Hemoglobin-derived porphyrins preserved in a Middle Eocene blood-engorged mosquito

Dale E. Greenwalta,1, Yulia S. Gorevab, Sandra M. Siljeströmb,c,d, Tim Roseb, and Ralph E. Harbache

Departments of aPaleobiology and bMineral Sciences, National Museum of Natural History, Washington, DC 20013; cGeophysical Laboratory, Carnegie Institution of Washington, Washington, DC 20015; dDepartment of Chemistry, Materials, and Surfaces, SP Technical Research Institute of Sweden, 501 11 Borås, Sweden; and eDepartment of Life Sciences, Natural History Museum, London SW7 5BD, United Kingdom

Edited by Michael S. Engel, University of Kansas, Lawrence, KS, and accepted by the Editorial Board September 18, 2013 (received for review June 7, 2013) Although hematophagy is found in ∼14,000 species of extant vertebrate hosts (15). Given the similarities of the fossilized insects, the fossil record of blood-feeding insects is extremely poor trypanosomes to known extant heteroxenous species, and the and largely confined to specimens identified as hematophagic hematophagic lifestyle of the extant relatives of the insect hosts, based on their taxonomic affinities with extant hematophagic Poinar has concluded that these fossils represent examples of insects; direct evidence of hematophagy is limited to four insect hematophagy. Even more direct evidence of hematophagy is the fossils in which trypanosomes and the malarial protozoan Plasmo- observation of nucleated erythrocytes containing putative para- dium have been found. Here, we describe a blood-engorged mos- sitophorous vacuoles in the gut of an amber-embedded sandfly(16). quito from the Middle Eocene Kishenehn Formation in Montana. Poinar has also reported the presence of Plasmodium spor- This unique specimen provided the opportunity to ask whether or ozoites in the salivary gland and salivary gland ducts of a fossil not hemoglobin, or biomolecules derived from hemoglobin, were female mosquito of the genus Culex, some extant species of preserved in the fossilized blood meal. The abdomen of the fossil which are vectors of avian malaria (17, 18). Hematophagy is an mosquito was shown to contain very high levels of iron, and mass obligate lifestyle for female mosquitoes that are blood-feeders spectrometry data provided a convincing identification of porphy- (many species are not blood-feeders), as a blood meal is required rin molecules derived from the oxygen-carrying heme moiety of for the completion of each gonatotrophic cycle and the resultant hemoglobin. These data confirm the existence of taphonomic production of fertile eggs (19). Given the rarity of mosquito

