Vegetation structure drives mosquito community composition in UK's largest managed lowland wetland.

PURPOSE: The rising burden of mosquito-borne diseases in Europe extends beyond urban areas, encompassing rural and semi-urban regions near managed and natural wetlands evidenced by recent outbreaks of Usutu and West Nile viruses. While wetland management policies focus on biodiversity and ecosystem services, few studies explore the impact on mosquito vectors. METHODS: Our research addresses this gap, examining juvenile mosquito and aquatic predator communities in 67 ditch sites within a South England coastal marsh subjected to different wetland management tiers. Using joint distribution models, we analyse how mosquito communities respond to abiotic and biotic factors influenced by wetland management. RESULTS: Of the 12 mosquito species identified, Culiseta annulata (Usutu virus vector) and Culex pipiens (Usutu and West Nile virus vector) constitute 47% of 6825 larval mosquitoes. Abundant predators include Coleoptera (water beetles) adults, Corixidae (water boatmen) and Zygoptera (Damselfy) larvae. Models reveal that tier 3 management sites (higher winter water levels, lower agricultural intensity) associated with shade and less floating vegetation are preferred by specific mosquito species. All mosquito species except Anopheles maculipennis s.l., are negatively impacted by potential predators. Culiseta annulata shows positive associations with shaded and turbid water, contrary to preferences of Corixidae predators. CONCLUSIONS: Tier 3 areas managed for biodiversity, characterised by higher seasonal water levels and reduced livestock grazing intensity, provide favourable habitats for key mosquito species that are known vectors of arboviruses, such as Usutu and West Nile. Our findings emphasise the impact of biodiversity-focused wetland management, altering mosquito breeding site vegetation to enhance vector suitability. Further exploration of these trade-offs is crucial for comprehending the broader implications of wetland management.

Phenotypic plasticity as a cause and consequence of population dynamics.

Predicting complex species-environment interactions is crucial for guiding conservation and mitigation strategies in a dynamically changing world. Phenotypic plasticity is a mechanism of trait variation that determines how individuals and populations adapt to changing and novel environments. For individuals, the effects of phenotypic plasticity can be quantified by measuring environment-trait relationships, but it is often difficult to predict how phenotypic plasticity affects populations. The assumption that environment-trait relationships validated for individuals indicate how populations respond to environmental change is commonly made without sufficient justification. Here we derive a novel general mathematical framework linking trait variation due to phenotypic plasticity to population dynamics. Applying the framework to the classical example of Nicholson's blowflies, we show how seemingly sensible predictions made from environment-trait relationships do not generalise to population responses. As a consequence, trait-based analyses that do not incorporate population feedbacks risk mischaracterising the effect of environmental change on populations.

Prey and predator density-dependent interactions under different water volumes.

Predation is a critical ecological process that directly and indirectly mediates population stabilities, as well as ecosystem structure and function. The strength of interactions between predators and prey may be mediated by multiple density dependences concerning numbers of predators and prey. In temporary wetland ecosystems in particular, fluctuating water volumes may alter predation rates through differing search space and prey encounter rates. Using a functional response approach, we examined the influence of predator and prey densities on interaction strengths of the temporary pond specialist copepod Lovenula raynerae preying on cladoceran prey, Daphnia pulex, under contrasting water volumes. Further, using a population dynamic modeling approach, we quantified multiple predator effects across differences in prey density and water volume. Predators exhibited type II functional responses under both water volumes, with significant antagonistic multiple predator effects (i.e., antagonisms) exhibited overall. The strengths of antagonistic interactions were, however, enhanced under reduced water volumes and at intermediate prey densities. These findings indicate important biotic and abiotic contexts that mediate predator-prey dynamics, whereby multiple predator effects are contingent on both prey density and search area characteristics. In particular, reduced search areas (i.e., water volumes) under intermediate prey densities could enhance antagonisms by heightening predator-predator interference effects.

