Xenobiotic resistance in mosquito eggs: current understanding and data gaps

Egg physiology and exposure to xenobiotics (antibiotics and insecticides)

Egg laying is an important aspect of female insects’ reproductive biology. Considering its significance, the “when” and “where” of egg laying is critically regulated and conserved in insect species. Insect species select the location to oviposit depending upon its ability to support the process of larval development. A non-exhaustive and generally accepted list of oviposition sites in insects includes soil (beetles, flies, etc.), plants (phytophagous insects), vertebrate wounds (flies), insect larvae (parasitic wasps, leafhoppers), body of insects or spiders (parasitic wasps), decaying organic matter (saprophagous insects), and water (mosquitoes) (Cury, Prud’homme & Gompel, 2019). The behavioral tendency of insect species, like mosquitoes, to return to similar egg laying sites over and over across generations is termed oviposition constancy. Oviposition constancy in insects is often a combination of innate and learnt preferences and helps females minimize their efforts being spent on unsuccessful oviposition (Nataraj, Hansson & Knaden, 2024). The inclination of gravid mosquitoes to select water bodies for oviposition, unlike many other insect species, is an important factor that increases the likelihood of exposure of eggs to xenobiotics. This makes the understanding of the survival mechanisms of mosquito eggs before the formation of SC all the more relevant.

As is the case with all insects, the production of eggs in mosquitoes is a complex and carefully regulated process. The initiation of follicle development by an accumulation of vitellogenin yolk proteins begins after a female mosquito takes a blood meal from a host. Most of the structural components of an eggshell are secreted by a single layer of follicular epithelial cell surrounding the oocyte. At the proteomic level, eight different proteins involved in the fecundity, melanization, and viability of mosquito eggs have been identified. These proteins include Nasrat, Closca, Polehole, Nudel, DCE2, DEC4, DCE5, and CATL3. Nasrat, Closca, Polehole, Nudel, and DCE2 proteins play a critical role in maintaining the integrity of the eggshell. Other proteins include structural proteins, enzymes, odorant binding proteins, and uncharacterized proteins of unknown functions (Isoe et al., 2019, 2023).

At the microscopic level, Mundim-Pombo et al. (2021) investigated the timeline of embryogenesis in detail. At the proteomic level, a non-exhaustive list of eggshell proteins and their functions is delineated in Table 1 (Marinotti et al., 2014).

A recent comprehensive bioinformatic analyses of putative protein-coding sequences of Aedes, Culex, and Anopheles genomes has resulted in the identification of another protein, i.e., the Eggshell Organizing Factor 1 (EOF1). The authors hypothesized that the EOF1 protein has conservatively evolved within the Culicidae family and affects eggshell formation and melanization (Isoe et al., 2019, 2023).

At the phenotypic level, the formation of the eggshell and SC during mid-to-late stages of embryogenesis plays a crucial role in the viability of the developing embryo. The formation of SC imparts desiccation resistance and ensures the survival of mosquito eggs during dry conditions. Considering the potential changes in climate globally including prolonged drought periods, the significance of SC in mosquito eggs cannot be underestimated. Even at the species level, the cuticle has been highly preserved among insects over the course of evolution. The cuticle offers protection by preventing the penetration of external compounds, like xenobiotics. The outermost layer of a cuticle is the epicuticle and is composed of hydrocarbons, proteins, and lipids. The lipids offer a hydrophobic barrier preventing the entry of hydrophilic compounds like insecticides (Balabanidou, Grigoraki & Vontas, 2018).

During early embryogenesis, the eggshell is only composed of exochorion and endochorion. The exochorion and endochorion are maternally derived and are secreted by the follicle cells. At this stage, the eggshell is highly permeable to water, and exposure to xenobiotics through water. The propensity of mosquitoes to oviposit in or near water sources exacerbates this exposure. The zygotically derived extraembryonic serosal cells secrete the SC over the course of embryogenesis. The SC eventually becomes the third and innermost layer and decreases the water permeability of eggs (Farnesi et al., 2017; Muthukrishnan et al., 2022; Isoe et al., 2023).

An important component of eggshells is a homopolymer of N-acetyl-D-glucosamine linked by β-1, 4-glycosidic bonds (i.e., chitin). The biosynthesis of chitin in insects starts with trehalose sugar found in the insect hemolymph and ends with the formation of chitin polymer. Chitin synthases (CHS) are membrane bound enzymes and use the activated “uridine diphosphate-N-acetylglucosamine” to form chitin polymer (Glaser & Brown, 1957). Chitin synthase A (CHSA) and chitin synthase B (CHSB) are the two most common chitin synthases found in insect species. Chitin synthase A is responsible for the formation of chitin incorporated in the cuticle, and is expressed mainly in the epidermal cells. Chitin synthase B is responsible for the formation of chitin in the peritrophic matrix, and is expressed in the midgut epithelial cells (Arakane et al., 2005; Zimoch et al., 2005). Chitin in eggshells offers protection from mechanical stress, dehydration, and xenobiotics (Muthukrishnan et al., 2022). The biosynthesis, transformation, and modification of chitin is an important requirement in the process of insect molting (Tetreau & Wang, 2019). Molting is crucial in the normal growth and development of insect species (Zhang et al., 2014).

