Spatial metabolomics reveal divergent cardenolide processing in the monarch (Danaus plexippus) and the common crow butterfly (Euploea core).

Although being famous for sequestering milkweed cardenolides, the mechanism of sequestration and where cardenolides are localized in caterpillars of the monarch butterfly (Danaus plexippus, Lepidoptera: Danaini) is still unknown. While monarchs tolerate cardenolides by a resistant Na+ /K+ -ATPase, it is unclear how closely related species such as the nonsequestering common crow butterfly (Euploea core, Lepidoptera: Danaini) cope with these toxins. Using novel atmospheric-pressure scanning microprobe matrix-assisted laser/desorption ionization mass spectrometry imaging, we compared the distribution of cardenolides in caterpillars of D. plexippus and E. core. Specifically, we tested at which physiological scale quantitative differences between both species are mediated and how cardenolides distribute across body tissues. Whereas D. plexippus sequestered most cardenolides from milkweed (Asclepias curassavica), no cardenolides were found in the tissues of E. core. Remarkably, quantitative differences already manifest in the gut lumen: while monarchs retain and accumulate cardenolides above plant concentrations, the toxins are degraded in the gut lumen of crows. We visualized cardenolide transport over the monarch midgut epithelium and identified integument cells as the final site of storage where defences might be perceived by predators. Our study provides molecular insight into cardenolide sequestration and highlights the great potential of mass spectrometry imaging for understanding the kinetics of multiple compounds including endogenous metabolites, plant toxins, or insecticides in insects.

Salivary corticosterone measurement in large-billed crows by enzyme-linked immunosorbent assay.

Salivary corticosteroid measurement, as a surrogate for plasma corticosteroid levels to evaluate an animal's stress or metabolic state, commonly used in mammals. However, the validity of salivary corticosterone (CORT) measurements in birds has not yet been reported. We aimed to measure salivary CORT in crows using a commercially available CORT enzyme-linked immunosorbent assay kit. An adrenocorticotropic hormone (ACTH) challenge experiment using synthetic cosyntropin, an ACTH analogue, was conducted to compare CORT level elevations between the serum and the saliva in a 10-60 min range. Both salivary and blood CORT was significantly elevated 10 min after injecting synthetic cosyntropin. The results supported the validation of salivary CORT as a surrogate for a blood CORT in crows.

A brain atlas of the carrion crow (Corvus corone).

Corvidae, passerine songbirds such as jays, crows, and ravens known as corvids, have become model systems for the study of avian cognition. The superior cognitive capabilities of corvids mainly emerge from a disproportionally large telencephalon found in these species. However, a systematic mapping of the neuroanatomy of the corvid brain, and the telencephalon in particular, is lacking so far. Here, we present a brain atlas of the carrion crow, Corvus corone, with special emphasis on the telencephalic pallium. We applied four staining techniques to brain slices (Nissl, myelin, combination of Nissl and myelin, and tyrosine hydroxylase targeting catecholaminergic neurons). This allowed us to identify brain nuclei throughout the brain and delineate the known pallial subdivisions termed hyperpallium, entopallium, mesopallium, nidopallium, arcopallium, and hippocampal complex. The extent of these subdivisions and brain nuclei are described according to stereotaxic coordinates. In addition, 3D depictions of pallial regions were reconstructed from these slices. While the overall organization of the carrion crow's brain matches other songbird brains, the relative proportions and expansions of associative pallial areas differ considerably in agreement with enhanced cognitive skills found in corvids. The presented global organization of the crow brain in stereotaxic coordinates will help to guide future neurobiological studies in corvids.

Behavior-specific occurrence patterns of Pinyon Jays (Gymnorhinus cyanocephalus) in three Great Basin study areas and significance for pinyon-juniper woodland management.