conditions conducive to the preservation of biomolecules through fossils, the fossil record’s bias against preservation of mosquitoes BIOCHEMISTRY deep time and support previous reports of the existence of heme- in amber, and the fragile nature of blood-engorged mosquitoes, derived porphyrins in terrestrial fossils. it is not surprising that a fossil of a blood-engorged mosquito has not been described; this despite the popular misconception of ematophagy is a feeding strategy that has arisen indepen- dinosaur DNA recovery from blood-engorged mosquitoes in Hdently numerous times and occurs in five orders of extant amber popularized by the 1993 film Jurassic Park. Here, we de- insects including fleas (Siphonaptera), lice (Phthiraptera), Lep- scribe a unique fossil of a 46-million-year-old blood-engorged idoptera, and true bugs [Hemiptera (e.g., Cimicidae or bed mosquito collected from the Kishenehn Formation in north- bugs)], but is most common in the family Diptera, where it is western Montana. The abdomen of the fossil mosquito is shown found in ∼9,000 species in 16 different families (1–4). One ex- to contain very high levels of iron, and time-of-flight secondary tinct family of Cretaceous scorpionflies (Mecoptera), Pseudo- ion mass spectrometry (ToF-SIMS) data provide unequivocal polycentropodidae, may have been blood feeding, but this identification of porphyrin molecules. The combination of these conclusion is controversial (5, 6). The mosquitoes (Culicidae) are two determinations indicates that the porphyrins are derived by far the most studied hematophagic insects as a consequence from the oxygen-carrying heme moiety of hemoglobin. of their ability to serve as vectors of widespread diseases such as malaria and yellow fever. As might be expected, the fossil record Significance of hematophagous insects, and mosquitoes in particular, is poor. The majority of all described extinct hematophagous insects are Fossils, in addition to documenting the existence of extinct biting midges (Ceratopogonidae) (7). Although there are roughly species, can often provide information on the behavior of an- similar numbers of extant species of Culicidae and Ceratopogo- cient organisms. The present study describes the fossil of nidae, there are ∼200 described extinct species of the latter family, a blood-engorged mosquito in oil shale from northwestern whereas only 25 species of fossil mosquitoes have been described Montana. The existence of this rare specimen extends the ex- (7–9). One important factor that undoubtedly contributes to this istence of blood-feeding behavior in this family of insects 46 disparity is that, whereas 70% of all fossil mosquito specimens are million years into the past. Heme, the oxygen-carrying group found in shale, more than 80% of Ceratopogonid fossils are of hemoglobin in the host’s blood, was identified in the ab- inclusions in amber (10). This difference is the result of a strong domen of the fossil mosquito by nondestructive mass-spec- taphonomic filter that segregates species and even entire families trometry analysis. Although large and fragile molecules such based on their propensity to populate a specific environmental as DNA cannot survive fossilization, other complex organic niche; in this case, a forest of resin-producing trees. molecules, in this case iron-stabilized heme, can survive intact Our ability to identify hematophagous insects is based on the and provide information relative to the mechanisms of the morphology of their mouthparts and their taxonomic affiliations. fossilization process. Fossils that contain direct evidence of hematophagy are ex- Author contributions: D.E.G. designed research; D.E.G., Y.S.G., S.M.S., T.R., and R.E.H. tremely rare. Poinar and Poinar have described two species of performed research; D.E.G., Y.S.G., S.M.S., T.R., and R.E.H. analyzed data; and D.E.G. trypanosome parasites from the gut and proboscis of sandflies wrote the paper. (Diptera: Psychodidae) embedded in amber from the Dominican The authors declare no conflict of interest. Trypanosoma fl Republic and Myanmar, and agellates in a fecal This article is a PNAS Direct Submission. M.S.E. is a guest editor invited by the Editorial pellet adjacent to an assassin bug (Hemiptera: Reduviidae) in Board. Dominican amber (11–14). Although many trypanosomes are 1To whom correspondence should be addressed. E-mail: [email protected]. restricted to a single insect host, a few genera are heteroxenous This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and have a life cycle that requires both blood-sucking insect and 1073/pnas.1310885110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1310885110 PNAS Early Edition | 1of5 Downloaded by guest on September 24, 2021 site was analyzed as a control and provided a carbon weight % value of 51.9 Wt %, nearly identical to that of the female thorax. These data suggest that both fossils retain a significant amount of their original compliment of carbon. As might be expected, the abdomen, once laden with protein-rich erythrocytes, is particu- larly carbon-rich. The weight % value for iron in the abdomen of the female mosquito was 8.97 ± 0.32%, 7.9 and 8.0 times higher than in the thorax of the female and the abdomen of the male mosquito, respectively (Table 1). EDS analysis of purified pig hemoglobin gave a weight % value of iron of 0.33%; the value reported for human hemoglobin is 0.34% (22). The percentage of iron in the abdomen of the female would be expected to in- crease as other more labile components decayed and were re- moved especially if the iron were bound in the very stable Fe– porphyrin complex. EDS analysis of permineralized elements suspected to be erythrocytes from the dinosaur Tarbosaurus bataar were reported to have a weight % iron of 8.09% (23). However, iron can be a major diagenetic component of the fossilization process, and high levels of iron, unrelated to a blood Fig. 1. Culiseta species (USNM 559050) (Diptera: Culicidae), a blood-engorged female from the Middle Eocene Kishenehn Formation of northwestern Mon- meal, have been demonstrated in other fossil insects (24). The tana. Note the distended and opaque dark-colored abdomen (compare to the iron component of the shales examined in the current study does male USNM 559051 in SI Text). not appear to be the result of pyrite (FeS2). Although sulfur was present in all samples examined, its concentrations did not cor- relate stoichiometrically with those of iron. The weight % sulfur Results varied only slightly, from 1.36% to 1.72% in the thorax and ab- To date, a total of 36 specimens of mosquitoes (Diptera: Culicidae) domen of the female and the abdomen of the male whereas the have been collected from the Coal Creek Member of the respective values for iron were highly disparate (Table 1). ToF- SIMS analyses of the two mosquito specimens detected neither Kishenehn Formation in northwestern Montana, an emerging + + Konservat–Lagerstätte of the early Middle Eocene assigned an FeS nor FeS2 . Neither EDS nor ToF-SIMS analyses detected age of 46 Ma (20). Ten of these specimens have been identified siderite (FeCO3). as two new species of the genus Culiseta, Cs. kishenehn and The two fossil mosquitoes were examined for the presence of Cs. lemniscata (8). Although the specimens discussed here are heme by ToF-SIMS. The ToF-SIMS spectrum obtained from the not preserved with the detail of the two fossil Culiseta species, the abdomen of the female was remarkably similar to the spectrum habitus of specimen USNM 559050 is obviously that of a female obtained from purified hemoglobin and decidedly different from blood-engorged mosquito with nonplumose antennae and a very that obtained from matrix adjacent to the fossil (Fig. 2). Analyses dark red/black distended abdomen compared with the non- were done at multiple sites on the female abdomen and thorax, hematophagous male USNM 559051 (Fig. 1, SI Text,andFig. S1). the matrix adjacent to the abdomen of the female mosquito, the In a study that demonstrated that structures identified as abdomen and thorax of the male mosquito, and matrix adjacent fossilized erythrocytes were in fact diagenetic in origin, it was to the male mosquito (Fig. 3 and Fig. S1). The spectrum char- suggestedthatheme,localizedtospecific structures, would acteristic of heme-derived porphyrins was absent at all sites ex- constitute incontrovertible evidence for the identification of these amined except for the female abdomen and analyses at three cells (21). Because heme consists of both iron and the prosthetic different sites on the female abdomen produced identical spectra group protoporphyrin, the fossil mosquitoes were analyzed for (Fig. 3). Both the female abdomen and hemoglobin control both. The presence of iron and other elements was determined spectra contain a large number of peaks in the mass region be- in situ by use of energy dispersive X-ray spectroscopy (EDS). tween mass/charge ratio (m/z) 350–520, forming Gaussian-like These analyses found high weight % values of carbon in the patterns (Fig. 2 and Table S1) separated by mass unit 14 (CH2). + abdomen and thorax of the female (66.9% and 51.1%, re- This fragmentation pattern, which is the result of Bi ion bom- spectively), whereas the value for the shale matrix was 21.1% bardment-mediated fragmentation, is characteristic of porphyrin (Table 1). Because male mosquitoes do not take blood meals, type structures including chlorophyll and heme and can serve the abdomen of a fossil male mosquito from the same collection as a fingerprint for identifying these molecules (25–31). The

