The impact of transgenesis on mosquito fitness: A review.

Transgenic mosquitoes developed by genetic manipulation, offer a promising strategy for the sustainable and effective control of mosquito-borne diseases. This strategy relies on the mass release of transgenic mosquitoes into the wild, where their transgene is expected to persist in the natural environment, either permanently or transiently, within the mosquito population. In such circumstances, the fitness of transgenic mosquitoes is an important factor in determining their survival in the wild. The impact of transgene expression, insertional mutagenesis, inbreeding depression related to laboratory adaptation, and the hitchhiking effect involved in developing homozygous mosquito lines can all have an effect on the fitness of transgenic mosquitoes. Therefore, real-time estimation of transgene-associated fitness cost is imperative for modeling and planning transgenic mosquito release programs. This can be achieved by directly comparing fitness parameters in individuals homozygous or hemizygous for the transgene and their wild-type counterparts, or by cage invasion experiments to monitor the frequency of the transgenic allele over multiple generations. Recent advancements such as site-specific integration systems and gene drives, provide platforms to address fitness issues in transgenic mosquitoes. More research on the fitness of transgenic individuals is required to develop transgenic mosquitoes with a low fitness cost.

Assessment of Developmental and Reproductive Fitness of Dengue-Resistant Transgenic Aedes aegypti and Improvement of Fitness Using Antibiotics.

BACKGROUND: Genetic modification offers opportunities to introduce artificially created molecular defence mechanisms to vector mosquitoes to counter diseases causing pathogens such as the dengue virus, malaria parasite, and Zika virus. RNA interference is such a molecular defence mechanism that could be used for this purpose to block the transmission of pathogens among human and animal populations. In our previous study, we engineered a dengue-resistant transgenic Ae. aegypti using RNAi to turn off the expression of dengue virus serotype genomes to reduce virus transmission, requiring assessment of the fitness of this mosquito with respect to its wild counterpart in the laboratory and semifield conditions. METHOD: Developmental and reproductive fitness parameters of TM and WM have assessed under the Arthropod Containment Level 2 conditions, and the antibiotic treatment assays were conducted using co-trimoxazole, amoxicillin, and doxycycline to assess the developmental and reproductive fitness parameters. RESULTS: A significant reduction of developmental and reproductive fitness parameters was observed in transgenic mosquito compared to wild mosquitoes. However, it was seen in laboratory-scale studies that the fitness of this mosquito has improved significantly in the presence of antibiotics such as co-trimoxazole, amoxicillin, and doxycycline in their feed. CONCLUSION: Our data indicate that the transgenic mosquito produced had a reduction of the fitness parameters and it may lead to a subsequent reduction of transgenic vector density over the generations in field applications. However, antibiotics of co-trimoxazole, amoxicillin, and doxycycline have shown the improvement of fitness parameters indicating the usefulness in field release of transgenic mosquitoes.

RNAi-mediated silencing of ARV1 in Setaria digitata impairs in-vitro microfilariae release, embryogenesis and adult parasite viability.

Setaria digitata is a nematode that resides in the peritoneal cavity of ruminants causing cerebrospinal nematodiasis disease affecting livestock and inflicting significant economic forfeitures in Asia. Further, this nematode can infect humans, causing abscesses, allergic reactions, enlarged lymph nodes, eye lesions and inflammation of the lungs. The 'ARE2 required for viability1' (ARV1) encodes for putative lipid transporter localized in the endoplasmic reticulum (ER) and Golgi complex membrane in humans and yeast. In the present study, the functional role of S. digitata ARV1 (SD-ARV1) was investigated using RNA interference (RNAi) reverse genetic tool. The targeted silencing SD-ARV1 transcripts by siRNA mediated RNAi resulted in a dramatic reduction of SD-ARV1 gene and protein expressions in S. digitata, which in turn modulated the parasitic motility, its production of eggs and microfilaria viability. Further, the same silencing caused severe phenotypic deformities such as distortion of eggs and embryonic development arrest in the intrauterine stages of adult female S. digitata. These results suggest that SD-ARV1 plays a pivotal role in worm embryogenesis, adult parasite motility and microfilariae viability. Finally, the ubiquitous presence of ARV1 in human filarial nematodes, its crucial functional roles in nematode biology and its remarkable diversity in primary protein structure compared to homologues in their hosts warrants further investigations to ascertain its candidacy in anthelmintic drug development.