Inter-Population Similarities and Differences in Predation Efficiency of a Mosquito Natural Enemy.

Predation is a critical factor that mediates population stability, community structure, and ecosystem function. Predatory natural enemies can contribute to the regulation of disease vector groups such as mosquitoes, particularly where they naturally co-occur across landscapes. However, we must understand inter-population variation in predatory efficiency if we are to enhance vector control. The present study thus employs a functional response (FR; resource use under different densities) approach to quantify and compare predatory interaction strengths among six populations of a predatory temporary pond specialist copepod, Lovenula raynerae, from the Eastern Cape of South Africa preying on second instar Culex pipiens complex mosquito larvae. All individuals from the sampled populations were predatory and drove significant mortality through per capita predation rates of 0.75-1.10 mosquitoes/h at maximum densities over a 5-h feeding time. Individuals from all copepod populations exhibited Type II FRs with no significant differences in attack rates. On the other hand, there were significant differences in handling times, and therefore also maximum feeding rates (maximum experimental prey density: 32), suggesting possible genetic differences among populations that influenced predation. Owing to a widespread distribution in arid landscapes, we propose that predatory calanoid copepods such as L. raynerae play a key regulatory role at the landscape scale in the control of disease vector mosquito populations. We propose that these ecosystems and their specialist biota should thus be conserved and enhanced (e.g., via selective breeding) owing to the ecosystem services they provide in the context of public health.

What the fluff is this? - Gammarus pulex prefer food sources without plastic microfibers.

Investigations into the impact of micro plastics (MP) and microfibers (MFs) upon the freshwater aquatic environment are still in their infancy despite our growing awareness of their importance. Gammarus pulex have long been used as a study organism for ecotoxicology and several studies have already used them to investigate the impact of MFs. One area of research which has not been exploited is the extent to which G. pulex can detect MFs and whether or not they avoid eating them. To answer this question we developed a reliable and accurate method of exposing Gammarus to known amounts of MF embedded in algal wafers. Here we show that when given the choice between control wafers and those contaminated with 2% or 3% MF Gammarus ingest fewer MF than would be expected if a random choice was made (2% W = 7 P = 0.01698, 3% W = 13 P = 0.03397). Their feeding behaviour also changes, with a significant reduction in time feeding (F1,18 = 21.3 P = 0.0002) as well as significantly fewer visits to contaminated wafers (F1,18 = 5.312 P = 0.0333). This suggests that G. pulex are able to detect MF in the 200-500 µm range and are partially repelled by them.

Impacts of polystyrene microplastics on Daphnia magna: A laboratory and a mesocosm study.

Most research into microplastics (MPs) in freshwaters has concentrated on measurements under controlled conditions without any link to the natural environment. Here we studied the effects of a 15 µm polystyrene MP on Daphnia magna survival, growth, and reproduction in the laboratory. We also exposed fifteen 25 L freshwater mesocosms to a high concentration of the same MPs. Five were controls seeded with five species found in all ponds (mosquito, water flea, midge, spire shell and water mite), five identical but treated with 15 µm polystyrene MPs and five seeded with only mosquitoes and water fleas. The laboratory chronic toxicity test for both adults and neonate Daphnia magna revealed that effects were more related to the availability of food rather than the toxicity of MPs. In the mesocosms most of the MPs settled in the sediment after the first week of exposure. After four weeks the D. magna population decreased significantly in the MP mesocosms compared to the control mesocosms, although it subsequently recovered. There was no impact on other organisms added to the mesocosms, other than a difference in timing of lesser water boatman (Corixa punctata) colonisation, which colonised the control mesocosms in week 4 and the treated 4 weeks later. The detrivorous, sediment sifting, mayfly Leptophlebia marginata appeared in mesocosms in the fourth week of sampling and with significantly higher numbers in the MP treated mesocosm. Their activity had no significant impact on MPs in the water column, although numbers did increase above zero. The significant decline of D. magna suggests that their effect in a natural situation is unpredictable where environmental conditions and invertebrate communities may add additional stresses.