The Pinyon Jay is a highly social, year-round inhabitant of pinyon-juniper and other coniferous woodlands in the western United States. Range-wide, Pinyon Jays have declined ~ 3-4% per year for at least the last half-century. Occurrence patterns and habitat use of Pinyon Jays have not been well characterized across much of the species' range, and obtaining this information is necessary for better understanding the causes of ongoing declines and determining useful conservation strategies. Additionally, it is important to better understand if and how targeted removal of pinyon-juniper woodland, a common and widespread vegetation management practice, affects Pinyon Jays. The goal of this study was to identify the characteristics of areas used by Pinyon Jays for several critical life history components in the Great Basin, which is home to nearly half of the species' global population, and to thereby facilitate the inclusion of Pinyon Jay conservation measures in the design of vegetation management projects. To accomplish this, we studied Pinyon Jays in three widely separated study areas using radio telemetry and direct observation and measured key attributes of their locations and a separate set of randomly-selected control sites using the U. S. Forest Service's Forest Inventory Analysis protocol. Data visualizations, principle components analysis, and logistic regressions of the resulting data indicated that Pinyon Jays used a distinct subset of available pinyon-juniper woodland habitat, and further suggested that Pinyon Jays used different but overlapping habitats for seed caching, foraging, and nesting. Caching was concentrated in low-elevation, relatively flat areas with low tree cover; foraging occurred at slightly higher elevations with generally moderate but variable tree cover; and nesting was concentrated in slightly higher areas with high tree and vegetation cover. All three of these Pinyon Jay behavior types were highly concentrated within the lower-elevation band of pinyon-juniper woodland close to the woodland-shrubland ecotone. Woodland removal projects in the Great Basin are often concentrated in these same areas, so it is potentially important to incorporate conservation measures informed by Pinyon Jay occurrence patterns into existing woodland management paradigms, protocols, and practices.

Cholecystokinin induces crowing in chickens.

Animals that communicate using sound are found throughout the animal kingdom. Interestingly, in contrast to human vocal learning, most animals can produce species-specific patterns of vocalization without learning them from their parents. This phenomenon is called innate vocalization. The underlying molecular basis of both vocal learning in humans and innate vocalization in animals remains unknown. The crowing of a rooster is also innately controlled, and the upstream center is thought to be localized in the nucleus intercollicularis (ICo) of the midbrain. Here, we show that the cholecystokinin B receptor (CCKBR) is a regulatory gene involved in inducing crowing in roosters. Crowing is known to be a testosterone (T)-dependent behavior, and it follows that roosters crow but not hens. Similarly, T-administration induces chicks to crow. By using RNA-sequencing to compare gene expression in the ICo between the two comparison groups that either crow or do not crow, we found that CCKBR expression was upregulated in T-containing groups. The expression of CCKBR and its ligand, cholecystokinin (CCK), a neurotransmitter, was observed in the ICo. We also showed that crowing was induced by intracerebroventricular administration of an agonist specific for CCKBR. Our findings therefore suggest that the CCK system induces innate vocalization in roosters.

The expression of tyrosine hydroxylase and DARPP-32 in the house crow (Corvus splendens) brain.

Birds of the family Corvidae which includes diverse species such as crows, rooks, ravens, magpies, jays, and jackdaws are known for their amazing abilities at problem-solving. Since the catecholaminergic system, especially the neurotransmitter dopamine, plays a role in cognition, we decided to study the distribution of tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines in the brain of house crows (Corvus splendens). We also studied the expression of DARPP-32 (dopamine and cAMP-regulated phosphoprotein), which is expressed in dopaminoceptive neurons. Our results demonstrated that as in other avian species, the expression of both TH and DARPP-32 was highest in the house crow striatum. The caudolateral nidopallium (NCL, the avian analogue of the mammalian prefrontal cortex) could be differentiated from the surrounding pallial regions based on a larger number of TH-positive "baskets" of fibers around neurons in this region and greater intensity of DARPP-32 staining in the neuropil in this region. House crows also possessed distinct nuclei in their brains which corresponded to song control regions in other songbirds. Whereas immunoreactivity for TH was higher in the vocal control region Area X compared to the surrounding MSt (medial striatum) in house crows, staining in RA and HVC was not as prominent. Furthermore, the arcopallial song control regions RA (nucleus robustus arcopallialis) and AId (intermediate arcopallium) were strikingly negative for DARPP-32 staining, in contrast to the surrounding arcopallium. Patterns of immunoreactivity for TH and DARPP-32 in "limbic" areas such as the hippocampus, septum, and extended amygdala have also been described.

The aryl hydrocarbon receptor 2 potentially mediates cytochrome P450 1A induction in the jungle crow (Corvus macrorhynchos).