Table 1. Energy dispersive X-ray analyses of specimens USNM 559050 (female) and USNM 559051 (male) Element Female abdomen (9) Female thorax (7) Male abdomen (8) Matrix (3)

Carbon 69.31 51.1 51.91 21.09 Oxygen 14.63 24.62 25.85 36.76 Magnesium 0.26 0.83 0.54 1.24 Aluminum 0.38 2.87 1.72 6.3 Silicon 1.06 7.19 4.50 16.1 Sulfur 1.6 1.36 1.72 0.38 Potassium 0.1 1.29 0.76 3.02 Calcium 3.66 8.80 10.7 12.00 Titanium 0.03 0.74 1.18 0.14 Iron 8.97 1.14 1.12 2.40

Values in parentheses = n, the number of different sites analyzed. Values are weight %.

2of5 | www.pnas.org/cgi/doi/10.1073/pnas.1310885110 Greenwalt et al. Downloaded by guest on September 24, 2021 Fig. 2. ToF-SIMS (positive detection mode) spectra of heme-derived porphyrins in the abdomen of the female blood-engorged mosquito (USNM 559050) and controls. (A) A spectrum of the abdomen of the blood-engorged mosquito. (B) A spectrum of the heme and heme-derived porphyrins in purified pig he- moglobin. Peaks m/z 455.04, m/z 469.05 and m/z 483.08 are three of numerous peaks present in both the abdomen of the female mosquito and pure he- moglobin (see list of heme-derived phorphyrins and their possible structures in Table S1). Intact heme has a mass/charge ratio (m/z) of 616.18. (C) A spectrum obtained from the matrix adjacent to the abdomen of the female mosquito. Peaks that denote specific porphyrins as well as the distinctive porphyrin spectral pattern are absent. (D–F) Enlarged insets of mass region 460.4–474.4 of the spectra on the left (A–C) further illustrate the Gaussian distribution of peaks and near identity between the abdominal and hemoglobin control spectra. See Table S1 for the proposed origins of peaks 467.03 and 469.05.