Sink trap: duckweed and dye attractant reduce mosquito populations.

Duckweeds, such as Lemna minor Linnaeus (Alismatales: Lemnaceae), are common in aquatic habitats and have been suggested to reduce larval mosquito survivorship via mechanical and chemical effects. Furthermore, pond dyes are used increasingly in aquatic habitats to enhance their aesthetics, although they have been shown to attract mosquito oviposition. The present study examined the coupled effects of L. minor and black pond dye on the oviposition selectivity of Culex pipiens Linnaeus (Diptera: Culicidae) mosquitoes in a series of laboratory choice tests. Subsequently, using outdoor mesocosms, the combined influence of duckweed and pond dye on mosquito abundances in aquatic habitats was quantified. Mosquitoes were strongly attracted to duckweed, and oviposited significantly greater numbers of egg rafts in duckweed-treated water compared with untreated controls, even when the duckweed was ground. The presence of pond dye interacted with the duckweed and further enhanced positive selectivity towards duckweed-treated water. The presence of duckweed caused significant and sustained reductions in larval mosquito numbers, whereas the relative effects of dye were not evident. The use of floating aquatic plants such as duckweed, combined with dye, may help reduce mosquito populations via the establishment of population sinks, characterized by high rates of oviposition coupled with high levels of larval mortality.

A novel metric reveals biotic resistance potential and informs predictions of invasion success.

Invasive species continue to proliferate and detrimentally impact ecosystems on a global scale. Whilst impacts are well-documented for many invaders, we lack tools to predict biotic resistance and invasion success. Biotic resistance from communities may be a particularly important determinant of the success of invaders. The present study develops traditional ecological concepts to better understand and quantify biotic resistance. We quantified predation towards the highly invasive Asian tiger mosquito Aedes albopictus and a representative native mosquito Culex pipiens by three native and widespread cyclopoid copepods, using functional response and prey switching experiments. All copepods demonstrated higher magnitude type II functional responses towards the invasive prey over the analogous native prey, aligned with higher attack and maximum feeding rates. All predators exhibited significant, frequency-independent prey preferences for the invader. With these results, we developed a novel metric for biotic resistance which integrates predator numerical response proxies, revealing differential biotic resistance potential among predators. Our results are consistent with field patterns of biotic resistance and invasion success, illustrating the predictive capacity of our methods. We thus propose the further development of traditional ecological concepts, such as functional responses, numerical responses and prey switching, in the evaluation of biotic resistance and invasion success.

Differential Interaction Strengths and Prey Preferences Across Larval Mosquito Ontogeny by a Cohabiting Predatory Midge.

Understandings of natural enemy efficacy are reliant on robust quantifications of interaction strengths under context-dependencies. For medically important mosquitoes, rapid growth during aquatic larval stages could impede natural enemy impacts through size refuge effects. The identification of biocontrol agents which are unimpeded by ontogenic size variability of prey is therefore vital. We use functional response and prey preference experiments to examine the interaction strengths and selectivity traits of larvae of the cohabiting predatory midge Chaoborus flavicans (Meigen 1830) (Diptera: Chaoboridae) towards larval stages of the Culex pipiens (Diptera: Culicidae) mosquito complex. Moreover, we examine the influence of search area variation on selectivity traits, given its importance in consumer-resource interactions. Chaoborids were able to capture and consume mosquito prey across their larval ontogeny. When prey types were available individually, a destabilizing Type II functional response was exhibited towards late instar mosquito prey, whereas a more stabilizing Type III functional response was displayed towards early instars. Accordingly, search efficiencies were lowest towards early instar prey, whereas, conversely, maximum feeding rates were highest towards this smaller prey type. However, when the prey types were present simultaneously, C. flavicans exhibited a significant positive preference for late instar prey, irrespective of water volume. Our results identify larval chaoborids as efficacious natural enemies of mosquito prey, with which they frequently coexist in aquatic environments. In particular, an ability to prey on mosquitoes across their larval stages, coupled with a preference for late instar prey, could enable high population-level offtake rates and negate compensatory reductions in intraspecific competition through size refuge.