To understand the role of aryl hydrocarbon receptor (AHR) isoforms in avian species, we investigated the functional characteristics of two AHR isoforms (designated as jcAHR1 and jcAHR2) of the jungle crow (Corvus macrorhynchos). Two amino acid residues corresponding to Ile324 and Ser380 (high sensitive type) in chicken AHR1 that are known to determine dioxin sensitivity were Ile325 and Ala381 (moderate sensitive type) in jcAHR1 and Val306 and Ala362 (low sensitive type) in jcAHR2. The quantitative comparison of the two jcAHR mRNA expression levels in a Tokyo jungle crow population showed that jcAHR2 accounted for 92.4% in the liver, while jcAHR1 accounted for only 7.6%. Both in vitro-expressed jcAHR1 and jcAHR2 proteins exhibited a specific binding to [3H]-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transactivation potencies for jcAHR1 and jcAHR2 in in vitro reporter gene assays were measured in jcAHR-expressed cells exposed to 16 dioxins and related compounds (DRCs). Both jcAHR1 and jcAHR2 were activated in a congener- and an isoform-specific manner. EC50 value of TCDD for jcAHR2 (0.61 nM) was six-fold higher than that for jcAHR1 (0.098 nM), but jcAHR2 had higher transactivation efficacy than jcAHR1 in terms of the magnitude of response. The high transactivation efficacy of jcAHR2 in DRCs is in contrast to that of AHR2s in other avian species with low transactivation efficacy. Molecular docking simulations of TCDD with in silico jcAHR1 and jcAHR2 homology models showed that the two sensitivity-decisive amino acids indirectly controlled TCDD-binding modes through their surrounding amino acids. Deletion assays of jcAHR2 revealed that 736-805 amino acid residues in the C-terminal region were critical for its transactivation. We suggest that jcAHR2 plays a critical role in regulating the AHR signaling pathway, at least in its highly expressed organs.

Constant light environment suppresses maturation and reduces complexity of new born neuron processes in the hippocampus and caudal nidopallium of a diurnal corvid: Implication for impairment of the learning and cognitive performance.

Periodic day-night environment shapes the temporal pattern in the behaviour and physiology (e.g. 24-h activity-rest and sleep-wake cycles) and the advanced brain function, such as learning, memory and decision making. In a previous study, we showed the abolition of 24-h rhythm in the activity-rest pattern, and an attenuated cognitive performance in diurnal Indian house crows (Corvus splendens) under constant light (no-night; LL) environment. Present study extended this, and investigated LL-induced effects on the neurogenesis (birth, maturation and neurite complexity of new born neurons) in the hippocampus and caudal nidopallium, the brain regions directly associated with learning and cognition in birds. We performed immunohistochemistry of doublecortin (DCX; a neurogenesis marker) and tyrosine hydroxylase (TH, a key enzyme of the dopamine biosynthesis) in the brain section containing hippocampus or caudal nidopallium of Indian house crows exposed for 2 weeks to LL, with controls maintained under 12L:12D. As expected, crows showed arrhythmicity with a significantly reduced rest period in the 24-h activity-rest pattern, and a decreased cognitive performance when tested for the spatial and pattern association learning tasks under LL. Importantly, there was a significant decrease in DCX-immunoreactive (ir) cells and, as shown by Sholl analysis, in the complexity of DCX-ir neurites in both, the hippocampus and caudal nidopallium of crows under LL, as compared to those under 12L:12D. The anatomical proximity of DCX-ir neurons with TH-ir fibers suggested a functional association of the new born hippocampal and caudal nidopallial neurons with the learning, and perhaps cognition in Indian house crows. These results give insights into possible impact of the loss of night on brain health and functions in an emerging ecosystem in which other diurnal species including humans may be inadvertently exposed to an illuminated night, such as in an overly lighted metropolitan urban habitat.

Relationship between Pb and Cd accumulations in house crow, their habitat, and food content from Klang area, Peninsular Malaysia.

