group to exploit cold environments.

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded southern California during 2003, successfully overwintered, amplified to epidemic levels, and then dispersed to every county in the state. Although surveillance programs successfully tracked and measured these events, mechanisms that allowed the efficient overwintering and subsequent amplification of WNV have not been elucidated. Our current research provided evidence for three mechanisms whereby WNV may have persisted in southern California during the winters of 2003-2004 and 2004-2005: 1) continued enzootic transmission, 2) vertical transmission by Culex mosquitoes, and 3) chronic infection in birds. WNV was detected in 140 dead birds comprising 32 species, including 60 dead American crows, thereby verifying transmission during the November-March winter period. Dead American crows provide evidence of recent transmission because this species always succumbs rapidly after infection. However, WNV RNA was not detected concurrently in 43,043 reproductively active female mosquitoes comprising 11 species and tested in 1,258 pools or antibody in sera from 190 sentinel chickens maintained in 19 flocks. Although efficient vertical transmission by WNV was demonstrated experimentally for Culex tarsalis Coquillett infected per os, 369 females collected diapausing in Kern County and tested in 32 pools were negative for WNV. Vertical transmission was detected in Culex pipiens quinquefasciatus Say adults reared from field-collected immatures collected from Kern County and Los Angeles during the summer transmission period. Chronic infection was detected by finding WNV RNA in 34 of 82 birds that were inoculated with WNV experimentally, held for >6 wk after infection, and then necropsied. Frequent detection of WNV RNA in kidney tissue in experimentally infected birds >6 wk postinfection may explain, in part, the repeated detection of WNV RNA in dead birds recovered during winter, especially in species such as mourning doves that typically do not die after experimental infection. In summary, our study provides limited evidence to support multiple modes of WNV persistence i n southern California. Continued transmission andvertical transmission by Culex p. quinquefasciatus Say seem likely candidates for further study.

Archaeopteryx has been regarded as an icon of evolution ever since its discovery from the Late Jurassic limestone deposits of solnhofen, Germany in 1861. Here we report the first evidence of colour from Archaeopteryx based on fossilized colour-imparting melanosomes discovered in this isolated feather specimen. using a phylogenetically diverse database of extant bird feathers, statistical analysis of melanosome morphology predicts that the original colour of this Archaeopteryx feather was black, with 95% probability. Furthermore, reexamination of the feather's morphology leads us to interpret it as an upper major primary covert, contrary to previous interpretations. Additional findings reveal that the specimen is preserved as an organosulphur residue, and that barbule microstructure identical to that of modern bird feathers had evolved as early as the Jurassic. As in extant birds, the extensive melanization would have provided structural advantages to the Archaeopteryx wing feather during this early evolutionary stage of dinosaur flight.

Feathers are amongst the most complex epidermal structures known, and they have a well-documented evolutionary trajectory across non-avian dinosaurs and basal birds. Moreover, melanosome-like microbodies preserved in association with fossil plumage have been used to reconstruct original colour, behaviour and physiology. However, these putative ancient melanosomes might alternatively represent microorganismal residues, a conflicting interpretation compounded by a lack of unambiguous chemical data. We therefore used sensitive molecular imaging, supported by multiple independent analytical tests, to demonstrate that the filamentous epidermal appendages in a new specimen of the Jurassic paravian Anchiornis comprise remnant eumelanosomes and fibril-like microstructures, preserved as endogenous eumelanin and authigenic calcium phosphate. These results provide novel insights into the early evolution of feathers at the sub-cellular level, and unequivocally determine that melanosomes can be preserved in fossil feathers.

