Unlike any previously studied mosquito species, Ur. sapphirina in our sample fed exclusively on annelid hosts. This finding explains the inability of previous investigations to identify Ur. sapphirina blood meals, as these studies were performed under the assumption, and corresponding laboratory methodology, that female mosquitoes take blood meals only from vertebrate animals. Annelids and vertebrates share enclosed circulatory systems and either extracellular (annelids) or intracellular (vertebrates) hemoglobin, which in both groups causes the characteristic red coloration of the blood. The presence of red blood in the guts of Ur. sapphirina females likely contributed to the confusion related to host use of this mosquito by other researchers. Future studies may need to consider the possibility that mosquitoes fed on other types of invertebrates may not display the red gut normally used to classify a mosquito as blood engorged.

The recognition of Ur. sapphirina as a specialist of annelid, not vertebrate, host animals has important implications in mosquito ecology and evolution, and in the epidemiology of mosquito-vectored pathogens. This finding demonstrates that the range of potential mosquito hosts is considerably broader than previously indicated. Uranotaenia sapphirina is a common species throughout eastern North America, where mosquitoes have been under extensive study since their involvement in pathogen transmission was recognized in 1881 (ref. 1). The presumption that adult female mosquitoes blood feed only from vertebrate hosts10 is a source of bias in the methodological framework used to study mosquito ecology. Mosquito blood meals are identified primarily through methods that, by design, selectively react with only vertebrate antigens or DNA. For this reason, estimating the extent to which invertebrate hosts are utilized by female mosquitoes is not possible given previously available methods. In the laboratory, caged mosquitoes, including mammalophilic Aedes and Anopheles species have been documented locating and feeding on lepidopteran larvae in no-choice experiments, and subsequently, in some cases, to produce viable eggs32,33,34,35. Anecdotal records of mosquitoes feeding on cicada nymphs, mantids, chironomid midges, and lepidopteran pupae reported in the early 1900s by Howard36, 37 and occasionally referenced in the literature26, 38 have been disputed by Downes10 as mistaken identifications and include few substantive details. Beyond these laboratory experiments and historic records, there is no previous evidence that suggests that such interactions occur in nature, although any instance of invertebrate host use would not be detected by the traditional methods of blood meal analysis. Interestingly, for some mosquito and Corethrellidae (the culicomorph sister taxon to mosquitoes + Chaoboridae39) species, blood meal analyses that are effective in other species have failed, which may indicate that these species also feed upon hosts that cannot be detected using the vertebrate-based methodology40,41,42,43. As new sequencing technologies are applied to blood meal analysis, the ability to detect unexpected hosts should improve, particularly with the recognition of the potential for invertebrate feeding in mosquitoes.

Understanding the extent to which mosquitoes, particularly pathogen vectors, interact with invertebrate hosts has epidemiological implications. Uranotaenia sapphirina has been implicated as a potential vector for several arboviruses. Field-collected Ur. sapphirina have tested positive for Eastern equine encephalitis virus2, 4, 44, 45 and West Nile virus3. Our results suggest that Ur. sapphirina is unlikely to become infected with these viruses through feeding on vertebrate hosts. It is possible that by feeding on hematophagous leeches, which themselves often parasitize competent arbovirus hosts46, 47, Ur. sapphirina could act as kleptoparasites, acquiring virus-infected vertebrate blood meals from their leech hosts. Similarly, interactions between Ur. sapphirina and leeches may affect the transmission of pathogens vectored by leeches48. In previous studies30, 31, a small proportion of examined Ur. sapphirina blood meals were derived from snakes and the ranid frog Lithobates catesbeianus. Some snake species (e.g., Agkistrodon piscivorus, Nerodia spp.) and ranid frogs are common at the margins of vegetated waterways at night, a microhabitat where Ur. sapphirina females were observed feeding on annelid hosts. In these microhabitats, snakes and frogs would be available to host-seeking Ur. sapphirina females, and may be fed on incidentally. An alternative possibility for the detection of arboviruses in Ur. sapphirina is that this mosquito occasionally feeds on vertebrate hosts that may be competent for some arboviruses. The identification of two snake-derived Ur. sapphirina blood meals30 is particularly noteworthy, as the role of snakes in arbovirus persistence and transmission has been increasingly supported31, 49,50,51,52. Future studies should elucidate how Ur. sapphirina comes into contact with these viruses, and investigate the possibility that Ur. sapphirina could represent a complex of cryptic species with varying host use patterns.