Heavy metal pollution has become a global concern due to accumulation in tissue and transferable effects to humans via the food chain. This study focused on monitoring the accumulation of cadmium (Cd) and lead (Pb) in surface soil and body content: bone, heart, brain, liver, lung, muscle, kidney, feathers, feces, and gizzard contents of house crow Corvus splendens in the Klang region, Malaysia. The results revealed the occurrence of Pb and Cd in all biological samples from house crows, food contents, and surface soil samples. Heart and kidney accrued high amounts of Cd, while high amounts of Pb were found to accumulate in bones and feathers. Major discrepancies were also discovered in the concentrations of metals between juvenile and adults, as well as female and male bird samples. Concentrations of Pb and Cd in house crow internal tissues correlated significantly with that of bird feathers, but none could be established with that of surface soil. In addition, a significant correlation was observed between Pb concentration in the internal tissues to that of the feces, but the same was not the case when compared with the surface soil concentration. Metal accrual in the house crows feathers and feces may be through a long-term transmission via the food chain, which are eliminated from feathers via molting. This may suggest the utility of molted breast feathers of house crow in the bio-monitoring of Cd and Pb contamination, whereas feces of house crow appear only to be suitable for the bio-monitoring of Pb contamination.

The use of feather as an indicator for heavy metal contamination in house crow (Corvus splendens) in the Klang area, Selangor, Malaysia.

The Klang area of Peninsular Malaysia has experienced rapid industrial growth with intense activities, which can increase the concentration of pollutants in the environment that significantly impact on habitats and the human health. The purpose of this study was to determine the levels of selected heavy metals (Cu, Zn, Ni, Fe, and Pb) in the heart, lung, brain, liver, kidney, muscle tissues, and feathers of house crow, Corvus splendens, in Klang, Peninsular Malaysia. House crow samples were collected from the Klang area through the Department of Public Health at Majlis Perbandaran Klang. Quantitative determination of heavy metals was carried out using atomic absorption spectrophotometer (AAS). The result shows the presence of heavy metals in all biological samples of house crows. For heavy metals in all the house crow tissues analyzed, Fe concentrations were the highest, followed by those of Zn, Cu, Pb, and Ni. The feathers and kidney accumulated high concentrations of Pb, whereas the liver accumulated high concentrations of essential heavy metals (Fe > Zn > Cu > Ni). Significant variations were also detected in the concentrations of Pb among adult and juvenile and male and female bird samples. The results also revealed significant positive correlations between Pb metal concentration in the breast feathers and all internal organs. Accumulation of toxic heavy metals in feathers reflected storing and elimination processes, while the accumulation of toxic heavy metals in the kidney can be consequential to chronic exposure. The present study clearly shows the usefulness of house crow breast feather as a suitable indicator for heavy metal accumulation in the internal organs of house crows in the Klang area.

In vitro and in silico evaluation of transactivation potencies of avian AHR1 and AHR2 by endogenous ligands: Implications for the physiological role of avian AHR2.

Aryl hydrocarbon receptor (AHR) is well conserved from invertebrates to vertebrates, and it mediates the toxic effects of exogenous ligands, including dioxins. Recent studies reported that AHRs activated by endogenous ligands play critical roles in mammalian physiological homeostasis. Avian species possess at least two AHR isoforms (AHR1 and AHR2), which exhibit species- and isoform-specific transactivation potencies to exogenous ligands, whereas mammals possess a single AHR. To delineate the profiles and roles of endogenous ligands for avian AHR isoforms, we investigated in vitro transactivation potencies of avian AHRs (AHR1 and AHR2 from the jungle crow, Corvus macrorhynchos; common cormorant, Phalacrocorax carbo; and black-footed albatross, Phoebastria nigripes) treated with the endogenous tryptophan metabolites 6-formylindolo [3,2-b] carbazole (FICZ), l-kynurenine (l-Kyn), kynurenic acid (KYNA), and indoxyl sulfate (IS). Furthermore, we analyzed the binding mode of these ligands to each avian AHR isoform by in silico docking simulations. The EC50 of FICZ (0.009-0.032nM) was similar regardless of the species or isoform of AHR. The estimated in silico binding mode of FICZ to AHRs was well conserved in both isoforms. The transactivation potencies of avian AHRs to other tryptophan metabolites were 10(5)-10(7) fold lower than those for FICZ, and EC50 values varied in a species- and isoform-specific manner. This was consistent with poor conservation of the binding mode of l-Kyn, KYNA, and IS predicted in in silico docking simulations. Our results suggest that in avian species, FICZ is the most potent endogenous AHR ligand, and that AHR1 and AHR2 are physiologically functional.

