Regulation of pheromone biosynthesis in the "Z strain" of the European corn borer, Ostrinia nubilalis

The regulation of pheromone biosynthesis by the neuropeptide PBAN in the Z strain of the European corn borer, Ostrinia nubilalis, was investigated using labeled intermediates. Injection of radiolabeled acetate showed PBAN did not influence the de novo synthesis of saturated fatty acids in the gland. When deuterium-labeled myristic acid was topically applied to the gland, females injected with PBAN produced more labeled pheromone than did control females, indicating that PBAN controls one of the later steps of pheromone biosynthesis. Although more myristic acid was Delta11-desaturated in the gland in the presence of PBAN, this was counterbalanced by less Delta11-desaturation of palmitic acid, indicating that desaturase activity did not change overall. This change in flux of myristic acid through to pheromone was shown to be caused by increased reduction of fatty acid pheromone precursors occurring in the presence of PBAN.     

Reduction of the Acyl Group: The Critical Step in Bombykol Biosynthesis That Is Regulated in Vitro by the Neuropeptide Hormone in the Pheromone Gland of Bombyx mori

The sex pheromone of Bombyx mori, bombykol [(10E,12Z)-10,12-hexadecadien-1-ol], can be biosynthesized in four steps: construction of a hexadecanoic moiety from acetyl CoA, ?-11-desaturation, .?-10,12-desaturation, and reduction of the acyl group. This biosynthesis is regulated by a hormone named the pheromone biosynthesis activating neuropeptide (PBAN). To examine the steps that are accelerated by this neurohormone, pheromone glands excised from decapitated females were incubated in vitro with either ¹⁴C-Iabeled sodium acetate or one of three fatty acids [hexadecanoic acid, (Z)-11-hexadecenoic acid, or (10E,12Z)-10,12-hexadecadienoic acid]. After analyzing the radioactivity that was incorporated from each precursor into bombykol and the biosynthetic precursors, it was observed that the first three steps proceeded in glands both treated and untreated with synthetic PBAN of B. mori; however, the last step proceeded only in the treated glands. From this in vitro experiment, it can be concluded that the main regulatory role of PBAN is in the reduction of the acyl group in B. mori, as was shown by our previous in vivo experiment.     

Sex pheromone biosynthesis in the Asian corn borer Ostrinia furnacalis (II): Biosynthesis of (E)- and (Z)-12-tetradecenyl acetate involves Δ14 desaturation

Sex pheromone biosynthesis in the Asian corn borer Ostrinia furnacalis was studied by topical application of deuterium labelled fatty acids to the pheromone gland. The incorporation of the labelled acids into pheromone components and precursors was determined by gas chromatography with flame ionization detection and mass spectrometry in the selected ion monitoring mode. The labelling experiments suggest that the pheromone components (E)- and (Z)-12-tetradecenyl acetates are biosynthesized from palmitic acid by δ14 desaturation, followed by chain shortening (β-oxidation), reduction, and acetylation. This is the first confirmation of a Δ14 desaturase in an eukaryotic system.     

Control of the biosynthetic pathway of Sesamia nonagrioides sex pheromone by the pheromone biosynthesis activating neuropeptide

(Z)-11-Hexadecenyl acetate, the main pheromone component of Sesamia nonagrioides sex pheromone, is biosynthesized from palmitic acid by Delta(11)-desaturation followed by reduction and acetylation. Production of (Z)-11-hexadecenyl acetate is regulated by the Pheromone Biosynthesis Activating Neuropeptide (PBAN). Transformation of (Z)-11-hexadecen-1-ol into the corresponding acetate is a target step for PBAN in the regulation of this biosynthetic sequence, thus being the first example of a PBAN-activated acetylation. The production of the minor component (Z)-11-hexadecenal is also stimulated by PBAN. The usefulness of pentafluorobenzyloxime-derivatives for the analysis of aldehyde pheromone constituents by gas chromatography coupled to mass spectrometry is also reported.     