The invasion of different southern California landscapes by West Nile virus (WNV) and its subsequent amplification to epidemic levels during 2004 enabled us to study the impact of differing corvid populations in three biomes: the hot Colorado desert with few corvids (Coachella Valley), the southern San Joaquin Valley (Kern County) with large western scrub-jay but small American crow populations, and the cool maritime coast (Los Angeles) with a large clustered American crow population. Similar surveillance programs in all three areas monitored infection rates in mosquitoes, seroconversion rates in sentinel chickens, seroprevalence in wild birds, numbers of dead birds reported by the public, and the occurrence of human cases. Infection rates in Culex tarsalis Coquillett and sentinel chicken seroconversion rates were statistically similar among all three areas, indicating that highly competent mosquito hosts were capable of maintaining enzootic WNV transmission among less competent and widely distributed avian hosts, most likely house sparrows and house finches. In contrast, infection rates in Culex pipiens quinquefasciatus Say were statistically higher in Kern and Los Angeles counties with elevated corvid populations than in Coachella Valley with few corvids. Spatial analyses of dead corvids showed significant clusters near known American crow roosts in Los Angeles that were congruent with clusters of human cases. In this area, the incidence of human and Cx. p. quinquefasciatus infection was significantly greater within corvid clusters than without, indicating their importance in virus amplification and as a risk factor for human infection. In contrast the uniform dispersion by territorial western scrub-jays resulted in a high, but evenly distributed, incidence of human disease in Kern County.

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded southern California during 2003, successfully overwintered, amplified to epidemic levels, and then dispersed to every county in the state. Although surveillance programs successfully tracked and measured these events, mechanisms that allowed the efficient overwintering and subsequent amplification of WNV have not been elucidated. Our current research provided evidence for three mechanisms whereby WNV may have persisted in southern California during the winters of 2003-2004 and 2004-2005: 1) continued enzootic transmission, 2) vertical transmission by Culex mosquitoes, and 3) chronic infection in birds. WNV was detected in 140 dead birds comprising 32 species, including 60 dead American crows, thereby verifying transmission during the November-March winter period. Dead American crows provide evidence of recent transmission because this species always succumbs rapidly after infection. However, WNV RNA was not detected concurrently in 43,043 reproductively active female mosquitoes comprising 11 species and tested in 1,258 pools or antibody in sera from 190 sentinel chickens maintained in 19 flocks. Although efficient vertical transmission by WNV was demonstrated experimentally for Culex tarsalis Coquillett infected per os, 369 females collected diapausing in Kern County and tested in 32 pools were negative for WNV. Vertical transmission was detected in Culex pipiens quinquefasciatus Say adults reared from field-collected immatures collected from Kern County and Los Angeles during the summer transmission period. Chronic infection was detected by finding WNV RNA in 34 of 82 birds that were inoculated with WNV experimentally, held for >6 wk after infection, and then necropsied. Frequent detection of WNV RNA in kidney tissue in experimentally infected birds >6 wk postinfection may explain, in part, the repeated detection of WNV RNA in dead birds recovered during winter, especially in species such as mourning doves that typically do not die after experimental infection. In summary, our study provides limited evidence to support multiple modes of WNV persistence i n southern California. Continued transmission andvertical transmission by Culex p. quinquefasciatus Say seem likely candidates for further study.

One-sentence summary for table of contents: Aerial spraying reduced incidence of human infection.

Three diagnostic assays for detecting West Nile virus (WNV) in avian oral swabs were evaluated in California in 2004 and 2005: two commercial antigen-capture assays, VecTest and Rapid Analyte Measurement Platform (RAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) of oral swabs in a specialized viral transport medium (VTM). Results from this study demonstrated that VTM was excellent for transportation and maintenance of WNV in avian oral swab samples and allowed for detection by RT-PCR and subsequent confirmation by virus isolation. Oral swabs and kidney tissue in VTM tested by RT-PCR were found to have similar accuracy in detecting WNV in corvids. The two antigen-capture assays, VecTest and RAMP, provided few false positives for corvids, with over 95% specificity. When performed by multiple local agencies throughout the state, VecTest and RAMP were similarly sensitive for oral swabs of American Crows (Corvus brachyrhynchos) (70% and 64%, respectively). Data from known WNV positive corvid oral swabs in VTM tested by antigen-capture assays at a diagnostic laboratory suggested that RAMP was more sensitive than VecTest. Due to high probability of false negatives, neither test is recommended for use on non-corvids. While WNV antigen-capture assays were effective screening tools for corvids, they were markedly less sensitive for Western Scrub Jays (Aphelocoma californica).