The presence of chitin in eggs, eggshells, and ovaries of mosquitoes has been well documented. Additionally, chitinase activity was observed not just in eggs as they near eclosion, but also in newly hatched larvae (Moreira et al., 2007). The chitinized SC decreases the permeability of eggs, preventing exposure to xenobiotics through water. Additionally, the chitinized SC also imparts desiccation resistance to eggs (Farnesi et al., 2015).

Desiccation significantly impacts the viability of a mosquito egg. Temperature and humidity are found to be the leading causes of desiccation in mosquito eggs (Juliano et al., 2002). Depending on the habitat into which the mosquito oviposits, the eggs of some species of mosquitoes are more prone to desiccation than others and exhibit desiccation tolerance accordingly. Aedes species oviposit their eggs in moist habitats where they are more prone to desiccation. Culex species on the other hand oviposit in water and are less prone to desiccation (Suman et al., 2013).

Increase in temperature accelerates the process of desiccation in mosquito eggs. Previous studies have established a relationship between temperature, the length of embryogenesis, and the acquisition of SC-medicated desiccation resistance. The length of embryogenesis and the formation of SC varies between different species of mosquitoes. At 28 °C, the total length of embryogenesis in Ae. aegypti eggs was reported to be 61.5 h post-oviposition. The SC was formed during the complete germ band extension phase at 11–13 h post-oviposition (Rezende et al., 2008). At 25 °C, embryogenesis was completed in Ae. aegypti, Anopheles aquasalis, and Culex quinquefasciatus eggs at 77.4, 51.3, and 34.3 h after oviposition, respectively. The SC-mediated desiccation resistance was acquired 14–16 h post-oviposition in Ae. aegypti, 9–11 h post-oviposition in An. aquasalis, and 10–12 h post-oviposition in Cx. quinquefasciatus (Vargas et al., 2014).

Table 2 summarizes the duration of embryogenesis and the time the SC forms, at two different temperatures in Ae. aegypti.

The time required for the formation of SC and the acquisition of SC-mediated desiccation resistance shortened with an increase in temperature. This is important to ensure the viability of eggs. In adult mosquitoes, a microbiome mediated response to heat stress was observed in Ae. aegypti. This involved the enrichment of midgut microbiome with heat-tolerant taxa such as Bacillus spp. in response to an increase in temperature (Onyango et al., 2020).

From the available literature, it is evident that the formation of SC does not happen for at least a few hours post-oviposition. This suggests that the eggs are vulnerable to exposure to insecticides and antibiotics present in the aquatic environments the mosquitoes lay eggs in. The current understanding of the effects of antibiotics and insecticides on mosquitoes and mosquito eggs is summarized in the next section.

Effect of xenobiotics (antibiotics and insecticides) on mosquitoes and mosquito eggs-current understanding

A 2018 survey by the World Organization for Animal Health estimated that between 69 and 76 kilotons of antimicrobial agents were used in the year 2018 for veterinary applications alone. The data was collected across 109 participating countries. A crucial finding was that antibiotics accounted for more than 54% of the total antimicrobials intended for use in animals (World Organization for Animal Health, 2022). The antibiotic runoff from these sources into nearby water bodies is a significant source of exposure of antibiotics to mosquito eggs.

Mosquitoes are at an increased risk of exposure to antibiotics across all life stages owing to the increased presence of antibiotics in the environment. Eggs of mosquitoes may often hatch in water contaminated with antibiotics, and the larvae and pupae also spend considerable time in water contaminated with antibiotics (Endersby-Harshman, Axford & Hoffmann, 2019). Adult mosquitoes ingest minute concentrations of antibiotics during a blood meal if the antibiotics are circulating in the host system (Gendrin et al., 2015).

Exposure of mosquito eggs to antibiotics is known to negatively affect embryogenesis and hatching success. A possible explanation is the inhibition of amino acid absorption resulting from exposure to antibiotics. Amino acids play a crucial role in egg production and larval development in mosquitoes (Ha et al., 2021). Exposure to antibiotics in the larval stage is also known to affect long-term survival and vector competence in mosquitoes. Exposure to antibiotics in the larval and subsequent stages disrupt the microbiome in mosquitoes (Garrigos et al., 2023). The effects of antibiotics on mosquito microbiome include alteration in the composition of microbiota, immune mediated responses, and nutrient absorption (Ferreira et al., 2023; Garrigos et al., 2023). Additionally, exposure to antibiotics is known to negatively affect the production of eggs in female mosquitoes. Antibiotics inhibit the lysis of red blood cells and decrease the digestion of blood proteins. This deprives the mosquito of essential nutrients and eventually results in a decrease in the number of eggs produced by female mosquitoes (Gaio et al., 2011).