Loner or socializer? Ravens' adrenocortical response to individual separation depends on social integration.

Non-breeding common ravens (Corvus corax) live in complex social groups with a high degree of fission-fusion dynamics. They form valuable relationships and alliances with some conspecifics, while taking coordinated action against others. In ravens, affiliates reconcile their conflicts, console each other after conflicts with a third party, and provide each other with social support - all behaviors that presumably reduce corticosterone levels and alleviate stress. However, how well an individual is socially integrated in a (sub)group might vary substantially. This raises the question whether the social integration of a raven affects its stress responses to fission-fusion dynamics. The present study aims to investigate this effect experimentally by separating single ravens (n=16) individually from their group for four days and subsequently reintroducing them. To determine stress response patterns in the separated individuals we measured the amounts of immunoreactive corticosterone metabolites (CM) in droppings. We compared two enzyme immunoassays, which we validated by conducting an ACTH challenge, and finally decided to apply an 11-oxoetiocholanolone enzyme immunoassay. Additionally, we determined levels of social integration using focal observations. Our findings suggest that a strong social integration is related to low CM levels when the individuals are within the group and high levels during separations, implying that separation leads to stress in these birds. In contrast, poorly socially integrated ravens seem to exhibit the opposite pattern, indicating that to them group living is more stressful than being temporarily separated. We, therefore, conclude that the birds' adrenocortical activity is modulated by their social integration.

Sex-reversed correlation between stress levels and dominance rank in a captive non-breeder flock of crows.

Group living has both benefits and costs to individuals; benefits include efficient acquisition of resources, and costs include stress from social conflicts among group members. Such social challenges result in hierarchical dominance ranking among group members as a solution to avoid escalating conflict that causes different levels of basal stress between individuals at different ranks. Stress-associated glucocorticoid (corticosterone in rodents and birds; CORT) levels are known to correlate with dominance rank in diverse taxa and to covary with various social factors, such as sex and dominance maintenance styles. Although there is much evidence for sex differences in the basal levels of CORT in various species, the correlation of sex differences in basal CORT with dominance rank is poorly understood. We investigated the correlation between CORT metabolites (CM) in the droppings and social factors, including rank and sex, in a captive non-breeder group of crows. In this group, all the single males dominated all the single females, and dominance ranks were stable among single males but relatively unstable among single females. CM levels and rank were significantly correlated in a sex-reversed fashion: males at higher rank (i.e., more dominant) had higher CM, whereas females at higher rank exhibited lower CM. This is the first evidence of sex-reversed patterns of CM-rank correlation in birds. The results suggest that different mechanisms of stress-dominance relationships operate on the sexes in non-breeder crow aggregations; in males, stress is associated with the cost of aggressive displays, whereas females experience subordination stress due to males' overt aggression.

The hooded crow (Corvus cornix) as an environmental bioindicator species of heavy metal contamination.

This study aims to examine the possible presence of lead and cadmium in the liver and kidneys of hooded crows (Corvus cornix). Liver and kidneys of hooded crow carcasses were collected in Province of Cuneo (Piedmont, Italy) in order to detect lead and cadmium content. Significant differences were found in lead and cadmium levels between areas of intensive cultivation versus areas where meadows are prevalent. Moreover, age greatly influenced the burden of heavy metals, while sex did not seem to affect the level of contamination. The source of contamination may be phosphate fertilizers used for intensive cultivation in the study area.

De novo assembly and analysis of crow lungs transcriptome.

The jungle crow (Corvus macrorhynchos) belongs to the order Passeriformes of bird species and is important for avian ecological and evolutionary genetics studies. However, there is limited information on the transcriptome data of this species. In the present study, we report the characterization of the lung transcriptome of the jungle crow using GS FLX Titanium XLR70. Altogether, 1,510,303 high-quality sequence reads with 581,198,230 bases was de novo assembled into 22,169 isotigs (isotig represents an individual transcript) and 784,009 singletons. Using these isotigs and 581,681 length-filtered (greater than 300 bp) singletons, 20,010 unique protein-coding genes were identified by BLASTx comparison against a nonredundant (nr) protein sequence database. Comparative analysis revealed that 46,604 (70.29%) and 51,642 (72.48%) of the assembled transcripts have significant similarity to zebra finch and chicken RefSeq proteins, respectively. As determined by GO annotation and KEGG pathway mapping, functional annotation of the unigenes recovered diverse biological functions and processes. Transcripts putatively involved in the immune response were identified. Furthermore, 20,599 single nucleotide polymorphisms (SNPs) and 7525 simple sequence repeats (SSRs) were retrieved from the assembled transcript database. This resource should lay an important base for future ecological, evolutionary, and conservation genetic studies on this species and in other related species.

