Over expression of ecdysone during development could explain why BPA causes mouth deformities and increased pupation time in C. riparius 11. In mosquitoes , the vitellogenin gene is a target for ecdysteroid receptor, which can be modulated by the xenoestrogen BPA. This suggests that BPA and other xenoestrogens could have an effect on the production of vitellogenin, the egg yolk protein. This also would lead to altered viability of offspring, which we did not assess. Acetaminophen and the mixture of PPCPs significantly slowed developmental time of C. quinquefasciatus, but antibiotics alone did not. In contrast, Chouaia et al.described delayed larval development of Anopheles stephensi after treatment with the antibiotic rifampicin at 120 µg/ mL. In our study, there was relatively little difference in development of immature mosquitoes between the antibiotic treatments and the controls for the Acetobacteraceae, to which Asaia belongs. It is possible that the antibiotics chosen for this study are not effective in eliminating Asaia. Alternatively, the antibiotics in our study were used at doses much lower than the rifampicin tested by Chouaia et al., and might have achieved similar effects if applied at higher doses. With a combination of antibiotics, hormones and other constituents that occur in PPCP-contaminated reclaimed water, it is difficult to know exactly which chemical is affecting which bacterial family and which bacteria were responsible for the deleterious effects on developmental time. Notably, the antibiotic treatments had approximately 1/3 of the total number of bacteria relative to the control. Therefore even the approximately 8500 counts of Rickettsiaceaein the antibiotic treatments are lower in number than the Enterobacteriaceaefound in control treatments. Surprisingly,macetas de plastico the substantial loss of bacterial counts in the antibiotic only treatments did not slow development.
Although the antibiotics decreased overall bacterial counts in C. quinquefasciatus, W. pipientis appeared to be relatively unaffected. Wolbachia pipientis is susceptible to doxycycline and rifampin. Of the chemicals we tested, oxytetracycline should have had the most impact on Wolbachia. However, because the bacterial counts were relatively similar for W. pipientis in all treatments, we suspect either the oxytetracycline was too dilute to have an effect, it allowed a non-susceptible strain of W. pipientis to dominate, or it is simply ineffective against W. pipientis. However the vast majority of the eight bacterial families were greatly reduced in the antibiotic treatments. Of these families, Enterobacteriaceae and Microbacteriaceae were the most reduced in both the antibiotic and mixture treatments. The family Enterobacteriaceae is highly associated with insect endosymbionts, such as Buchnera in pea aphids. Buchnera bacteria are known to aid the aphid by supplying essential amino acids lacking from a nutritionally deficient diet. Without these endosymbionts the aphids do not develop properly, but the microorganisms apparently have no other direct biological effect on the aphid. The size of the Enterobacteriaceae populations probably had a minimal effect on larval developmental time, as thecounts in the acetaminophen treatment are relatively similar to the hormone-treated group. Additionally, the hormone treated group has a substantially reduced count of Enterobacteriaceae bacteria compared to the control, but showed no significant changes in developmental time. We suspect, therefore, that the acetaminophen is negatively impacting some other biological system in the larvae, but determination will require additional research. The use of reclaimed water for crop irrigation and the release of water from waste treatment plants and farm waste ponds into surface waters is occurring and likely to escalate as demand for fresh water increases. While, the research reported here suggests that PPCPs contaminating reclaimed water will have potentially useful effects for mosquito control, if the data can be extrapolated to other insect species, PPCPs will also have unintended negative effects on other aquatic insects. Very little is known regarding how these contaminants might bio-magnify or change chemically as they move through the food web. The eventual impact on populations is also unknown. Further, combinations of PPCPs may be more important for some insects than individual components.
Additional research is needed not only on aquatic insects living in surface waters, but also on uptake by plants and associated herbivores in terrestrial environments.Endosymbionts, bacterial species known to grow, develop, and vertically transmit in an organisms’ cells, usually for mutualistic symbiosis, are essential to the growth, development, and fecundity of many insect species. Many aphid species, such as Acyrthosiphon pisum, Megoura viciae, and Myzus persicae, rely heavily on endosymbionts to survive on the unbalanced diet of phloem sap. Their bacteriocyte endosymbiont, Buchnera spp, provides essential amino acids. Without Buchnera, aphids demonstrate reduced growth rates and development and produce few or no offspring. However, some endosymbionts in the genus Wolbachia are known to manipulate insects for their own benefit and can also lead to increased vector competency for transmission of human diseases such as West Nile Virus. Wolbachia species are common vertically transmitted endosymbionts in many mosquito species and typically infect reproductive tissue. There are many species of Wolbachia that influence a variety of insect behaviours and life-history traits. Some endosymbionts can also act defensively by killing parasitoid eggs after they are laid in the host . Because endosymbionts play major roles in insect development, they have been proposed for use in insect control . Due to the importance of endosymbionts for development in many insect species, some species vertically transfer endosymbionts . For example, Estes et al. describes the microbiome of dung beetles’ brood balls, which are used to nourish their offspring until they are adults. When the microbiota in the beetle offspring and their female parent were compared in sterilized dung and soil, they had proportionally identical 16S rRNA sequences from their microbiome. Interestingly, over their life stages, the proportions of the families of bacteria in the beetles’ microbiome changed. The predominant family of the first three instars varied by individual. However, from the pupal stage onwards, Enterobacteriaceae was the most common family in the dung beetle. Bees acquire important bacteria through social interaction and also from the environment . Without some of these bacteria, it is thought that honey bees could become more susceptible to outside diseases and increase incidents of colony collapse .
