Leah Houri-Zeevi

Complex traits that span multiple tissues and systems often integrate large numbers of genes across development, physiology, and behavior, making it challenging to identify their essential components. Blood feeding in mosquitoes is one such trait. It is ancestral to the mosquito family, maintained in most species for ~200 million years, and was independently lost in three lineages. These convergent losses offer a natural experiment to discover the genetic, physiological, and neural features required for blood feeding. We assembled high-quality, chromosome-level genomes for seven mosquito species, along with whole-brain tomographic reconstructions. Our study spanned the three known non-blood-feeding lineages (Toxorhynchites rutilus, Topomyia yanbarensis, and Malaya genurostris), blood-feeding relatives, and the variable blood feeder Wyeomyia smithii. Comparing orthologous gene clades, we detected convergent gene loss specific to the three lineages that had lost blood feeding. The losses include the salivary platelet-aggregation inhibitor Aegyptin, blood-activated serine proteases such as Chymotrypsin-1 and 2, and a carboxylesterase expressed in the female fat body and brain glia. The loss of blood feeding also extended to chemosensation. Non-blood feeders lack two odorant-binding protein clades, two ionotropic receptor clades associated with blood-component taste detection, and odorant receptor clades expressed in a discrete, strongly female-biased population of antennal neurons. Female-biased head gene expression was reduced in non-blood feeders. Finally, examination of whole-brain tomographic reconstructions across the species revealed smaller antennal lobes in non-blood-feeding females, consistent with reduced olfactory input. Together, these findings identify a compact set of genes, expression patterns, and brain regions associated with blood feeding, offering an evolutionary entry point for functional dissection of how this complex and dangerous trait is built and dismantled.

Amino acid composition within a proteome is conventionally viewed as a property of individual protein function. However, specific nutrient scarcity has been shown to reshape which amino acids are encoded in the genome of microbes. Mosquitoes have fed on blood for 200 million years, relying on blood amino acids for reproduction. We wondered whether this ancient dependency has left an imprint on the mosquito proteome. Here we show a proteome-wide shift to lower isoleucine usage in blood-feeding mosquitoes, mirroring the known isoleucine deficiency of adult human hemoglobin. In comparing amino acid composition across the proteomes of blood-feeding and non-blood-feeding mosquitoes, we found that blood feeders show systematically lower isoleucine usage and that highly expressed gut enzymes that digest the blood meal are nearly isoleucine free. Analysis of 707 vertebrate genomes reveals that isoleucine deficiency in major hemoglobin subunits is shared among mammals. Specifically, all non-Malagasy primates, including humans, lack isoleucine in their adult hemoglobin subunits while retaining it in fetal and embryonic subunits. We find that two other major blood proteins, serum albumin and immunoglobulin G, also have reduced isoleucine levels (∼1.4%) compared to the human proteome-wide average of 4.38%. Finally, we reasoned that this widespread isoleucine restriction could serve to defend adult red blood cells against isoleucine-dependent blood parasites that feed on them. We tested this hypothesis with the malaria parasite Plasmodium falciparum , which is known to arrest growth in adult red blood cells when extracellular isoleucine is withdrawn. We found that neonatal red blood cells purified from umbilical cord blood, which contain isoleucine-rich fetal hemoglobin, permit growth without extracellular isoleucine. This work demonstrates that nutritional scarcity is reflected in the genomes of animals, and that amino acid depletion of host protein sequences may serve a protective function against blood parasites.

Summary: Mosquitoes, the world’s deadliest animal, exemplify single-mating systems where females mate only once in their lifetime, making mate choice critically important for reproductive success and mosquito control. Despite this importance, the mechanisms of female mating control and what prevents additional matings remain poorly understood. To address this gap, we developed a dual-color fluorescent sperm system in invasive Aedes aegypti mosquitoes and quantified mating patterns, confirming that 86-96% of females mate only once. Using behavioral tracking of mating pairs, deep learning, and quantitative analysis at increasing resolution, we discovered that females actively control mating initiation through a previously undescribed behavior: genital tip elongation. This female response is triggered by rapidly evolving male genital structures, creating a precise lock-and-key mechanism that determines mating success. Comparative analysis revealed that Aedes albopictus , separated from Aedes aegypti by 35 million years of evolution, employs a similar female-controlled system. However, Aedes albopictus males uniquely bypass female control when attempting cross-species matings with Aedes aegypti females but not with conspecific females. This “lock-picking” ability, combined with the known sterility of cross-species matings, may explain how Aedes albopictus competitively displaces Aedes aegypti populations in overlapping territories. Our findings redefine mosquito reproduction as a female-controlled process and establish a quantitative framework for investigating the molecular and neurobiological mechanisms underlying mating control and species competition in these globally important disease vectors.

Summary: The female Aedes aegypti mosquito’s remarkable ability to hunt humans and transmit pathogens relies on her unique biology. Here, we present the Aedes aegypti Mosquito Cell Atlas, a comprehensive single-nucleus RNA sequencing dataset of more than 367,000 nuclei from 19 dissected tissues of adult female and male Aedes aegypti , providing cellular-level resolution of mosquito biology. We identify novel cell types and expand our understanding of sensory neuron organization of chemoreceptors to all sensory tissues. Our analysis uncovers male-specific cells and sexually dimorphic gene expression in the antenna and brain. In female mosquitoes, we find that glial cells in the brain, rather than neurons, undergo the most extensive transcriptional changes following blood feeding. Our findings provide insights into the cellular basis of mosquito behavior and sexual dimorphism. The Aedes aegypti Mosquito Cell Atlas resource enables systematic investigation of cell type-specific expression across all mosquito tissues.