Variation in the susceptibility of Anopheles gambiae to botanicals across a metropolitan region of Nigeria.

Pesticide resistance is normally associated with genetic changes, resulting in varied responses to insecticides between different populations. There is little evidence of resistance to plant allelochemicals; it is likely that their efficacy varies between genetically diverse populations, which may lead to the development of resistance in the future. This study evaluated the response of Anopheles gambiae (larvae and adults) from spatially different populations to acetone extracts of two botanicals, Piper guineense and Eugenia aromatica. Mosquito samples from 10 locations within Akure metropolis in Southwest Nigeria were tested for variation in susceptibility to the toxic effect of botanical extracts. The spatial distribution of the tolerance magnitude (T.M.) of the mosquito populations to the botanicals was also mapped. The populations of An. gambiae manifested significant differences in their level of tolerance to the botanicals. The centre of the metropolis was the hot spot of tolerance to the botanicals. There was a significant positive correlation between the adulticidal activities of both botanicals and initial knockdown. Hence, knockdown by these botanicals could be a predictor of their subsequent mortality. In revealing variation in response to botanical pesticides, our work has demonstrated that any future use of botanicals as alternative environmentally friendly vector control chemicals needs to be closely monitored to ensure that resistance does not develop.

The influence of microplastics on trophic interaction strengths and oviposition preferences of dipterans.

Microplastic (MP) pollution continues to proliferate in freshwater, marine and terrestrial environments, but with their biotic implications remaining poorly understood. Biotic interactions such as predation can profoundly influence ecosystem structuring, stability and functioning. However, we currently lack quantitative understandings of how trophic interaction strengths and associated behaviours are influenced by MP pollution, and how transference of MPs between trophic levels relates to consumptive traits. We also lack understanding of key life-history effects of MPs, for example, reproductive strategies such as oviposition. The present study examines the predatory ability of non-biting midge larvae, Chaoborus flavicans, towards larvae of Culex pipiens mosquitoes when the latter are exposed to MPs, using a functional response (FR) approach. Transfer of MPs occurred from larval mosquitoes to larval midges via predation. Microplastics transfer was significantly positively related to predation rates. Predation by C. flavicans followed a Type II FR, with average maximum feeding rates of 6.2 mosquito larvae per hour. These and other FR parameters (attack rates and handling times) were not significantly influenced by the presence of MPs. Further, C. pipiens adults did not avoid ovipositing in habitats with high concentrations of MPs. We thus demonstrate that MPs can move readily through freshwater food webs via biotic processes such as predation, and that uptake correlates strongly with consumption rates. Further, as MPs do not deter adult mosquitoes from ovipositing, our experiments reveal high potential for MP exposure and transference through ecosystems.

Examining effects of ontogenic microplastic transference on Culex mosquito mortality and adult weight.

Microplastics (MPs) continue to proliferate and pollute aquatic and terrestrial environments globally. The impacts of MP pollution on ecosystems and their functioning remain poorly quantified, with most research hitherto focusing on marine ecosystems. There is a paucity of information on the impacts of MPs in freshwater ecosystems, despite the broad range of pathways through which MPs can proliferate and the extensive range of species which actively ingest MPs in these systems. Of particular interest are organisms that bridge aquatic and terrestrial habitats. The present study thus examines the uptake, ontogenic transference and effect of different concentrations (0, 50, 100 and 200 MPs mL-1) and sizes (2 and 15 µm) of polystyrene MPs between aquatic and terrestrial life stages of Culex pipiens complex mosquitoes. Both 2 and 15 µm MPs transferred from the aquatic larval to terrestrial adult stage of Culex mosquitoes, and uptake correlated tightly with initial exposure concentration. However, neither concentration nor size of MPs significantly influenced mortality rates between the aquatic larval and terrestrial adult stage. There was also no impact of MPs on the weight of emerging mosquito adults. We thus demonstrate that MPs can be transferred ontogenically through organisms with complex life histories, presenting a potential pathway for dispersal of MPs into terrestrial environments. We also show that MPs exposure does not affect mortality rates between life stages of freshwater Culex populations. This suggests that MPs do not impact nutritional uptakes, with unhampered development to adulthood facilitating subsequent dispersal of MPs aerially and between freshwater and terrestrial habitats.