Functional analysis of a novel parasitic nematode-specific protein of Setaria digitata larvae in Culex quinquefasciatus by siRNA mediated RNA interference.

BACKGROUND: Functional analysis of animal parasitic nematode genes is often quite challenging due to the unavailability of standardised in vitro culture conditions and lack of adequate tools to manipulate these genes. Therefore, this study was undertaken to investigate the suitability of Culex quinquefasciatus, as an in vivo culture platform for Setaria digitata larvae and RNA interference (RNAi), as a post-transcriptional gene silencing tool to study the roles of a vital gene that encodes a novel parasitic nematode-specific protein (SDNP). RESULTS: The red colour fluorescence detected following RNAi injection to the thorax of C. quinquefasciatus indicated the uptake of dsRNA by S. digitata larvae. The reduction of SDNP transcripts in siRNA treated larvae compared to non-treated larvae, as determined by qPCR, indicated that the siRNA pathway is operational in S. digitata larvae. The observation of motility reductions and deformities during the development indicated the association of SDNP in larvae locomotion and development processes, respectively. The irregularities in the migration of larvae in mosquitoes and elevated survival rates of mosquitoes compared to their untreated counterparts indicated reduced parasitism of S. digitata larvae in mosquitoes upon targeted downregulation of SDNP by siRNA treatment. CONCLUSION: SDNP plays vital roles in muscle contraction, locomotion, development processes, larval development and parasitism of S. digitata. Its ubiquitous presence in parasitic nematodes and its absence in their hosts provide a tantalising prospect of the possibility of targeting SDNP for future development of anthelmintic drugs. The susceptibility of the larval stages of S. digitata for RNAi in Culex quinquefasciatus was also demonstrated for the first time in this study.

ITS-2 secondary structures and phylogeny of Anopheles culicifacies species.

BACKGROUND: Second internal transcribed spacer (ITS2) has proven to contain useful biological information at higher taxonomic levels. OBJECTIVES: This study was carried out to unravel the biological information in the ITS2 region of An. culicifacies and the internal relationships between the five species of Anopheles culicifacies. METHODOLOGY: In achieving these objectives, twenty two ITS2 sequences (approximately 370bp) of An. culicifacies species were retrieved from GenBank and secondary structures were generated. For the refinement of the primary structures, i.e. nucleotide sequence of ITS2 sequences, generated secondary structures were used. The improved ITS2 primary structures sequences were then aligned and used for the construction of phylogenetic trees. RESULTS AND DISCUSSIONS: ITS2 secondary structures of culicifacies closely resembled near universal eukaryotes secondary structure and had three helices, and the structures of helix II and distal region of helix III of ITS2 of An. culicifacies were strikingly similar to those regions of other organisms strengthening possible involvement of these regions in rRNA biogenesis. Phylogenetic analysis of improved ITS2 sequences revealed two main clades one representing sibling B, C and E and A and D in the other. CONCLUSIONS: Near sequence identity of ITS2 regions of the members in a particular clade indicate that this region is undergoing parallel evolution to perform clade specific RNA biogenesis. The divergence of certain isolates of An. culicifacies from main clades in phylogenetic analyses suggests the possible existence of camouflaged sub-species within the complex of culicifacies. Using the fixed nucleotide differences, we estimate that these two clades have diverged nearly 3.3 million years ago, while the sibling species in clade 2 are under less evolutionary pressure, which may have evolved much later than the members in clade 1.