Plastics, synthetic organic polymers, are essential in modern society for their strength, flexibility, and durability, but since the 1960s their production and disposal have contributed significantly to global waste, posing environmental risks. Recently, attention has turned to small plastics, both intentionally produced and as byproducts of larger plastics. These particles, ranging from macro- to nanoplastics, are dispersed by wind, and accumulate in marine and freshwater ecosystems, threatening species, ecosystems, and human health. Various organisms, including mammals, birds, fish, and zooplankton, can ingest micro- and nanoplastics (MNPLs), contributing to their transport and transformation, raising ecological concerns. Mosquitoes also play a role in MNPL dispersal. Their larvae and pupae live in aquatic habitats that may be MNPL-contaminated and can emerge as adults carrying these particles, potentially facilitating bioaccumulation across trophic levels. Mosquitoes can lay eggs in urban water reservoirs and domestic wastewater, areas with high MNPL concentrations. The Asian tiger mosquito, Aedes albopictus, which thrives in these habitats, is a key model for studying the impact of MNPLs on mosquito biology, with implications for public health as it is a primary vector for arboviruses like dengue, Zika, and chikungunya. This thesis aims to investigate the effects of MNPL exposure on Ae. albopictus larvae using a multidisciplinary approach combining insect physiology, fluorescence and confocal microscopy, and metabolomics. The study traces the fate of polystyrene MNPLs after ingestion by mosquito larvae across various organs and developmental stages and explores the potential impact of MNPL exposure on larval metabolism, providing preliminary insights into the relationship between plastic pollutants and mosquito biology. First-instar larvae were exposed to fluorescent polystyrene micro- (MPs, 2 μm) and nanoplastics (NPs, 0.5 μm). Their ability to ingest and internalize these particles was assessed using fluorescence and confocal microscopy to trace the MNPLs in whole larvae, dissected organs, and tissues. This allowed for detailed observation of plastic uptake, transport, and accumulation. The study also examined the transfer of MNPLs from larvae to pupae and adults. Additionally, a preliminary metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS) was conducted to explore potential metabolic changes in larvae exposed to MNPLs. Results confirmed that Ae. albopictus larvae actively ingest both MP and NP beads, which were found throughout the alimentary tract. Microscopy revealed that MNPLs crossed the intestinal barrier, accumulating in the Malpighian tubules, the hemocoel, and the hemolymph. MNPLs were also detected throughout the developmental stages, indicating that the particles were transferred and persisted in adult tissues. Despite the uptake and persistence of MNPLs, no significant impact on mosquito development was observed. The duration of larval and pupal stages, as well as survival rates to adulthood, did not significantly differ between MNPL-treated groups and controls. However, metabolomics analysis revealed that MNPL exposure induced metabolic changes in larvae. 81 metabolites were found to be significantly altered, with many downregulated metabolites involved in central carbon metabolism, including glycolysis and the pentose phosphate pathway. These findings suggest that while MNPLs do not seem to affect mosquito development, they may have important effects on metabolic pathways. This study underscores the potential of plastic pollution to impact mosquito biology. Investigating the metabolic effects of MNPLs paves the way for future research on the molecular mechanisms underlying plastic-mosquito interactions, offering insights into vector biology and environmental toxicology.
Le materie plastiche, polimeri organici sintetici, sono cruciali nella società moderna per la loro resistenza e versatilità, ma il loro uso provoca gravi impatti ambientali. In particolare, le micro- e nanoplastiche (MNPL), disperse nell'ambiente, si accumulano negli ecosistemi marini e d'acqua dolce, minacciando specie e biodiversità. Vari organismi, inclusi mammiferi, pesci e uccelli, ingeriscono queste particelle, contribuendo alla loro diffusione. Anche le zanzare possono essere esposte alle MNPL, specialmente negli stadi immaturi, cioè larve e pupe, che vivono in habitat acquatici. Questi organismi possono accumulare MNPL nei loro tessuti, trasportandole tra diversi livelli trofici. Inoltre, è noto che le zanzare adulte possono deporre le uova in acque contaminate da MNPL. I bacini urbani, caratterizzati da alte concentrazioni di MNPL, rappresentano habitat ideali per la zanzara tigre asiatica Aedes albopictus, un vettore di malattie come dengue, Zika e chikungunya. Questo lavoro di tesi si concentra sugli effetti dell'esposizione alle MNPL sulle larve di Ae. albopictus, utilizzando un approccio multidisciplinare che integra fisiologia, microscopia a fluorescenza e confocale, ed analisi metabolomiche preliminari. Lo scopo principale è tracciare il destino di MNPL di polistirene dopo l’ingestione da parte delle larve attraverso vari organi e stadi di sviluppo, e investigare il loro potenziale impatto sul metabolismo larvale. L'approccio sperimentale ha previsto l’esposizione di larve di primo stadio a micro- (MP, 2 μm) e nanoplastiche (NP, 0,5 μm) fluorescenti, monitorando la capacità delle larve di ingerirle e internalizzarle. Tecniche di microscopia a fluorescenza e confocale sono state utilizzate per tracciare le MNPL nei tessuti, osservando il loro assorbimento, trasporto e accumulo. Lo studio ha anche indagato il trasferimento delle MNPL dalle larve alle pupe e infine agli adulti. Inoltre, è stata condotta un'analisi metabolomica basata su cromatografia