From an origin in tropical forests where it blood fed on animals, Aedes aegypti adopted a preference for developing near human habitations and blood feeding on humans, spreading widely to become the principal vector of important arboviral diseases including dengue, chikungunya, yellow fever, and Zika (Ramasamy, et al., 2021). It was regarded as an obligate fresh water (FW) mosquito that lays eggs and undergoes larval and pupal development in natural (e.g. rainwater pools, leaf axils) and anthropogenic (e.g. water storage tanks, discarded containers) FW collections near human habitations. Larval source reduction efforts, critically important for controlling arboviral diseases, presently only target such FW habitats of Ae. aegypti and the secondary arboviral vector Aedes albopictus. These two Aedes vectors were recently shown to develop in coastal anthropogenic brackish water (BW) habitats with a salt concentration up to 50% sea water (e.g. beach litter, coastal wells) (Ramasamy, et al, 2011; Jude, et al, 2012; Surendran, et al 2012). BW Aedes showed inheritably greater salinity tolerance, changes in the structure of the osmoregulatory anal papillae, differences adult and larval cuticles, and susceptibility to dengue virus infection (Ramasamy, et al 2014; Surendran et al, 2018a&b; Ramasamy, et al, 2021) Development of Ae. aegypti and Ae. albopictus in BW increases the potential for arboviral disease transmission which can be exacerbated by rising sea levels due to global warming causing greater salinization of inland waters (Ramasamy and Surendran, 2011, 2012 & 2016). Genetic changes and physiological mechanisms that permit FW Aedes and also FW anopheline malaria vectors to develop in BW field habitats were previously unknown and were therefore investigated in BW-adapted Ae. aegypti from the coastal Jaffna peninsula in North Sri Lanka (Ramasamy, et al, 2021).
BW- and FW-Ae. aegypti were compared by (i) RNA-seq analysis on the gut, anal papillae and rest of the carcass in fourth instar larvae (L4); (ii) protein composition of the cuticles shed when L4 metamorphose into pupae; (iii) transmission electron microscopy of cuticles in L4 and adult females (Ramasamy, et al, 2021).
Genes for specific cuticle proteins, signalling proteins, moulting hormone-related proteins, membrane transporters, enzymes involved in cuticle metabolism, and cytochrome P450 were expressed with different mRNA levels in BW and FW L4 tissues. Salinity-tolerant Ae. aegypti were also characterised by altered L4 cuticle protein composition and changes in cuticle ultrastructure of L4 and adults (Ramasamy, et al, 2021).
RNA-seq analysis resulted in 30,485 transcripts being mapped in the gut, anal papilla and carcass of Ae. aegypti L4. Transcript levels from a gene varied between the three structures and sometimes between BW and FW L4. The ratio of transcript expression in BW to FW L4 termed fold change (FC) were calculated for every transcript. All transcripts with highly increased (FC>100) or decreased (FC≤0.01) levels in L4 of BW Ae. aegypti, and the detection of corresponding proteins in shed L4 cuticles were particularly scrutinised.
Transcripts, including multiple transcripts from the same gene, for several cuticle proteins (CPs) were increased in BW with FC>100 in all three structures. Aedes aegypti CPs were classified into families by homology with CP families in Anopheles gambiae. Transcripts for CPs formed a significant proportion of all transcripts with FC>100 in carcass (49%), anal papilla (31%) and gut (44%). Transcripts for the RR2 family of CPs formed a large majority of the CP transcripts with FC>100 in carcass (74%) and anal papilla (79%). Analysis of CP composition in shed L4 cuticles showed an increase in specific CPs in BW, including members of the RR1 and RR2 families. RNA-seq also revealed marked changes in BW Ae. aegypti in long non-coding RNAs (lncRNAs) levels that may regulate gene expression at the chromosome, transcription and post-transcription levels, as well as mRNA levels for several other proteins associated with cuticles (termed OPACs). Prominent expression changes in mRNAs for membrane receptors, transcription regulatory proteins, signalling pathway proteins, moulting-related hormones and associated proteins, cytochrome P450, membrane transporters, enzymes involved in chitin metabolism, and enzymes concerned with the synthesis of components of the waxy larval epicuticle, also occurred in BW Ae. aegypti. Changes in Ae. aegypti were consistent with those seen in An. gambiae larvae subject to short-term salinity exposure in the laboratory.
Electron microscopy of adult Ae. aegypti abdomen suggested that (i) the whole cuticle was thicker in BW than FW, and (ii) the endocuticle and the exocuticle were also thicker in BW adults. The cuticle also appeared thicker in BW Ae. aegypti L4 abdomens but thinner in BW L4 anal papillae. Additionally, parallel sheets termed lamellae and helicoidally twisted sheets termed Bouligands that are formed from chitin microfibrils and chitin-binding cuticle proteins tended to be more prominent in BW L4 than FW L4 cuticles (Ramasamy, et al, 2021).
The changes in mRNA levels for CPs and OPACs as well as enzymes concerned with chitin metabolism and cuticle synthesis strongly suggested that BW adaptation in Ae. aegypti L4 is accompanied by changes in cuticle structure. This is consistent with the changes in L4 cuticle structure observed by electron microscopy and protein composition analysis. The epicuticle and its waxy envelope, containing respectively tanned cuticulins and both straight chain and methyl-branched long chain hydrocarbons, make a large contribution to water impermeability in arthropod cuticles. Increased synthesis of long chain hydrocarbons in BW L4 is supported by the large increases observed in transcripts for fatty acid synthase, very long chain fatty acid elongase, fatty acid acyl CoA reductase and a specific cytochrome. Together with the marked increase in cuticulin (an OPAC) transcripts, these transcriptomic findings suggest that augmentation of the water proofing epicuticle and its waxy envelope in the body wall, and possibly also the tracheal system, is important for salinity tolerance in Ae. aegypti larvae.
Cuticle protein changes have been suggested to contribute to thicker cuticles in adult pyrethroid-resistant An. gambiae. The thickening and other cuticle changes in BW L4 and adult female Ae. aegypti, can potentially result in greater resistance to larval and adult insecticides. A cuticle that reduces water and ion permeability in salinity-tolerant larvae may also reduce absorption of the organophosphate Temephos, the most widely-used larvicide for larval source reduction of FW Ae. aegypti worldwide.
Similar BW-adaptive changes to those in Ae. aegypti occurring in FW anophelines accompanied by reproductive isolation in coastal areas may have been the origin of salinity-tolerant species like Anopheles merus in Africa, Anopheles sundaicus in Asia and Anopheles aquasalis in America. However, salinity tolerance in Ae. aegypti which involves heritable changes had not yet prevented interbreeding and gene flow with FW Ae. aegypti in the rapidly salinizing Jaffna peninsula in northern Sri Lanka (Ramasamy, et al, 2014). The spread of the salinity-tolerant trait in the peninsula is shown by Ae. aegypti collected in FW ovitraps in the peninsula demonstrating a higher LC50 for salinity than those collected from mainland Sri Lanka. Salinity-tolerant Ae. aegypti in the Jaffna peninsula have the potential expand their range to coastal areas of mainland Sri Lanka and India (Ramasamy and Surendran, 2016).
These findings show the need for additional investigations on cuticle structure and function in relation to insecticide resistance and the genomic biology of salinity tolerance in Ae. aegypti. The observations in the principal global arboviral vector Ae. aegypti have fundamental biological and multiple epidemiological implications in the context of rising sea levels caused by climate change expanding coastal brackish water habitats (Ramasamy, et al, 2021) as well as for other BW-adapting FW mosquito vectors and the diseases they transmit (Jude, et al, 2010).
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