The Effect of the Alternative Prey, Paramecium caudatum (Peniculida: Parameciidae), on the Predation of Culex pipiens (Diptera: Culicidae) by the Copepods Macrocyclops albidus and Megacyclops viridis (Cyclopoida: Cyclopidae).

Biological control can be an effective tool to combat public health risks associated with mosquito-borne disease. However, target impacts of biological control agents may be reduced by biotic contexts such as the presence of alternative prey. In turn, this can impede our ability to realistically assess biocontrol agent efficacy. Here, we examine the effects of alternative ciliate prey on the predation potential of two cyclopoid copepods, Macrocyclops albidus Jurine (Cyclopoida: Cyclopidae) and Megacyclops viridis Jurine (Cyclopoida: Cyclopidae), toward larvae of the West Nile virus vector mosquito Culex pipiens Linnaeus (Diptera: Culicidae). Using functional responses (FRs; resource use under different resource densities), we demonstrate that both copepods exhibit potentially destabilizing type II FRs toward mosquito prey. However, where the alternative prey was present, we observed species-specific modulations to FR form and magnitude. For M. albidus, FRs remained type II where ciliate prey were present, however, maximum feeding rates on mosquito larvae were reduced. Conversely, for M. viridis, FRs moved toward more stabilizing type III, while maximum feeding rates on mosquito larvae were not significantly reduced. While both species of cyclopoid copepod were able to effectively target and consume larval mosquitoes in the presence of alternative prey, we demonstrate that overall efficacies may be reduced in aquatic habitats which contain multiple prey types. We thus advocate that biotic contexts such as prey selectivity should be integrated into predatory biocontrol agent examinations for mosquitoes which vector pathogens and parasites, to more holistically assess their efficacy.

Intermediate predator naïveté and sex-skewed vulnerability predict the impact of an invasive higher predator.

The spread of invasive species continues to reduce biodiversity across all regions and habitat types globally. However, invader impact prediction can be nebulous, and approaches often fail to integrate coupled direct and indirect invader effects. Here, we examine the ecological impacts of an invasive higher predator on lower trophic groups, further developing methodologies to more holistically quantify invader impact. We employ functional response (FR, resource use under different densities) and prey switching experiments to examine the trait- and density-mediated impacts of the invasive mosquitofish Gambusia affinis on an endemic intermediate predator Lovenula raynerae (Copepoda). Lovenula raynerae effectively consumed larval mosquitoes, but was naïve to mosquitofish cues, with attack rates and handling times of the intermediate predator unaffected by mosquitofish cue-treated water. Mosquitofish did not switch between male and female prey, consistently displaying a strong preference for female copepods. We thus demonstrate a lack of risk-reduction activity in the presence of invasive fish by L. raynerae and, in turn, high susceptibility of such intermediate trophic groups to invader impact. Further, we show that mosquitofish demonstrate sex-skewed predator selectivity towards intermediate predators of mosquito larvae, which may affect predator population demographics and, perversely, increase disease vector proliferations. We advocate the utility of FRs and prey switching combined to holistically quantify invasive species impact potential on native organisms at multiple trophic levels.

Up and away: ontogenic transference as a pathway for aerial dispersal of microplastics.