Evidence for two-step regulation of pheromone biosynthesis by the pheromone biosynthesis-activating neuropeptide in the moth Heliothis virescens

The control of pheromone biosynthesis by the neuropeptide PBAN was investigated in the moth Heliothis virescens. When decapitated females were injected with [2-(14)C] acetate, females co-injected with PBAN produced significantly greater quantities of radiolabeled fatty acids in their pheromone gland than females co-injected with saline. This indicates that PBAN controls an enzyme involved in the synthesis of fatty acids, probably acetyl CoA carboxylase. Decapitated females injected with PBAN showed a rapid increase in native pheromone, and a slower increase in the pheromone precursor, (Z)-11-hexadecenoate. Total native palmitate and stearate (both pheromone intermediates) showed a significant decrease after PBAN injection, before their titers were later restored to initial levels. In contrast, the acyl-CoA thioesters of these two saturated fatty acids increased during the period when their total titers decreased. When a mixture of labeled palmitic and heptadecanoic (an acid that cannot be converted to pheromone) acids was applied to the gland, PBAN-injected females produced greater quantities of labeled pheromone and precursor than did saline-injected ones. The two acids showed similar time-course patterns, with no difference in total titers of each of the respective acids between saline- and PBAN-injected females. When labeled heptadecanoic acid was applied to the gland alone, there was no difference in titers of either total heptadecanoate or of heptadecanoyl-CoA between PBAN- and saline-injected females, suggesting that PBAN does not directly control the storage or liberation of fatty acids in the gland, at least for this fatty acid. Overall, these data indicate that PBAN also controls a later step involved in pheromone biosynthesis, perhaps the reduction of acyl-CoA moieties. The control by PBAN of two enzymes, near the beginning and end of the pheromone biosynthetic process, would seem to allow for more efficient utilization of fatty acids and pheromone than control of only one enzyme.     

Hormonal control of female sex pheromone biosynthesis in the redbanded leafroller moth,Argyrotaenia velutinana

Pheromone biosynthesis in female redbanded leafroller moths (RBLR) is under control of a neuropeptide produced in the brain. A bioassay consisting of isolated abdomens was developed to test the mode of action of the pheromone biosynthesis activating neuropetide (PBAN). Pheromone titer and incorporation of radiolabeled acetate into pheromone could be monitored with this bioassay. Synthetic PBAN with sequences identical to PBAN isolated from Heliothis zea and Bombyx mori were active in inducing synthesis of pheromone in RBLR. Removal of the ventral nerve cord in isolated abdomens did not inhibit the action of PBAN. Small amounts of PBAN-like activity was found in hemolymph collected from normal females but not from decapitated females. Severing the VNC in vivo in normal females did not lower the pheromone titer. These data indicate that PBAN is released into the hemolymph and then travels to its site of action. A two-fold increase in both pheromone titer and radiolabeled acetate incorporation upon incubation with PBAN was shown with isolated pheromone glands. However, the differences between control and PBAN-induced values were smaller than those obtained with the isolated abdomen culture bioassay where a seven-fold increase was observed. A decrease in pheromone titer was seen upon the in vivo removal of the corpus bursae from normal females. Removal of the corpus bursae in the isolated abdomen cultures also abolished the activity of PBAN. However, cutting the cervix bursae and leaving the corpus bursae in the abdomen culture increased both titer and radiolabeled acetate incorporation into pheromone without the presence of PBAN. An aqueous extract made from the corpus bursae of 5-day-old females was also active by itself in inducing pheromone biosynthesis in the isolated abdomen cultures. Experiments performed using newly emerged females confirmed that the corpus bursae extracts will induce pheromone biosynthesis. These results indicate that both PBAN and the corpus bursae are involved in controlling pheromone biosynthesis in RBLR.     

Sex pheromone biosynthesis in the tortricid moth Planotortrix excessana (Walker) involves chain-shortening of palmitoleate and oleate