On the other hand, the global use of pesticides in agriculture in the year 2022 was estimated to be 3.7 million tons of active ingredient. This represented a 4% increase from the year 2021, a 13% increase from the previous decade, and a 200% increase from the 1990s. Insecticides used around the world include chlorinated hydrocarbons, organophosphates, carbamates, pyrethroids, botanical and biological products. Compared to the 1990s, the most recent decade saw a 48% increase in the use of insecticides (FAO, 2024). Most of the insecticides are water soluble and their runoff into water bodies is a significant source of exposure of mosquito eggs to these insecticides. Exposure of adult mosquitoes to insecticides frequently occurs due to regional vector control programs. Additionally, eggs may also hatch in waters contaminated with run-off insecticides from various domestic and regional vector control operations.

The toxic effects of chemicals used in vector control programs on adult mosquitoes and larvae are well documented, and the ovicidal activity of some of those chemicals has also been studied. Exposure of eggs to insect growth regulators like pyriproxyfen, azadirachtin, and diflubenzuron results in abnormal embryonic development. Pyriproxyfen and azadirachtin arrest embryonic development, and diflubenzuron affects chitin development of the embryonic cuticle (Suman et al., 2013). Exposure to sublethal concentrations of cypermethrin has resulted in not only fewer eggs laid by female mosquitoes, but also in reduced hatching success (Sunday, Kayode & Ashamo, 2016).

The ovicidal effects of xenobiotics clearly affect mosquitoes at a population level. However, it is not clear if eggs are evolving to combat the exposure to xenobiotics prior to the formation of SC. This is a glaring knowledge gap in mosquito biology that needs to be investigated. The next section attempts to present a rational explanation based on existing evidence that could lead to further research in this direction.

Xenobiotic resistance in eggs and adult mosquitoes: current understanding and knowledge gaps?

Role of male mosquitoes in the propagation of xenobiotic resistance

Male mosquitoes are often not considered for any xenobiotic susceptibility/resistance studies. A big reason is their lack of vectorial capacity, and their lack of ability to transmit diseases (Alphey et al., 2010). However, a few observations from recent studies and critical data gaps pertaining to the role of male mosquitoes in the propagation of xenobiotic resistance are highlighted below:

1. Mains, Brelsfoard & Dobson (2015) have investigated the use of male mosquitoes to complement the current vector control approaches to deliver larvicides like pyriproxyfen to mosquito breeding sites. They termed this approach, “Auto-Dissemination Augmented by Males (ADAM)”. Male mosquitoes either contaminated the breeding sites or cross-contaminated females delivering lethal doses of larvicides, in both the cage and open field trials. Male mosquitoes also demonstrated tolerance to larvicidal doses of pyriproxyfen. Most importantly, it can be inferred that male mosquitoes act as another source of exposure of xenobiotics to mosquito eggs.

2. A recent study by Ojianwuna et al. (2024) in Nigeria revealed that male Anopheles gambiae mosquitoes are demonstrating resistance to dichlorodiphenyltrichloroethane (DDT) and lambda-cyhalothrin, and male Aedes aegypti mosquitoes are demonstrating resistance to lambda-cyhalothrin.

3. The genetic engineering of male mosquitoes is being considered as another vector control approach lately. Two techniques that are being considered for this purpose are the Sterile Insect Technique (SIT) and the Incompatible Insect Technique (IIT). Both SIT and IIT techniques resulted in reduced mosquito densities and incidence of mosquito-borne diseases like Dengue, as reviewed by Rahul et al. (2024).

From the above observations, it can be inferred that male mosquitoes are demonstrating xenobiotic resistance. An important question in this regard is the mode of acquisition of xenobiotic resistance, especially in male mosquitoes. One explanation is the transovarial transmission of xenobiotic resistance genes, which enable both eggs and adults survive exposure to xenobiotics. The presence of xenobiotic resistance genes in the egg also facilitates its survival until the formation of SC. Another data gap is the role of male mosquitoes in the propagation of xenobiotic resistance. If the male mosquitoes are surviving exposure to xenobiotics due to the presence of resistance genes, it is very likely they are also passing them to the next generations. Unlike females, in which the process of oviposition comes at considerable fitness/energy costs that make them more susceptible to external stressors, male mosquitoes are likely to survive more severe exposure to xenobiotics. It needs to be investigated if males are passing more resistance genes than females to the offspring, through the egg. A straightforward way to investigate the presence of xenobiotic resistance genes in male mosquitoes is to extract their DNA and screen for the presence of known resistance genes.