essential identity of the spectra taken from the hemoglobin masses of analyzed peaks with the hemoglobin control and cal- BIOCHEMISTRY control and the abdomen of the female mosquito is clearer when culated masses of porphyrin derivatives (Table S1), as well as the peaks are enlarged (Fig. 2 D and E). Comparison of the exact previous ToF-SIMS studies on heme, strongly indicate that the iron is still part of the porphyrin structure (25, 28–30). The most significant difference between the spectra of the female mos- quito abdomen and the spectra of the purified pig hemoglobin is + the absence of the peak for the intact heme ion [M] at m/z 616.18 and the fragment ions centering at m/z 557.14, [M- + CH2COOH] . The lack of these peaks is likely due to partial degradation of the heme molecule in the female mosquito, pos- sibly occurring during diagenesis. A thin layer of potassium aluminum silicate overlaying the fossils was removed before analysis. Scanning electron microscopy of the female abdomen clearly distinguishes between the opaque silicate and the underlying smooth carbonaceous fossil (Fig. 4A). ToF-SIMS ion images show colocalization of iron, several different − porphyrins, and CN derived from the heterocyclic pyrrole groups of the porphyrin within exposed areas of the abdomen of the fe- male mosquito (Fig. 4 D–F). These areas are easily distinguishable from the surrounding silicate matrix by the high silicon and po- tassium content of the matrix (Fig. 4 B and C). Apparent coloc- alization of Fe and porphyrins in ToF-SIMS ion images supports spectral evidence of the Fe being bound to the porphyrin struc- tures. Variability in the area exposed for analysis and the inherent differences in heme concentration between the standard and the fossil account for the peak intensity differences in Figs. 2 and 3. Discussion The preservation of fossil female mosquito USNM 559050 was an extremely improbable event. The insect had to take a blood meal, be blown to the water’s surface, and sink to the bottom of a pond or similar lacustrine structure to be quickly embedded in fine anaerobic sediment, all without disruption of its fragile distended blood-filled abdomen. This fossil has provided a unique opportunity to ask whether or not a portion of the he- Fig. 3. ToF-SIMS spectra from different areas of the female (USNM 559050) moglobin molecule could be preserved after tens of millions of and male (USNM 559051) mosquitoes. (A) Spectra taken from three different areas of the abdomen of the female mosquito. (B) Control spectra obtained years; heme was the most obvious target for our analysis. De- tection of heme-derived porphyrins in the female specimen from two different areas of matrix adjacent to the female mosquito, two fi different areas of the thorax of the female mosquito, two different areas of con rms that it is indeed a blood-engorged mosquito and pro- the abdomen of the male mosquito and a single spectrum of the matrix vides direct evidence of hematophagy in the fossil record. The adjacent to the male mosquito. two species of mosquitoes known from the Kishenehn basin,