Biosynthesis of the sex pheromone components, (Z)-5-tetradecenyl acetate (Z5-14:OAc) and (Z)-7-tetradecenyl acetate (Z7-14:OAc), was investigated in the New Zealand tortricid moth Planotortrix excessana (Walker) by fatty acid methyl ester (FAME) analysis of base-methanolyzed extracts of lipids in the sex pheromone gland and through application of various labelled fatty acids. Analysis of the base-methanolyzed gland extracts revealed common FAMEs, including methyl oleate and methyl palmitoleate, as well as the FAMEs of the putative precursors, methyl (Z)-5-tetradecenoate and methyl (Z)-7-tetradecenoate. Application of labelled, saturated fatty acids, myristic, palmitic, and stearic did not result in any significant incorporation of label into either of the unsaturated pheromone components, although label was incorporated into tetradecyl acetate (14:OAc). In contrast, application of labelled oleic acid resulted in incorporation of label into Z5-14:OAc but not into Z7-14:OAc or into 14:OAc, whereas application of labelled palmitoleic acid resulted in incorporation of label into Z7-14:OAc but not into Z5-14:OAc or 14:OAc. These data support a route for biosynthesis of Z5-14:OAc and Z7-14:OAc in this species by limited β-oxidation of the common fatty acyl moieties, respectively, oleate (involving two cycles of 2-carbon chain-shortening) and palmitoleate (involving only one cycle of 2-carbon chain-shortening), and apparently involving no desaturase (other than the common Δ9) specific to sex pheromone biosynthesis. Interestingly, P. excessana females biosynthesize the same component (Z5-14:OAc) from an entirely different route from that of the related species Ctenopseustis obliquana (which biosynthesizes Z5-14:OAc by Δ5-desaturation of myristate). Additionally, the pheromone biosynthesis activating neuropeptide (PBAN) stimulates pheromone biosynthesis in this species. Arch. Insect Biochem. Physiol. 37:158–167, 1998. © 1998 Wiley-Liss, Inc.     

Effect of the pheromone biosynthesis activating neuropeptide on sex pheromone biosynthesis in Spodoptera littoralis isolated glands

The control of Spodoptera littoralis sex pheromone biosynthesis has been investigated with synthetic pheromone biosynthesis activating neuropeptide (PBAN) and different labeled tracers using an in vitro isolated gland system. Responsiveness of the glands to PBAN stimulation was impaired by careless tissue manipulation. The fact that PBAN is active in the isolated gland system suggests that this might be a target organ for this peptide in S. littoralis. As reported previously with Br-SOG extracts and intact females, label incorporation into the pheromone increased in glands treated with PBAN from all the precursors tested. However, the formation of labeled intermediates from d₅E11–14:Acid also occurred in glands incubated in the absence of the peptide, but the amounts of d₅Z9, E11–14:Acid were lower in PBAN treated glands than in controls. These results indicate that PBAN controls pheromone biosynthesis in S. littoralis by regulating the reduction of acyl moieties. © 1994 Wiley-Liss, Inc.     

Structure and activity of Bombyx PBAN

Two structurally related molecular species of pheromone biosynthesis activating neuropeptides (PBANs), PBAN-I and -II, were isolated from adult heads of the silkworm, Bombyx mori, and characterized. PBAN-I is a carboxyl-terminally amidated 33-residue peptide. Structure-activity relationship studies revealed that 1) its carboxyl-terminal pentapeptide is the smallest size showing activity, 2) the carboxyl-terminal amide is indispensable for activity, and 3) oxidation of three Met residues in PBAN-I to Met(O) (methionine sulfoxide) caused marked enhancement of activity, and the three Met(O) residues contribute equally to the enhancement of activity. Molecular design of PBAN analogs using a carboxyl-terminal hexapeptide showed that modification of the amino-terminal amino group brought about a dramatic increase in activity. This increase was presumed to be mainly due to the increased stability in hemolymph. PBANs share the common carboxyl-terminal sequence, -Phe-Xaa-Pro-Arg-Leu-NH₂, with myotropic peptides isolated from locust and cockroach. Examination of cross-activity of these two groups of peptides revealed that PBAN and its analogs exhibited myotropic activity comparable to myotropic peptides, while myotropic peptides showed extremely high pheromonotropic activity. In B. mori, PBAN activates sex pheromone (bombykol) production presumably by promoting the reduction reaction from acyl to alcohol, which is the last step in the biosynthesis of bombykol. © 1994 Wiley-Liss, Inc.     

