Discovery and description of a novel mode of oviposition in the mosquito genus Culex

Mosquitoes (Diptera: Culicidae) are a remarkably diverse group of blood-feeding flies that possess distinct oviposition behaviors, host use patterns, and microhabitat associations that vary considerably among the 112 genera and 3,550 species currently recognized worldwide^1,2,3,4,5. The continued study of these insects should be of great interest to researchers, not only due to the medical significance of some species (approximately 2.5% of all mosquito species are known vectors of medically important pathogens⁶ but also for the fascinating adaptations and strategies that have enabled their widespread success^7,8,9. Varied, often specialized oviposition behaviors among genera represent one such strategy that allows female mosquitoes to ensure larval survival and maximize subsequent offspring performance. Mosquitoes rely on numerous sensory cues, including chemotactile, visual, and olfactory signals, to evaluate the suitability of oviposition sites^2,8. Once a gravid female locates a sufficient lentic body of water, the species-specific process of egg-laying typically involves one of several mechanisms. These include ovipositing a mass of eggs (“rafts”) on the water surface, depositing a single egg between different sites (directly on the water or above the waterline), attaching a cluster of eggs to submerged vegetation, launching single eggs into a water-filled container while in flight^10,11, depositing eggs in clusters on wet ground or container surfaces where water is expected to collect, and even ovipositing the eggs onto the hindlegs¹² and carrying the eggs to an appropriate water source when ready to hatch^2,8,13. The diversity of oviposition behaviors exhibited by mosquito species has allowed for successful larval development in a wide range of aquatic habitats across nearly every continent, except Antarctica^4,14,15.

Despite the wide variation in oviposition strategies across the Culicidae, the predominant egg-laying behavior observed in well-studied species, particularly vectors of pathogens, has led to the generalization of these complex strategies across entire genera. A prime example is mosquitoes in the genus Culex, which contains > 800 species in 28 subgenera¹⁶. Several species of Culex subgenus Culex are the primary vectors of West Nile virus, St. Louis encephalitis virus, and other diseases. Within the genus Culex, these vector species (e.g., Culex pipiens, Culex quinquefasciatus, Culex tarsalis) have remained the primary focus of research attention. Largely based on the oviposition habits of these medically important subgenus Culex species, it is commonly believed that all genus Culex species exclusively lay characteristic egg masses (“rafts”) that float on the water surface^{8,15,17,18,19}. Even though oviposition of many subgenera Culex species is poorly documented, this oversimplification persists^{8,15,17,18,19,20,21,22}. Previous studies have found exceptions to the Culex “egg raft paradigm” and suggest that it must be contested and revised, while further concluding that an examination of understudied Culex subgenera (Anoedioporpa, Micraedes, and Microculex, for example) will uncover a broader spectrum of oviposition behaviors in the genus^18,19,20.

Throughout the 1970s, Mattingly¹⁹ compiled anecdotal notes on mosquito eggs and documented examples that may contradict the conventional Culex oviposition behavior. One particularly unusual case involved Culex antillummagnorum (Dyar) (Fig. 1), which is the most widely distributed mosquito species on the island of Puerto Rico²³ and has been documented on several other Caribbean islands^24,25,26. This species is one of six in the subgenus Micraedes, a poorly studied group for which essentially no ecological or behavioral information is available^14,19. Culex antillummagnorum larvae can be found in both natural and man-made containers, where they filter feed on organic detritus and microorganisms, but they are most frequently collected from phytotelmata (plant-held aquatic microhabitats) such as bromeliad tanks, bamboo internodes, “malanga” (Colocasia esculenta) leaf axils, and Heliconia caribaea flower bracts^23,26,27. Until recently, the host associations of Cx. antillummagnorum were unknown, but photographs from Reeves et al.²⁸ show adults feeding on two anole species, Anolis gundlachi and A. cristatellus, in Puerto Rico. From these observations, the species is now considered a candidate vector of the three Plasmodium species known to circulate among Anolis lizard communities on the island.

In his original note, Mattingly¹⁹ was intrigued by the unusual egg morphology of Cx. antillummagnorum (Fig. 4) and hypothesized that the species might display a mode of oviposition not previously documented in Culex: the emission of single eggs while hovering above a water-filled container. Skip-oviposition (a “bet hedging” oviposition strategy where female mosquitoes scatter eggs among various sites) has been observed in Anopheles spp., Deinocerites spp., and Aedini tribe species⁸. Oviposition in flight has only been reported in Anopheles spp⁸., Toxorhynchites spp¹¹., and Wyeomyia spp^1,29. and other species in the Sabethini tribe³⁰. No Culex species has been previously recorded ovipositing while in flight or is known to skip-oviposit^{8,11,14,19,29,30}. Verifying this atypical mode of oviposition within Culex would raise compelling questions about the evolution and ecological drivers of such a behavior, as well as suggest that the understanding of Culex egg-laying remains incomplete^8,15,18,20.

(a) Adult female Culex antillummagnorum. The length of the long, slender palpi (0.35 of the proboscis) can be seen; this character is most often used to positively morphologically identify the species. (b) Cx. antillummagnorum larvae collected from a Heliconia caribaea bract. The larvae possess coarse, stellate tufts of abdominal hairs, uniformly shaped antennae, and extremely long, thin siphons; the 4th instar larvae of the species are easily distinguished from other mosquito larvae found in Puerto Rico (Barrera). Both specimens collected in the Luquillo Experimental Forest, Puerto Rico..

Preliminary sampling of natural containers, including C. esculenta leaf axils, H. caribaea flower bracts, epiphytic bromeliad tanks, and fallen palm spathes, throughout the Luquillo Experimental Forest (LEF) in Puerto Rico showed higher numbers of larval Cx. antillummagnorum individuals in the water-filled bracts of H. caribaea racemes (Fig. 6), suggesting a potential preference among gravid females for these oviposition sites in the LEF (data from natural container sampling were used to assess previous research and were not quantified). This finding aligned with research by Richardson et al.² and Yee²³. Gravid Cx. antillummagnorum females were then observed and filmed for several nights as they deposited eggs within H. caribaea bracts near El Verde Field Station in the LEF. With this study, we aimed to resolve the nearly 50-year-old question regarding the oviposition behavior of Cx. antillummagnorum, first raised by Mattingly¹⁹, and in doing so, learn more about this understudied species and candidate lizard malaria vector in Puerto Rico^23,28. By conducting field observations and recording numerous videos of gravid Cx. antillummagnorum in the LEF, we documented the first instance, to our knowledge, of a Culex mosquito aerially and skip-ovipositing.