More studies are needed to fully understand the importance of microbiota in mosquitoes as they have been linked to increased transmission of pathogens from mosquitoes to humans. Mosquitoes are common disease vectors, which spend their juvenile stages in aquatic environments . Bacteria from the water, both symbiotic and free-living, have been shown to influence the microbiome of mosquitoes, suggesting that some of their possible endosymbionts are collected from the environment . Consequently, if the environment is altered, and some of these necessary bacteria are reduced/eliminated, there could be detrimental effects on the development of mosquito larvae. Such a removal effect may occur as the result of antibiotic runoff or other pollution,macetas rectangulares and/or environmental changes. For example, Rosi-Marshall et al. showed that common pharmaceuticals in streams will alter the respiration and diversity of biofilms. Pennington et al.reported differences in whole-body micro-biomes and increased developmental times for Culex quinquefasiatustreated with various pharmaceuticals and personal care products .Chemicals intended for human use often occur in aquatic environments and/or enter water supplies through water treatment plant overflow or from use of reclaimed water in water scarce areas. These chemicals can then affect bacterial communities in the water and the associated aquatic insect community. Presence of these contaminants can alter effectiveness of Bacillus thuringiensis subsp. israelensisa pesticide commonly applied for mosquito control . However, little is known about how such contaminants will influence the microbiome of such insects. Similarly, there is a lack of data determining if mosquito bacterial communities vary during the course of larval development; all available studies we are aware of examine only late instar larvae or mixed lower in stars and species, and information regarding mosquitoes’ endosymbionts and their function is very limited or non-existent outside of Wolbachia. Therefore, we describe the differences in the bacterial communities of the mosquito C. quinquefasciatus in multiple distinct larval stages, as well as when these mosquitoes are reared in environments contaminated with ecologically relevant concentrations of PPCPs that commonly occur in combinations in order to provide a baseline for more in-depth studies. In preparation for sequencing, three mosquitoes from each PPCP treatment were collected as second, third and fourth instars, as first instars were too small to collect without damage, and twice washed with 95% ethanol to remove any external microorganisms. Larvae were then transferred to individual sterile 2 mL microcentrifuge tubes with 95% ethanol and stored at -60 ± 3°C in an ultra cold freezer until DNA extraction. DNA was extracted using a Qiagen DNeasy® Blood and Tissue Kit following the manufacturers protocols amended as in Pennington et al.. In addition to mosquitoes, samples of water and water plus diet were also extracted using identical protocols, with the additional step of centrifugation at 2900 rpm in an IEC HN-SII tabletop centrifuge for 1 h to create a pellet. Upon extraction, nucleic acid concentration was quantified using a Nanodrop ND- 2000c Spectrophotometer , to confirm enough genetic material for sequencing. This process revealed no DNA in water or water and diet samples when no mosquitoes werepresent and thus they were not subjected to further analysis because any bacteria would have originated from the mosquitoes, their egg-rafts, or from the air after the water had been altered by the mosquitoes’ various biological processes. Roche 454 bacteria barcoded amplicon pyrosequencing was performed by a commercial sequencing facility .
The procedure used the forward primer 27Fmod and the reverse primer 519Rmodbio in a single-step 30 cycle PCR using HotStarTaq Plus Master Mix . PCR was performed using the following cycle conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s; 53°C for 40 s, 72°C for 1 min; and a final elongation at 72°C for 5 min. After PCR, all amplicons were mixed in equal concentrations and purified using Agencourt Ampure beads . Samples were then sequenced with Roche 454 FLX titanium instruments and reagents following the manufacturer’s guidelines.Operational taxonomic units were chosen by the default 97% identity threshold, which roughly correlates to species , via the UCLUST method as implemented in the pick_otus.py script . Representative OTUs were chosen using the pick_rep_set.py script and default settings. The Greengenes reference database clustered at 97% identity was used to assign taxonomy using the assign_taxonomy.py script. OTUs were counted and summarized using the make_otu_table.py and summarize_taxa.py scripts respectively.There were 658 distinct OTUs at the species level with 58 distinct families; OTUs failed to match any contained within the database and could not be assigned taxonomically. Fifteen families were chosen by their proportionality being greater than or equal to 1% in at least one sample. The cut-off was chosen at 1% as this was assumed to be the minimum to influence larval development. For alpha diversity, multiple rarefactions were performed using the multiple_rarefaction.py script with the lowest rarefaction depth of 2000, the highest rarefaction depth of 21000, a step size of 1000, and a replicate number of ten, which normalizes the data at each depth. Alpha diversity was calculated using the alpha_diversity.py script with the metrics observed species and Shannon Indices. Alpha diversity data was not averaged between replicate mosquitoes as they have been averaged by resampling-replicates and the complications and validity of this is still being considered. Metrics were summarized using the collate_alpha.py script. Statistical analyses were performed using R . Following processing through the QIIME pipeline, “Permutational MANOVA” in the Vegan package was used to compare the OTU data. Independent variables were instar , PPCP treatment and the interaction of the two, with three replicates of each instar in each PPCP treatment and control . PERMANOVA is analogous to MANOVA but is suited to address the non-normality that is commonly associated with count data in ecological community and genetic data . Microbial community data were further examined via principal component analysis performed in the FactoMineR package . PCA and PERMANOVA were conducted on each instar in the individual PPCP and control treatment groups. Following PCA, variables were examined for their contributions and correlation to each of the first two dimensions. Those variables that were ≥ 85% correlated were included in subsequent pairwise comparisons by instar in their respective treatment.