Microplastics (MPs) are ubiquitous pollutants found in marine, freshwater and terrestrial ecosystems. With so many MPs in aquatic systems, it is inevitable that they will be ingested by aquatic organisms and be transferred up through the food chain. However, to date, no study has considered whether MPs can be transmitted by means of ontogenic transference, i.e. between life stages that use different habitats. Here, we determine whether fluorescent polystyrene beads could transfer between Culex mosquito life stages and, particularly, could move into the flying adult stage. We show for the first time that MPs can be transferred ontogenically from a feeding (larva) into a non-feeding (pupa) life stage and subsequently into the adult terrestrial life stage. However, transference is dependent on particle size, with smaller 2 µm MPs transferring readily into pupae and adult stages, while 15 µm MPs transferred at a significantly reduced rate. MPs appear to accumulate in the Malpighian tubule renal excretion system. The transfer of MPs to the adults represents a potential aerial pathway to contamination of new environments. Thus, any organism that feeds on terrestrial life phases of freshwater insects could be impacted by MPs found in aquatic ecosystems.

Calanoid Copepods: An Overlooked Tool in the Control of Disease Vector Mosquitoes.

Biological control can assist in the management of disease vector mosquitoes. However, we urgently require the identification of novel and effective agents to aid population management strategies. Previously, predatory biocontrol of disease vector mosquito species has focused extensively on cyclopoid copepods, but prey size refuge effects have been identified as a hindrance to their predatory efficacy. Calanoid copepods have yet to be examined in the context of mosquito control, despite their high prevalence, diversity, and distribution. Here, we apply functional responses (FRs; resource use as a function of resource density) to examine predation efficiencies of a recently described ephemeral pond specialist species, the freshwater calanoid copepod Lovenula raynerae Suárez-Morales, Wasserman & Dalu 2015 (Calanoida: Diaptomidae), using different size classes of larvae of the disease vector complex Culex pipiens (Diptera: Culicidae) as prey. Lovenula raynerae effectively consumed Cx. pipiens larvae across their ontogeny. A potentially population destabilizing type II FR was exhibited toward both early and late instar mosquitoes, indicative of a lack of prey refuge across ontogenetic stages. Attack rates were greatest and handling times lowest for early instar larvae compared to late instar larvae. These traits contrast to other copepods commonly applied in biocontrol, which are only able to handle early instars, and in much smaller numbers. We thus advocate that calanoid copepods can exert particularly marked predatory impact on lower trophic groups, and that their use in disease vector mosquito control strategies should be further explored.

Impact of polystyrene microplastics on Daphnia magna mortality and reproduction in relation to food availability.

Microplastics (MPs) in the environment continue to be a growing area of concern in terms of acute and chronic impacts on aquatic life. Whilst increasing numbers of studies are providing important insights into microparticle behaviour and impacts in the marine environment, a paucity of information exists regarding the freshwater environment. This study focusses on the uptake, retention and the impact of 2 µm polystyrene MPs in the freshwater cladoceran Daphnia magna in relation to food intake (algae Chlorella vulgaris), with MP size chosen to approximately match the cell size of the algae. Daphnia were exposed to varied concentrations of MPs and algae. When exposed to a single concentration of MPs Daphnia almost immediately ate them in large quantities. However, the presence of algae, even at low concentrations, had a significant negative impact on MP uptake that was not in proportion to relative availability. As MP concentrations increased, intake did not if algae were present, even at higher concentrations of MPs. This suggests that Daphnia are selectively avoiding eating plastics. Adult Daphnia exposed to MPs for 21 days showed mortality after seven days of exposure in all treatments compared to the control. However significant differences were all related to algal concentration rather than to MP concentration. This suggests that where ample food is present, MPs have little effect on adults. There was also no impact on their reproduction. The neonate toxicity test confirmed previous results that mortality and reproduction was linked to availability of food rather than MP concentrations. This would make sense in light of our suggestion that Daphnia are selectively avoiding eating microplastics.

