Ae. aegypti and Ae. albopictus share a common trend in CTP Synthase fluctuation patterns, but do not conform to expected trends in developmental growth
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Should confer two points: (a) that aalCTPsyn fc fluctuations is greater than that of aaeCTPsyn, and (b) that despite such contrasting ranges in fc values, the two species display similar CTPsyn fluctuation trends, with many overlapping high and low peaks of expression throughout development. However, quantitative PCR (RT-qPCR) results for the most part clashed with our hypotheses of when aeCTPsyn should be expressed most greatly. These preconceived notions were derived based off of established CTPsyn expression level changes seen in Drosophila melanogaster at different points of their development (Azzam & Liu, 2013). Accordingly, we hypothesized high-expression points to be (a) the first twelve-hours in embryos, (b) the period spanning larval and pupal phases, (c) in blood-fed females, and (d) in fast-dividing cell culture. Expected low-points are (a) middling embryonic periods, (b) virgin adult females, and (c) adult males. The opposite is observed to be true for most of these hypotheses.
A developmental transcriptome for Ae. aegypti has not only been detailed exhaustively (Akbari et al., 2013), but the esteemed authors have made their findings available as histograms on an accompanying webpage (Hay Group, 2013). Our discussion herewith will thus heavily involve CTPsyn expression trends presented by the platform, with data specifically for Ae. aegypti extrapolated for Ae albopictus on the principle of their common genus relationship. Given the points discussed in the previous section, we do realize that this approach may therefore be flawed as well, and Ae. aegypti transcriptomic data for its CTPsyn may not necessarily reflect aalCTPsyn expressional patterns. Nevertheless, until the Ae. albopictus developmental transcriptome becomes as thoroughly described as it has been for Ae. aegypti, we are compelled to employ the sources we have in-hand.
Lower-than-average levels of CTPsyn are recorded from zero to ninth hour embryos. We suspect the flaw to our hypothesis to be in the discounting of the time-lapse required for a newly-laid embryo to begin actively expressing genes on its own. Inherited maternal factors e.g. CTPsyn proteins supporting the growth of the young embryo would not have been detected in qPCR. A steady upward trend in expression is nonetheless maintained as the embryos progress into its twelfth hour. However, our observations here are absolutely un-complementary to Akbari’s, whereby a spike in CTPsyn was seen between zero to four hours of embryo deposition.
We also projected larvae and pupae tissue to both show high CTPsyn expression levels. This stretch of development witnesses the fastest growth in terms of body size (Association, 2017). Upon pupation, genes required to facilitate the pupae to adult transition will also be abundantly transcribed (Heyland & Moroz, 2006). High levels of cellular division and genetic expression translates into high nucleotide demand, and inevitably great rapidity in CTPsyn protein production. Akbari’s data accordingly showed that first instar larvae in particular would carry higher levels of CTPsyn than pre-hatching embryos. Our data instead showed an abrupt drop in the transition from 48 to 72 hour embryos to first instar larvae. We cannot provide a scientifically solid explanation as to why. Even so, both ours and Akbari’s data do support that CTPsyn levels are higher than average in 72-hour embryos. This makes sense: prior to entering diapause, it is possible that the embryos more actively express CTPsyn in preparation for a prolonged period of dormancy (Diniz et al., 2017). We thus assume that the incongruence observed later can largely be attributed to sample differences. First instar larvae were collected within three hours of hatching in our study. Similar to zero-hour embryos, it is highly likely that a period of recovery for active genetic expression is required for freshly-hatched larvae as well, and the low CTPsyn levels could be due to the fact that our sample collection practices did not accommodate this need. Regardless, our hypothesis for succeeding larval stages do coincide with Akbari’s data. Though low-than-average levels are still seen in pupal-staged Ae. aegypti, both species showed significant upswings in CTPsyn transcripts from fourth instar larvae, and thus our hypothesis for pupal samples is upheld.
In adult tissues, we had predicted high CTPsyn expression for blood-fed females. Low levels were expected in females and males raised on a sucrose-exclusive diet. Our rationale for these hypotheses was simple. Other than for proteins required in processes necessary for continued survival, very little genomic transcription is expected in non-blood fed females and males. It does not make sense to retain high expression levels of CTPsyn when its protein will be of little use. Once a female has acquired blood however, oogenesis reactivation and the ensuing processes should kick-start a period of rapid gene transcription, and therefore a greater need for CTPsyn activity.