Annelid host specialization by Ur. sapphirina raises important questions about the origin and evolution of blood feeding in mosquitoes. Foremost is whether feeding on invertebrates is an ancestral or derived trait. Divergence and radiation time estimates of Culicomorpha and vertebrate host lineages suggest that mosquitoes, or their culicomorph ancestors, adapted to vertebrate host groups after their diversification. The relationship between host and mosquito/Culicomorpha phylogenies has yet to be assessed, but other hematophagous insects have undergone stepwise transitions, with diversification of hematophagous insect lineages paralleling their host phylogenies53. Understanding how invertebrates factor into the evolution of host use by mosquitoes and other Culicomorpha will ultimately depend on a more complete accounting of mosquito host use patterns and the extent of invertebrate host use, and a well-resolved mosquito phylogeny. Until more information becomes available, understanding the origins and evolution of blood feeding in mosquitoes will remain speculative. However, the evolutionary history of Culicomorpha and host animals, and the host use patterns of basal mosquitoes may provide clues.

Birds and mammals are the major hosts of many modern mosquitoes, particularly among the more derived lineages (Fig. 3). However, it is unlikely that birds or mammals were the initial hosts of ancestral mosquitoes, as the earliest known fossil mosquito, Burmaculex antiquus54, precedes the diversification of birds55 and mammals56 by 30–40 million years. Modern frog-biting midges (Corethrellidae), sister to the mosquitoes + phantom midges (Chaoboridae)39, are known to feed only on anuran hosts (Fig. 3), and this association dates to the Lower Cretaceous57, pre-dating Burmaculex by 75 million years. The antiquity of anuran host use by Corethrella and the use of endothermic hosts by modern mosquitoes suggests a relationship between the vertebrate and mosquito phylogenies. However, this hypothesis is not supported by the basal placement of Anopheles, the human malaria vectors, that are generally considered specialists of mammalian hosts (Fig. 3). The split between Anophelinae and Culicinae is estimated at 45–126 million years before Burmaculex58, 59, suggesting either that mammal specialization in Anophelinae is not the ancestral trait, or that the basal placement of Anophelinae is incorrect. While phylogenetic analyses based on molecular data have not yet fully resolved deeper (genus-level) divisions within Culicidae26, they have estimated the time of divergence of Uranotaenia from other genera at >150 mya58. This event would have been concurrent with the diversification of major anuran groups60 and the first actual fossils of frog-biting midges57, and 50 million years older than Burmaculex. Uranotaenia, with its sister group Aedeomyia, is placed in a basal position within Culicinae58, 61 (Fig. 3), a diverse clade containing the majority of mosquito genera, implying an ancient origin for Uranotaenia. This might suggest that, if invertebrate feeding is the pleisiomorphic state, host affinities within Uranotaenia are indicative of early patterns of host use within Culicidae that were lost in other basal lineages (e.g., Anophelinae).

Fig. 3 Patterns of blood-feeding on vertebrate host classes for Culicidae and related lineages (Corethrellidae and Chaoboridae). Composite image adapted using phylogeny from Harbach and Kitching61 and Borkent and Grimaldi54. Numbers in parentheses following mosquito taxa indicate number of species within each taxon14, 57, 79. Shading of squares indicates the degree to which mosquito or related lineages interact with vertebrate classes14, 15, 24, 27, 28, 30, 31, 40, 43, 54, 57, 62, 79,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95. Uranotaenia sapphirina is contained within tribe Uranotaeniini*. All mosquito lineages are extant except Burmaculex antiquus#, the oldest known fossil mosquito, from the middle Cretaceous (110 mya). Burmaculex antiquus is presumed to have been a blood feeder, but had a moderately elongate proboscis and lacked the palpal sensillae that function as CO 2 receptors for host location in modern mosquitoes54 Full size image