Termination of sex pheromone production in mated females of the silkworm moth

A mating duration of more than 6 h was necessary to permanently terminate the production of the sex pheromone (bombykol) in the silkworm moth, Bombyx mori L. (Lepidoptera: Bombycidae), although the female formed a bursa copulatrix including a spermatophore and laid fertilized eggs even after mating for only 0.5 h. The 6-h mated female again produced bombykol if given an injection of synthetic pheromonotropic neuropeptide (PBAN), which is known to activate pheromone biosynthesis in a virgin female. Extracts of brain-suboesophageal ganglion (SG) complexes, which were removed from 6- and 24-h mated females, showed strong pheromonotropic activities. These results indicated that the pheromone gland of the mated female maintained its ability to biosynthesize bombykol; however, it could not produce pheromone due to a suppression of PBAN secretion from the SG. Furthermore, bombykol titers did not decrease after mating in females with a transected ventral nerve cord, even after the injection of a spermatophore extract, suggesting that the suppression of PBAN secretion was mediated by a neural signal and not by a substance in the spermatophore. The mated females accumulated (10E, 12Z)-10,12-hexadecadienoic acid, a precursor of bombykol biosynthesis, in their pheromone glands as did decapitated females. © 1996 Wiley-Liss, Inc.     

RNA interference of PBAN receptor suppresses expression of two fatty acid desaturases in female Plutella xylostella

Pheromone biosynthesis-activating neuropeptide (PBAN) stimulates sex pheromone biosynthesis by activating PBAN receptor (PBANr), which triggers a specific signal transduction in the pheromone gland cells. We have shown that RNA interference (RNAi) of PBANr of Plutella xylostella significantly suppressed pheromone biosynthesis and subsequent mating behavior. In order to assess molecular events occurring downstream of PBAN signaling, we cloned partial sequences of Δ9 and Δ11 fatty acid desaturases of P. xylostella. Phylogenetic analysis indicated that these two desaturase genes were highly clustered with other desaturases associated with sex pheromone biosynthesis in other insects. RT-PCR analysis showed that Δ9 desaturase was dominantly expressed in adult females, whereas Δ11 desaturase was expressed in all P. xylostella developmental stages. When PBANr expression was suppressed by PBANr-RNAi, the treated females also showed significant suppression of expression of both desaturases. These results suggest that expressions of the two desaturases are controlled by PBAN and that the two desaturases may be involved as downstream components in sex pheromone biosynthesis of P. xylostella.     

Comparative sex pherome biosynthesis in Thaumetopoea pityocampa and T. processionea: a rationale for the phenotypic variation in the sex pherome within the genus Thaumetopoea

The female sex pheromones of the Mediterranean processionary moths (Thaumetopoea sp.) are conjugated dienes or enynes of 16 carbon atoms with the unsaturations located at C11 and C13. To investigate the biochemical basis of this phenotypic variation, the biosynthetic pathway of T. processionea sex pheromone, a diene acetate, has been elucidated and compared to that reported for the enyne-producing species T. pityocampa. Mass labeling experiments showed that T. processionea sex pheromone is biosynthesized from palmitic acid, by subsequent (Z)-11 and (Z)-13 desaturations and final reduction and acetylation. The Pheromone Biosynthesis Activating Neuropeptide (PBAN) activates this biosynthetic pathway downstream of the dienoate intermediate. When either 11-hexadecynoic acid or (Z)-13-hexadecen-11-ynoic acid were administered to T. processionea, this species was able to produce the enyne sex pheromone of T. pityocampa upon PBAN stimulation. In contrast, T. pityocampa does not produce either 11-hexadecynyl acetate or (Z,Z)-11,13-hexadecadienyl acetate, despite having the corresponding precursors in the pheromone gland. However, both acetates are detected after administration of the corresponding alcohols. These overall results suggest that the absence of delta(11) acetylenase and the existence of an enynoate specific reductase in the diene and enyne-producing Thaumetopeae, respectively, account for the different sex pheromones produced by the two groups.     