Procedures for identifying infectious prions after passage through the digestive system of an avian species.

Infectious prion (PrP(Res)) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases(1). Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal(2,3) as well as from environmental sources(4-6). Scavengers and carnivores have potential to translocate PrP(Res) material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrP(Res) material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger(7). We describe procedures used to document passage of PrP(Res) material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Prion remains infectious after passage through digestive system of American crows (Corvus brachyrhynchos).

Avian scavengers, such as American crows (Corvus brachyrhynchos), have potential to translocate infectious agents (prions) of transmissible spongiform encephalopathy (TSE) diseases including chronic wasting disease, scrapie, and bovine spongiform encephalopathy. We inoculated mice with fecal extracts obtained from 20 American crows that were force-fed material infected with RML-strain scrapie prions. These mice all evinced severe neurological dysfunction 196-231 d postinoculation (x =198; 95% CI: 210-216) and tested positive for prion disease. Our results suggest a large proportion of crows that consume prion-positive tissue are capable of passing infectious prions in their feces (ˆp=1.0; 95% CI: 0.8-1.0). Therefore, this common, migratory North American scavenger could play a role in the geographic spread of TSE diseases.

Essential and nonessential elements in nestling rooks Corvus frugilegus from eastern Poland with a special emphasis on their high cadmium contamination.

Concentration of minerals (sodium, potassium, calcium [Ca], magnesium, iron [Fe], copper, zinc [Zn], manganese [Mn], and cobalt) as well as toxic metals (cadmium [Cd], lead [Pb]) were determined in five tissues (liver, lung, kidney, muscle, and bone) of nestling rooks (Corvus frugilegus; 1 to 13 days old) found dead in seven breeding colonies in eastern Poland. Cd concentration in all analyzed tissues was in the narrow range of 17.0-17.2 mg/kg dry weight (dw) Cd, which in the light of the literature data indicates acute contamination by this toxic metal. Similarly, we found increased levels of Pb, which in all tissues ranged between 5.0 and 6.2 mg/kg dw. Results of multivariate general linear model (GLM) testing of the effect of three variables (tissue type, colony, and nestling age) on tissue concentrations of various metals showed significance for Fe, Cu, Zn, and Mn. Only concentrations of Ca, Fe, and Zn differed significantly between the analyzed tissues. GLM analysis did not show any statistically significant differences in tissue levels of minerals and both toxic metals among examined rookeries, which indicates the widespread presence of nonpoint Cd and Pb pollution linked to agricultural activity and similar levels of these inorganic contaminants on crop fields (feeding grounds) around breeding colonies. We concluded that high levels of both toxic metals, Cd and Pb, probably resulting from the diet of nestling rooks, are based mainly on a diet of ground-dwelling beetles gathered on crop fields.

Microstructure of the feather in Japanese Jungle Crows (Corvus macrorhynchos) with distinguishing gender differences.

Assessing gender difference in Japanese Jungle Crows (Corvus macrorhynchos) is difficult by gross observation because both sexes have black plumage colors. Careful observation of the plumage, however, reveals that it is actually iridescent glossy purple and dark-green in color, and that these colors are more marked in adult males than in females. In birds, such iridescent structural colors are generally produced in the feather barbules, where light is scattered constructively by laminar arrays consisting of alternating layers of materials with different refractive indices, namely keratin, melanin and air. We have investigated differences in the microstructure of the feathers of male and female Jungle Crows by means of scanning and transmission electron microscopy. Male birds had more barbs than females, and the length of the prongs was shorter in males than in females. The density of the melanin granules in the cross-section of barbules was higher in males than in females. Moreover, only in males did the melanin granules show an ordered arrangement beneath a keratin cortex layer at the edges of barbules. These results demonstrate that there are microstructural differences in the feathers of male and female Jungle Crows and suggest that the Jungle Crows' feathers may have iridescent coloring that differs according to gender.