SUMMARY: Sensory compensation is a process that allows individuals with a loss of one sense, for instance hearing or vision, to adapt to changes in their sensory abilities. Where this phenomenon has been observed, there is enhanced perception by another sense to compensate for deficiency of the lost sense. Such compensation is important for humans and non-human animals that use multisensory integration for effective navigation and the execution of vital tasks. Among these, female mosquitoes are sensory specialists that rely heavily on integrating multiple human-emitted cues in their quest for a suitable host to obtain a blood meal. Here, we identify a previously undescribed mechanism of sensory compensation in female Aedes aegypti mosquitoes. Mutant mosquitoes lacking the odorant receptor co-receptor Orco show specific enhancement in heat-seeking behavior. This s compensation does not require the antenna, which was previously assumed to be the primary mosquito thermosensitive organ. Instead, we found that the tips of the forelegs are required to detect heat, and that the heightened sensitivity in heat detection is mediated by increased neuronal activity in foreleg sensory neurons, which are distant from the head appendage neurons that express Orco . By comparative gene expression analysis in wildtype and Orco mutant legs, we identify Ir140 , a foreleg-enriched member of the Ionotropic Receptor (IR) superfamily of sensory receptors, as strongly upregulated in Orco mutant legs. Emphasizing the important role of IRs in thermosensation, we find that mutant mosquitoes lacking the IR co-receptor, Ir25a , lose all responses to heat, and Ir140 mutants show strong deficits in responding to human skin temperatures. We generated an Ir140 , Orco double mutant and show that these animals lose the remarkable sensory compensation seen in Orco mutants. This strongly suggests that upregulation of Ir140 in the foreleg is the mechanism of sensory compensation in Orco mutants. Odorant receptor expression is sparse in legs, suggesting an indirect, long-range mechanism of sensory compensation. Our findings reveal a novel compensatory mechanism in which loss of one sensory modality in female Aedes aegypti mosquitoes results in greater sensitivity in another to maintain the overall effectiveness of their host-seeking behavior, further enhancing their status as the most dangerous predator of humans.

Transgenerational inheritance of small RNAs challenges basic concepts of heredity. In Caenorhabditis elegans nematodes, small RNAs are transmitted across generations to establish a transgenerational memory trace of ancestral environments and distinguish self-genes from non-self-elements. Carryover of aberrant heritable small RNA responses was shown to be maladaptive and to lead to sterility. Here, we show that various types of stress (starvation, high temperatures, and high osmolarity) induce resetting of ancestral small RNA responses and a genome-wide reduction in heritable small RNA levels. We found that mutants that are defective in various stress pathways exhibit irregular RNAi inheritance dynamics even in the absence of stress. Moreover, we discovered that resetting of ancestral RNAi responses is specifically orchestrated by factors that function in the p38 MAPK pathway and the transcription factor SKN-1/Nrf2. Stress-dependent termination of small RNA inheritance could protect from run-on of environment-irrelevant heritable gene regulation.

Life experiences trigger transgenerational small RNA-based responses inC. elegansnematodes. Dedicated machinery ensures that heritable effects would re-set, typically after a few generations. Here we show that isogenic individuals differ dramatically in the persistence of transgenerational responses. By examining lineages composed of >20,000 worms we reveal 3 inheritance rules: (1) Once a response is initiated, each isogenic mother stochastically assumes an “inheritance state”, establishing a commitment that determines the fate of the inheritance. (2) The response that each mother transfers is uniform in each generation of her descendants. (3) The likelihood that an RNAi response would transmit to the progeny increases the more generations the response lasts, according to a “hot hand” principle. Mechanistically, the different parental “inheritance states” correspond to global changes in the expression levels of endogenous small RNAs, immune response genes, and targets of the conserved transcription factor HSF-1. We show that these rules predict the descendants’ developmental rate and resistance to stress.

Transgenerational inheritance of small RNAs is challenging basic concepts of heredity and achieving control over such responses is of great interest. In C. elegans nematodes, small RNAs are transmitted across generations to establish a transgenerational memory trace of ancestral environments and distinguish self from non-self genes. Inheritance of small RNAs is regulated by dedicated machinery and carryover of aberrant heritable small RNA responses was shown to be maladaptive and to induce sterility. Here we show that various types of stress (starvation, high temperatures, and high osmolarity) but not non-stressful changes in cultivation conditions, lead to resetting of small RNA inheritance. We found that stress leads to a genome-wide reduction in heritable small RNA levels and that mutants defective in different stress pathways exhibit irregular RNAi inheritance dynamics. Moreover, we discovered that resetting of heritable RNAi is orchestrated by MAPK pathway factors, the transcription factor SKN-1, and the MET-2 methyltransferase. Termination of small RNA inheritance, and the fact that this process depends on stress, could protect from run-on of environment-irrelevant heritable gene regulation.