These expectations were based off of our own immunostaining results, where we have demonstrated the great possibility that CTPsyn levels and distribution are indeed affected positively by a blood-meal. Analysis of RT-qPCR data along with Akbari’s data conversely proved these hypotheses to be completely misplaced. We are again uncertain as to why such opposing results are observed instead. Our speculation is that CTPsyn function, and therefore the continued expression of its gene(s), is necessary for energy metabolism in males and non-blood-fed females. In a caged population, it is easy to distinguish these individuals from blood-fed females. They display more voracity, always active, on the move, and spend much time mid-flight. Egg-producing females are very dormant by contrast. Though aeCTPsyn expression levels do appear to support this theory, it is of course an unproven assumption. An energy measuring assay in conjunction with CTP quantification could provide further certainty regarding its validity.
We have suitably hypothesized the upregulation of the gene for both Aag2 and C6/36 cells. The point of discussion here is therefore in fc variances between the two culture populations. Observed discrepancies could be due to a number of reasons. For one, these cell lines are derived from different tissue types. Aag2 is embryonic in origin, whereas C6/36 is larval (Peleg, 1968; Singh, 1967). Yet, based on our RT-qPCR data, this should not result in such a great divide in fc values between the two. An alternative theory is populational differences in growth vigour. Our own Aag2 culture is relatively weaker and propagates at a slightly slower rate than C6/36 cells; CTPsyn expression levels may have thus been compromised, as the health of Aag2 is also compromised. Nevertheless, this does not change the fact that greater frequencies of cytoophidia is seen in C6/36 cells, even when Aag2 is treated with the same DON concentrations. Increments in cytoophidia numbers accompanying increments in applied DON in C6/36 is also exponential, rather than proportional as seen in Aag2. These observations thus justifies our belief that CTPsyn is indeed more highly expressed in Ae. albopictus cells.
Overall, RT-qPCR readings showed that a great deal of expressional fluctuation occurs throughout the growth progression of an individual mosquito. This is a trait displayed by both species. More interesting to note is that they display almost perfectly congruent trend-lines in fluctuation, with shared low and high peaks across the board. A transgression between the two is the fold change or fc disparity. From our data, we cannot have this attributed to differences in mRNA levels from species to species; geomean-ed Cq was similar at 22.94 and 24.55 for Ae. aegypti and Ae. albopictus, respectively. This means that the observed fold changes are due to species-unique, self-contained transitional differences in mRNA levels amongst the developmental stages. In one of the previous sections, we did elaborate on how more CTPsyn variants are reported for Ae. albopictus than its cousin species. This is perhaps why the fc dissimilarities are so stark; the expression levels of these ‘additional’ isoforms are probably in itself modulated between the developmental time points in Ae. albopictus. As our qPCR primers were indiscriminate, these minute differences are detected, and they eventually influence the fc values applied to generation of the presented figures. Variant-specific transcript quantification followed closely by protein isolation and characterization on a variant-to-variant basis could be routes taken in the future to better understand the expressional behaviours of aeCTPsyn, both as a gene and as a protein.
With this chapter we had had all the intention to provide the complete picture of localization and distribution patterns of aeCTPsyn, in as many tissue types as was manageable. This was to include embryo and larvae samples as well; unfortunately, our attempts did not result in satisfactory immunostaining outcomes. We suspect that this is primarily due to the usage of an unsuitable protocol. On the basis of tissue-composition similarity, we had adapted a detailed methodology proven to stain zebrafish embryos effectively for both mosquito embryo and larvae mosquito samples (Goody & Henry, 2013 ; Martins et al., 2011).
However, there could be points in the procedure which may not be as suited to neither Ae. aegypti nor Ae. albopictus tissue. The first suspect is during fixation. The original zebrafish protocol called for fixing in 4% PFA at 4°C overnight. During this process, proteins are crosslinked and stabilized to prevent degradation, and to keep them in situ. Both time and temperature are influential factors in determining the efficacy of fixation (Eltoum, Fredenburgh, & Grizzle, 2001; Montero, 2003). We believe that the duration of fixing recommended for zebrafish embryos was far too long for Aedes embryos and larvae. Tissues show signs of over-fixation, such as total opacity. This is caused by excessive crosslinking from overexposure to PFA, which subsequently also masks antigen epitopes from primary antibodies (Webster et al., 2009). Many other alternative methods, each suggesting different time lengths and temperatures, were attempted to no avail, eventhough one in particular was actually tailored for Ae. aegypti (Clemons et al., 2010). Quick rectification is imperative; without the establishment of a suitable methodology, the knowledge void in CTPsyn behavioural patterns during such an informatively crucial time point as these developmental stages are will not be fulfilled.
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