liquida-spettrometria di massa (LC-MS) per iniziare ad investigare le potenziali alterazioni metaboliche nelle larve esposte alle MNPL. I risultati hanno confermato che le larve di Aedes albopictus possono ingerire sia MP che NP, osservabili lungo tutto il tratto alimentare. Le MNPL non solo si accumulano nell'intestino, ma attraversano anche la barriera intestinale, raggiungendo i tubuli malpighiani, l'emocele e l'emolinfa. Le MNPL sono state identificate in tutti gli stadi di sviluppo, suggerendo che vengono trasferite dalle larve agli adulti e rimangono nei tessuti adulti. Nonostante l'assunzione e la persistenza delle MNPL nei tessuti delle zanzare, non sono stati osservati effetti significativi sul loro sviluppo. La durata degli stadi larvali e pupali e la probabilità di sopravvivenza fino all'età adulta non si sono mostrate significativamente differenti tra i gruppi trattati con MNPL ed i controlli. Tuttavia, l'analisi metabolomica ha evidenziato alterazioni nei pathways metabolici delle larve esposte alle MNPL. In particolare, 81 metaboliti hanno mostrato variazioni significative, con molti composti centrali del metabolismo del carbonio, come quelli coinvolti nella glicolisi e nella via del pentoso fosfato, sotto-regolati in seguito all'esposizione alle MNPL. Questi risultati suggeriscono che, pur non influenzando direttamente lo sviluppo delle zanzare, le MNPL possono avere un impatto significativo sul loro metabolismo. La comprensione dei meccanismi biochimici e molecolari che regolano le interazioni tra MNPL e zanzare è cruciale per ampliare le conoscenze sulla tossicologia ambientale e per valutare l'eventuale impatto dell’esposizione a inquinamento da plastiche sulla capacità vettoriale.
Somministrazione di micro- e nano- plastiche a larvae di Aedes albopictus (Diptera: Culicidae): un'analisi preliminare basata su microscopia a fluorescenza e metabolomica
SOLDANO, SARA
2023/2024
Abstract
Plastics, synthetic organic polymers, are essential in modern society for their strength, flexibility, and durability, but since the 1960s their production and disposal have contributed significantly to global waste, posing environmental risks. Recently, attention has turned to small plastics, both intentionally produced and as byproducts of larger plastics. These particles, ranging from macro- to nanoplastics, are dispersed by wind, and accumulate in marine and freshwater ecosystems, threatening species, ecosystems, and human health. Various organisms, including mammals, birds, fish, and zooplankton, can ingest micro- and nanoplastics (MNPLs), contributing to their transport and transformation, raising ecological concerns. Mosquitoes also play a role in MNPL dispersal. Their larvae and pupae live in aquatic habitats that may be MNPL-contaminated and can emerge as adults carrying these particles, potentially facilitating bioaccumulation across trophic levels. Mosquitoes can lay eggs in urban water reservoirs and domestic wastewater, areas with high MNPL concentrations. The Asian tiger mosquito, Aedes albopictus, which thrives in these habitats, is a key model for studying the impact of MNPLs on mosquito biology, with implications for public health as it is a primary vector for arboviruses like dengue, Zika, and chikungunya. This thesis aims to investigate the effects of MNPL exposure on Ae. albopictus larvae using a multidisciplinary approach combining insect physiology, fluorescence and confocal microscopy, and metabolomics. The study traces the fate of polystyrene MNPLs after ingestion by mosquito larvae across various organs and developmental stages and explores the potential impact of MNPL exposure on larval metabolism, providing preliminary insights into the relationship between plastic pollutants and mosquito biology. First-instar larvae were exposed to fluorescent polystyrene micro- (MPs, 2 μm) and nanoplastics (NPs, 0.5 μm). Their ability to ingest and internalize these particles was assessed using fluorescence and confocal microscopy to trace the MNPLs in whole larvae, dissected organs, and tissues. This allowed for detailed observation of plastic uptake, transport, and accumulation. The study also examined the transfer of MNPLs from larvae to pupae and adults. Additionally, a preliminary metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS) was conducted to explore potential metabolic changes in larvae exposed to MNPLs. Results confirmed that Ae. albopictus larvae actively ingest both MP and NP beads, which were found throughout the alimentary tract. Microscopy revealed that MNPLs crossed the intestinal barrier, accumulating in the Malpighian tubules, the hemocoel, and the hemolymph. MNPLs were also detected throughout the developmental stages, indicating that the particles were transferred and persisted in adult tissues. Despite the uptake and persistence of MNPLs, no significant impact on mosquito development was observed. The duration of larval and pupal stages, as well as survival rates to adulthood, did not significantly differ between MNPL-treated groups and controls. However, metabolomics analysis revealed that MNPL exposure induced metabolic changes in larvae. 81 metabolites were found to be significantly altered, with many downregulated metabolites involved in central carbon metabolism, including glycolysis and the pentose phosphate pathway. These findings suggest that while MNPLs do not seem to affect mosquito development, they may have important effects on metabolic pathways. This study underscores the potential of plastic pollution to impact mosquito biology. Investigating the metabolic effects of MNPLs paves the way for future research on the molecular mechanisms underlying plastic-mosquito interactions, offering insights into vector biology and environmental toxicology.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28433