Greenwalt et al. PNAS Early Edition | 3of5 Downloaded by guest on September 24, 2021 molecules in situ. Blood vessels isolated from trabecular bone of the dinosaur Brachylophosaurus canadensis bound hemoglobin- specific antiserum in an immunosorbant assay, albeit with a sig- nal only twice that of background levels (34). In immunoblot assays, antiserum raised against extracts of Tyrannosaurus rex trabecular bone reacted with purified hemoglobin but antiserum against hemoglobin did not react with extracts of B. canadensis bone (34, 35). Both NMR and Raman spectroscopy have been applied to extracts of T. rex trabecular bone tissue, and data from both techniques were suggestive of the presence of heme and/or porphyrin (35). The data reported herein provide incontrovertible documentation of the presence of heme- and arguably hemoglo- bin-derived porphyrins in a 46-million-year-old compression fossil and localize the porphyrins to a specific anatomical structure within that fossil. As previously suggested, ToF-SIMS has significant potential for the unequivocal detection and nondestructive in situ locali- Fig. 4. Colocalization of heme-derived porphyrins and iron. (A) A scanning zation of heme and related porphyrins in fossilized life forms electron micrograph (backscattered electron mode) of a portion of the ab- (29). Although not readily applicable to amber inclusions, wider domen of the blood-engorged female mosquito (USNM 559050). The actual application of this technology to compression fossils may lead to fossil surface is black with multiple minute cracks due to dehydration. Large fi portions of the fossil surface are covered by an opaque layer of potassium the identi cation of other biomolecules and provide insight as magnesium aluminum silicate as indicated by ToF-SIMS ion image localiza- to their heretofore unsuspected preservation. + + tion of Si (m/z 27.98; B) and K (m/z 38.97; C). (D–F) Colocalization of heme- − Materials and Methods derived porphyrins (m/z 460.40–474.40), CN (m/z 26.00) characteristic of the + heterocyclic pyrrole groups of porphyrin, and Fe (m/z 55.93), respectively, in Specimens are housed at the Department of Paleobiology, National Museum the area depicted in A. (Scale bar, 50 μm.) of Natural History (NMNH), Smithsonian Institution (Washington, DC). Fossils were immersed in 95% ethanol for examination and photographed with an Olympus SZX12 microscope equipped with a Q-Color5 Olympus camera. Culiseta kishenehn and Cs. lemniscata, most closely resemble Image-Pro Plus 7.0 software was used to capture and record the images and extant species of the subgenera Climacura and Culicella,re- measurements. A thin layer of silicate covering the fossil mosquito speci- spectively (8). Both subgenera are small, with 5 and 14 species, mens was removed through use of a Faber–Castell eraser pencil (no. 185698) respectively, and both contain species that feed almost exclu- and/or a Becton Dickinson 1 cc insulin syringe with attached 29-gauge sively on birds [e.g., Culiseta (Culicella) morsitans and Culiseta needle (no. 309311). Samples were rinsed with ethanol and mounted for in (Climacura) melanura situ ToF-SIMS analysis in a laminar flow hood. Microanalysis and imaging ] (32). The host of the blood-engorged were performed at the NMNH Department of Mineral Sciences on uncoated mosquito described herein, which is similar to Cs. lemniscata (SI Text samples using an FEI NOVA NanoSEM 600 FEG Variable Pressure Analytical ), is unknown. Scanning Electron Microscope (SEM) outfitted with a ThermoNoran energy Porphyrins are energetically very stable molecules and have dispersive spectrometer (EDS) and a ToF-SIMS IV (ION-TOF) spectrometer. been shown to be preserved through geological time as common Standardless EDS analyses were performed at 15 kV and 1–2 nA and have an components of many oil shales (28, 29). Although the majority of analytical uncertainty of less than 5%. ToF-SIMS ion imaging was done using + such geoporphyrins are derived from chlorophyll and bacterio- a 25 keV Bi3 beam at a pulsed current of 0.3 pA, rastered over an area of chlorophyll, some are thought to be derived from either heme ∼300 × 300 μm for 180–600 s. The accumulated primary ion dose never × 10 2 or its precursors found in microbial cytochromes and related exceeded 1.25 10 ions per cm , which is below the static limit (the point where the same location is statistically sampled more than once) of 1012 ions proteins. A microbial origin of the porphyrins found in the blood- per cm2 for organic molecules (36). Analyses were performed with the in- engorged mosquito is unlikely based on the following observa- Δ i strument optimized for high mass resolution (bunched mode: m/ m of at tions: ( ) Scanning electron microscopy did not reveal any mi- least ∼5,000 at m/z 30). Pig hemoglobin (SigmaAldrich H4131-1G), dissolved crobial structures, either intact or fragmentary, on the surface of in water at 5 mg/mL and evaporated onto the surface of an aluminum stub the fossil. (ii) If microbes were the source of the porphyrin, their and a glass slide at 55 °C, was used as a positive control in EDS and ToF-SIMS growth would not be expected to be completely limited to a por- experiments, respectively. tion (i.e., the abdomen) of the body of the fossil mosquito. (iii) The anaerobic environment required for the preservation of the ACKNOWLEDGMENTS. We thank Kurt, Leona, and Norm Constenius for the gift of their collection of fossil insects from the Kishenehn Formation to the Kishenehn fossil insects would have supported only anaerobic NMNH. We also thank Conrad Labandeira for reviewing the manuscript, microbes. Because such anaerobic organisms have lost their ability Finnegan Marsh for administrative support, Keana Scott for her input with to use molecular oxygen as a terminal electron transporter in EDS, and three anonymous reviewers for constructive input. This work was anaerobic environments, they express very low levels of porphyrin- funded in part by the Deep Carbon Observatory (A. Steele, Principle In- containing cytochromes (33). vestigator), the Postdoctoral fellowship program of Geophysical Laboratory, fi Carnegie Institution of Washington, and the Swedish National Space Board Reports of the identi cation of hemoglobin and/or heme in (Contract 121/11). This is Contribution 283 of the Evolution of Terrestrial fossils are few in number, equivocal and do not localize these Ecosystems Consortium of the US National Museum.

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