Molecular cloning of pheromone biosynthesis activating neuropeptide in silkworm, Bombyx mori

Pheromone biosynthesis activating neuropeptide (PBAN) is a suboesophageal ganglion secretory polypeptide of insect, which activates the pheromone gland to produce sex pheromone biosynthesis in female silkworm, Bombyx mori. A Bombyx genomic library was screened by the method of plaque hybridization using the 32P-labeled BomDH cDNA as a probe. The genomic sequence encoding PBAN has been cloned and its structure is analyzed. The PBAN gene comprises two exons interspersed by a single intron 697 bp in length. Preceding the PBAN amino acid sequence is a 32-amino acid sequence containing two FXPRL amide peptides, which are α-SGNP (Ile-Ile-Phe-Thr-Pro-Lys-Leu) and β-SGNP (Ser-Val-Ala-Asn-Pro-Arg-Thr-His-Glu-Ser-Leu-Glu-Phe-Ile-Pro-Arg-Leu), which is followed by a Gly-Arg processing site. Immediately, after the PBAN amino acid sequence is a Gly-Arg processing site and a FXPRL amide peptide γ-SGNP (Thr-Met-Ser-Phe-Ser-Pro-Arg-Leu). It is suggested that besides PBAN, 7-, 8-, and 17-residue amidated peptides wer     

Ant Trail Pheromone Biosynthesis Is Triggered by a Neuropeptide Hormone

Our understanding of insect chemical communication including pheromone identification, synthesis, and their role in behavior has advanced tremendously over the last half-century. However, endocrine regulation of pheromone biosynthesis has progressed slowly due to the complexity of direct and/or indirect hormonal activation of the biosynthetic cascades resulting in insect pheromones. Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN) was identified that stimulated sex pheromone biosynthesis in a lepidopteran moth. Since then, the physiological role, target site, and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in moths. Despite that PBAN-like peptides (∼200) have been identified from various insect Orders, their role in pheromone regulation had not expanded to the other insect groups except for Lepidoptera. Here, we report that trail pheromone biosynthesis in the Dufour''s gland (DG) of the fire ant, Solenopsis invicta, is regulated by PBAN. RNAi knock down of PBAN gene (in subesophageal ganglia) or PBAN receptor gene (in DG) expression inhibited trail pheromone biosynthesis. Reduced trail pheromone was documented analytically and through a behavioral bioassay. Extension of PBAN''s role in pheromone biosynthesis to a new target insect, mode of action, and behavioral function will renew research efforts on the involvement of PBAN in pheromone biosynthesis in Insecta.     

Pheromonotropic stimulation of moth pheromone gland cultures in vitro

The direct neurohormonal control of pheromone biosynthesis by pheromone biosynthesis activating neuropeptide (PBAN) was demonstrated in Helicoverpa (Heliothis) spp. using pheromone gland cultures in vitro. Pheromone gland activation involved the de novo production of the main pheromone component (Z)-11-hexadecenal as revealed by radio-TLC, radio-HPLC, and radio-GC. Activation was found to be a specific response attributed to pheromone gland cultures alone. Specificity of pheromonotropic activation was demonstrated to be limited to nervous tissue extracts. A sensitive and specific radioimmunoassay was developed using [³H]-PBAN, and the spatial and temporal distribution of PBAN-immunore-activity was studied. PBAN-immunoreactivity in brain complexes was found throughout the photoperiod and in all ages. From the distribution of PBAN-immunoreactivity it appears that PBAN release is affected by photoperiod. Pheromone gland cultures were found to be competent to pheromone production irrespective of age and photoperiod. Therefore, the neuroendocrine control of pheromone production operates at the level of neuropeptide synthesis and/or release and not at the level of the target tissue itself. The involvement of cyclic-AMP as a second messenger system was demonstrated. Brain extracts and PBAN were shown to stimulate dose- and time-dependent changes in intracellular cyclic-AMP levels. The role of cyclic-AMP in this mechanism was further verified by the ability of cyclic-AMP mimetics to mimic the pheromonotropic effect of brain extracts and PBAN. However, dose-response studies using PBAN and a hexapeptide C-terminal fragment of PBAN suggested that PBAN induces a two mechanism response, one occurring at low PBAN concentrations (high affinity receptor) and another at higher PBAN concentrations (low affinity receptor). Further evidence indicating a dual receptor system was obtained with the observation that the active phorbol ester (phorbol-12-myristate 13-acetate), the diacyl-glycerol analog (1,2-dioleolyl-sn-glycerol), and the intracellular calcium ionophore (ionomycin) mimicked the physiological action of PBAN and that lithium chloride had a pheromonostatic effect. The results indicate that pheromone glands also possess receptors that are linked to inositol phosphate hydolysis. © 1994 Wiley-Liss, Inc.     