The effect of pond dyes on oviposition and survival in wild UK Culex mosquitoes.

British Culex pipiens complex [Culex pipiens sensu lato) mosquito distribution, abundance, and potential for disease transmission are intimately linked to their environment. Pond and lake dyes that block light to restrict algal photosynthesis are a relatively new product assumed to be an environmentally friendly since they are based on food dyes. Their use in urban garden ponds raises questions linked to mosquito oviposition, since coloured water can be an attractant. Culex (mostly pipiens) is commonly found in UK gardens and is a potential vector of viruses including the West Nile Virus (WNV). Any factors that significantly change the distribution and population of Cx pipiens could impact future risks of disease transmission. A gravid trap was used to catch female Cx pipiens mosquitoes for use in oviposition choice tests in laboratory and semi-field conditions. Two types of pond dye, blue and shadow (which looks slightly red), were tested for their impact on oviposition and survival of wild caught Cx pipiens. There were no significant differences in the number of egg batches laid when gravid mosquitoes were given a choice between either blue dye and clear water or shadow dye and clear water indicating that these dyes are not attractants. Larvae hatched from egg batches laid by wild-caught gravid females were used to measure survival to adulthood with or without dye, in a habitat controlled to prevent further colonisation. The experiment was run twice, once in the summer and again in the autumn, whereas the dyes had no impact on emergence in the summer, there were highly significant reductions in emergence of adults in both dye treated habitats in the autumn. Containers with or without shadow dye were placed outside to colonise naturally and were sampled weekly for larvae and pupae over a 6 month period through summer and autumn. There was a significant negative effect of shadow dye on pupal abundance in a three week period over the summer, but otherwise there was no effect. It is likely that population abundance and food was a more powerful factor for mosquito survival than the dye.

Pond dyes are Culex mosquito oviposition attractants.

BACKGROUND: British mosquito population distribution, abundance, species composition and potential for mosquito disease transmission are intimately linked to the physical environment. The presence of ponds and water storage can significantly increase the density of particular mosquito species in the garden. Culex pipiens is the mosquito most commonly found in UK gardens and a potential vector of West Nile Virus WNV, although the current risk of transmission is low. However any factors that significantly change the distribution and population of C. pipiens are likely to impact subsequent risk of disease transmission. Pond dyes are used to control algal growth and improve aesthetics of still water reflecting surrounding planting. However, it is well documented that females of some species of mosquito prefer to lay eggs in dark water and/or containers of different colours and we predict that dyed ponds will be attractive to Culex mosquitoes. METHODS: Black pond dye was used in oviposition choice tests using wild-caught gravid C. pipiens. Larvae from wild-caught C. pipiens were also reared in the pond dye to determine whether it had any impact on survival. An emergence trap caught any adults that emerged from the water. Water butts (80 L) were positioned around university glasshouses and woodland and treated with black pond dye or left undyed. Weekly sampling over a six month period through summer and autumn was performed to quantified numbers of larvae and pupae in each treatment and habitat. RESULTS: Gravid female Culex mosquitoes preferred to lay eggs in dyed water. This was highly significant in tests conducted under laboratory conditions and in a semi-field choice test. Despite this, survivorship in black dyed water was significantly reduced compared to undyed water. Seasonal analysis of wild larval and pupal numbers in two habitats with and without dye showed no impact of dye but a significant impact of season and habitat. Mosquitoes were more successful, with significantly higher numbers of pupae, in the habitat where they had vegetation cover and shade. DISCUSSION: Our study has raised some interesting possibilities; one is that where used, pond dyes may be encouraging mosquitoes to breed in gardens in close proximity to people. Considering the concerns over potential future spread of disease in urban environments, this as well as shading of ponds and water butts, should inform future advice over reducing mosquito breeding and spread.