The limited evidence available indicates that the earliest lineages of Culicomorpha fed on the body fluids (hemolymph) of the open circulatory systems of insects62. This trait is retained in multiple culicomorph families, including some extant chironomids and ceratopogonids, and mammalophilic Anopheles33 and Aedes32, 34 mosquitoes are able to locate insect hosts and utilize hemolymph to mature eggs. The closed circulatory systems of annelids, by contrast, are analogous to those of vertebrates, with phenotypically similar components, including heart(s), blood vessels, and hemoglobin63, 64. Annelid feeding may represent an evolutionary link between by an ancestral culicomorph feeding on free hemolymph in the body cavity and modern mosquitoes feeding on vertebrates with closed circulatory systems. While annelid circulatory systems are analogous to those of vertebrates, annelid immune systems and hemostatic responses are less sophisticated65, 66, suggesting that annelid feeding mosquitoes may encounter fewer defenses. In that context, annelid feeding may have primed the development of physiological, biochemical, behavioral, and morphological adaptations that would enable mosquitoes to eventually circumvent barriers to blood feeding from diverse vertebrate hosts, ultimately leading to the tremendous human health impacts caused by pathogen transmission in modern mosquitoes.

The use of annelid hosts in Ur. sapphirina alternatively could be a trait derived from frog feeding ancestors switching to worms and leeches that were encountered in habitats similar to anuran hosts. The phylogenetic position (Fig. 3) of the frog-biting midges, and their presumed ancient association with frogs57 may suggest that amphibians were the hosts of the common ancestor of Corethrellidae and Culicidae + Chaoboridae. However, a recent study of the hosts of Corethrella found that PCR assays successfully identified only <30% of blood meals, despite the use of primers that amplify a broad range of vertebrate hosts43. The authors of that study43 compared their low success rate with that of Ur. sapphirina from other published works, leading them to conclude that female Corethrella may feed on additional, undetermined hosts.

The function of annelid host use by Ur. sapphirina is not yet established, and the molecular analyses and field observations we report cannot discount the possibility that annelid host use by Ur. sapphirina could be a derived trait that evolved to serve a non-reproductive function, such as preventing dehydration or supplementing energetic reserves. Blood feeding by mosquitoes serves a function that is primarily reproductive: the females of most mosquito species require nutrients, particularly proteins, from host blood to provision developing eggs10, although blood-derived resources can also be diverted to meet metabolic needs67, 68 or in response to dehydration69. Dehydration can alter the behavior of mosquitoes by prompting them to increase host seeking and blood feeding69. Carbohydrates, obtained directly or indirectly from plants, serve primarily as a metabolic resource to both male and female mosquitoes, but can also enhance the reproductive potential of a female mosquito70. Our findings do not exclude the potential that female Ur. sapphirina utilize annelid blood as an energetic resource or a means of maintaining hydration; however, circumstantial evidence supports the idea that annelid feeding plays a reproductive role for this mosquito. For example, despite the collection of numerous male Ur. sapphirina, blood meals were only found in females in our samples. In addition, both males and females have been observed nectaring on flowers at field locations (L.E.R., personal observation), and collected engorged with nectar71. The details of egg production following annelid feeding, and the potential for annelid blood to serve a function other than egg maturation, needs to be explored by subsequent research to better understand the evolutionary context of these findings, as our data are limited to the identification of annelids as the hosts of Ur. sapphirina.

Specialization of Ur. sapphirina on annelid hosts demonstrates that the host breadth of mosquitoes is substantially broader than previously understood. Prior research on the host interactions of mosquitoes has centered around a minor subset of mosquito species, particularly the Aedes, Anopheles, and Culex pathogen vectors. For many genera, particularly those restricted to tropical regions, host use patterns have not been investigated, leaving substantial gaps in the understanding of mosquito–host relations. Combined with the large diversity of mosquitoes, there is potential that invertebrate host specialization extends beyond Ur. sapphirina. The fact that a common North American mosquito specializing on annelid hosts has gone undocumented as far as we are aware for more than a century suggests that invertebrate host use by mosquitoes is easily overlooked. Future work towards a more complete understanding of mosquito host use patterns should consider this possibility and, ideally, make use of novel molecular technologies that are compatible with the detection of invertebrate hosts.