Neuroendocrine control of pheromone production in moths

In many moth species regulation of pheromone production has been attributed to the timely release of a pheromone biosynthesis activating neuropeptide (PBAN). The gene encoding PBAN has been sequenced in two moth species. Immunochemical studies as well asin situ hybridization and Northern analysis of PBAN encoding mRNA have localized the neuroendocrine cells responsible for the production of PBAN and have traced the neuronal network of PBAN immunoreactivity. Release into the bloodstream has been demonstrated, the target tissue delineated, and the signal transduction pathway and its modulation analyzed. This paper reviews the current status of research concerning the neuroendocrine control of pheromone production in Lepidopterans and presents some recent developments concerning the receptors involved in the pheromonotropic activity. In this study, we report on the use of a biologically active photoaffinity-biotin-labeled derivative of PBAN N-[N-(4-azido-tetrafluorobenzoyl-biocytinyloxyl-succinimide) and show the presence of a protein (estimated molecular weight of 50 kDa) which specifically binds to PBAN in membrane preparations of pheromone glands. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No.2279-E, 1997 series     

Mechanisms involved in the control of pheromone production in female moths: recent developments

Species‐specific pheromone blends of nocturnal female moths, derived from fatty acid precursors, are produced and released for mate‐finding, and are initiated by the circadian, trophic hormone, Pheromone Biosynthesis Activating Neuropeptide (PBAN). PBAN, produced in the sub‐oesophageal ganglion, is a 33 amino acid neuropeptide with a minimum active core in its FXPRLamide C‐terminal. PBAN acts directly on pheromone gland cells of mature females by binding to a specific G‐protein‐coupled membrane receptor (GPCR), and thereby initiating a signal transduction cascade involving calcium and cAMP. This discussion will review recent developments concerning the identification of the PBAN GPCR, its regulation by juvenile hormone (JH), and its mode of action at the level of the pheromone biosynthetic pathway. The discussion will also include recent developments concerning events occurring as a result of the transfer of pheromonostatic compounds of male origin after mating.     

PBAN regulation of pheromone biosynthesis in female tobacco hornworm moths,Manduca sexta (L.)

Manduca sexta females that were decapitated produced no pheromone during the scotophase following decapitation, indicating that they were free of pheromone biosynthesis activating neuropeptide (PBAN). When deuterated hexadecanoic or (Z)-11-hexadecenoic acid was applied to the sex pheromone glands of decapitated or intact females of the same age, and allowed to incubate in vivo for 24 h, deuterium labeled Δ-11- and Δ-10, 12-unsaturated 16-carbon fatty acids were produced in both types of females. Injection of PBAN into intact or decapitated females 23 h after application of labeled acids had no effect on the production of unsaturated labeled fatty acids. However, deuterium labeled aldehydes were produced only in females that were injected with PBAN. Therefore, in this species, PBAN activates the process by which fatty acyl precursors in the pheromone gland are converted into the pheromonal aldehydes. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Effects of decapitation and PBAN injection on amounts of triacylglycerols in the sex pheromone gland of Manduca sexta (L.)

The correlation between triacylglycerols containing conjugated diene fatty acyl moieties and pheromone aldehydes in the sex pheromone glands of females of Manduca sexta was investigated. Females decapitated 15 h after adult emergence neither called nor produced pheromone during the natural period of pheromone production on the subsequent two nights. However, these females could be stimulated to produce sex pheromone for prolonged periods by repeated injection of synthetic pheromone biosynthesis activating neuropeptide (PBAN). Gas chromatographic analysis of methanolysis products of lipids extracted from the pheromone glands of decapitated and intact females showed no differences in the amounts of fatty acyl precursors of pheromone. High performance liquid chromatographic analysis of the triacylglycerols containing conjugated diene analogues of the pheromone components (diene TG), obtained 24 and 48 h after decapitation, showed that the total amounts of these components were not affected by decapitation. The amounts of all diene TG peaks declined significantly when decapitated females were stimulated to produce pheromone during a 7 h period by repeated injection of PBAN at 3 h intervals but recovered when pheromone production subsided. These results indicate that PBAN induces liberation of pheromone precursors from the triacylglycerols during pheromone biosynthesis but does not induce replenishment of this storage pool. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America