Generalized linear hypotheses was used to perform pairwise comparisons in the multcomp . P values were adjusted using the p.adjust command. Alpha diversity data was analysed using a negative binomial generalized linear models at a sequence depth of 3000 sequences/sample to normalize data to the highest number where all sample mosquitoes were present. The alpha level for all tests was 0.05.PCA demonstrates that various instars separate from each other in the four treatment groups with third and fourth instar loading similarly on the first dimension and all three instars loading distinctly in the second dimension. Bacterial families Oxalobacteraceae and Aeromonadaceae closely follow the separation pattern in the first dimension and Cryomorphaceae follows in the second . In the antibiotic treatments second instar loaded separately from third and fourth on the second dimension . The bacterial family Propionibacteriaceae also follows this trend and was the only family with at least 85 % correlation in either of the first two dimensions . For hormone treated mosquitoes, second instars loaded separately from fourth instars on the first dimension . The bacterial families, which follow this trend, are Enterobacteriaceae and Pseudomonadaceae. As shown in Figure 3.2 and Table 3.2, control treatments’ bacterial families changed with instar; starting with Cytophagaceaein second instars, changing to Enterobacteriaceaein third instar and finally the control fourth instars’ most predominant family was Microbacteriaceae . There were a total of eight bacterial families with proportionalities greater then 1% in at least one instar of the control treatment. In fourth instars there is a resurgence of bacterial families from second instars,macetas cuadradas which were overshadowed by the third instar bacterial families . Interestingly, the family Rickettsiaceae was the second most predominant family in all control instars.
Operational taxonomic units assigned to the family Rickettsiaceae were found in most treatments and instars, although they were reduced in many hormone treated samples relative to the controls and increased in the antibiotic treatments. Notably, antibiotic and mixture treatment groups’ most predominant family was Rickettsiaceae over all instars. The second most predominant family in antibiotic and mixture treatment groups was Sphingobacteriaceae in all instars. Hormone treatments changed bacterial communities between instars but not as drastically as the control group. Second instars exposed to hormones predominately contained Oxalobacteraceae, which changed to Microbacteriaceae and Rickettsiaceae in the third instar. The predominant family of fourth instar hormones was Microbacteriaceae, although some proportion of Rickettsiaceae was still present.In the alpha diversity analysis, richness was examined as mean observed species and evenness was measured by mean Shannon’s index . For mean observed species at a sequencing depth of 3000 sequences/sample there was a significant difference between treatments , instars and a significant interaction of treatment and instar . Mosquitoes treated with antibiotics had lower richness and fewer total sequences per sample than all other treatments with the richness decreasing as larvae age. This is evident in Fig. 3.2, as there are proportionally fewer bacterial families outside of Rickettsiaceae and Sphingobacteriaceae than in other treatments. In contrast, mosquitoes reared in the mixture of hormones, antibiotics and the common contaminants, acetaminophen and caffeine; demonstrate a relatively constant richness over time .
The control groups and hormone treatments fluctuate more than the mixture and antibiotic treatment groups but demonstrate consistently higher richness . The mean Shannon’s diversity index suggests that antibiotics alone substantially reduced diversity. The mixture treatments also display reduced diversity, however, they are more diverse than their antibiotic treatment counterparts. The control groups display a greater diversity than both the antibiotic alone and mixture treatments when compared by increasing instar, where as the hormone treatment group, had no discernable pattern. The mixture also displays no discernable pattern compared by instar, which is likely due to the effects of the hormones added to antibiotics. Finally, it is notable that in some treatments, the mean species number failed to reach an asymptote. Here we have demonstrated that the microbiome of larval Culex mosquitoes changes throughout development, and variation between instars is affected when exposed to various PPCPs. It has previously been demonstrated that mosquitoes rely on their microbiomes to aid in development and that removing certain symbionts can significantly slow larval development. Pennington et al. demonstrated that PPCPs at environmentally relevant concentrations, which are significantly lower than those used in most laboratory studies, can alter the microbiome of mosquitoes and slow their development. In the field, Duguma et al. showed pooled Culex species’ microbiomes will change from early to late instars. Coon et al. and Wang et al. showed that the microbiome of mosquitoes will change as the insects advance from fourth instar larvae, to pupae, to the adult stage, and after adults fed on a blood meal. We have shown that the microbiome of early instars’ will change from one instar to the next even without exposure to PPCPs .
In the second, third, and fourth instars, predominant families change from Cytophagaceae to Enterobacteriaceae and finally to Microbacteriaceae. However similar to Pennington et. al , third and fourth instar were not significantly different and our findings also correlate to what Duguma et al. found in their laboratory reared Culex tarsalis late instars. However, as their third and fourth instars were pooled, only the Enterobacteriaceae family predominates. These families were all removed in the antibiotic and mixture treatments. Fourth instar larvae in the control group match what was described in Aedes aegypti by Coon et al. . Coon et al. also described the microbiome of two other mosquito species during the fourth instar. Their microbiomes had different proportions of familial microbiota between each other and both were different from the findings in our C. quinquefasciatus fourth instars. To our knowledge ours is the first study to look at the microbiome changes of individual early instars in mosquitoes. This suggests the possibility of a new strategy for mosquito control targeting the critical microorganisms essential for development at specific stages. Specifically, additional research targeting key symbionts found in earlier instars would determine if the younger larvae can be controlled more effectively, as has been seen with pesticides such as Bti . A number of mosquitoes are common carriers of the bacterial genus Wolbachia, which usually acts as a reproductive parasite in the ovaries of the females, and is suspected to be in at least 20% of all insect species . As in Pennington et al. , Rickettsiaceae, the family containing Wolbachia pipientis, continuously holds the majority count of the antibiotic and mixture treatments’ microbiome. When the OTUs mapped to the family Rickettsiaceae was examined at the level of genus the predominant and sole genus detected was Wolbachia. Rickettsiaceae is vertically transmitted from mother to offspring ; however, for many of the other bacterial families present, it is difficult to discern the source or how they are incorporated into the insects’ microbiome. This is made further complicated since these traits may vary by species or genus and mapping OTUs to finer taxonomic levels was generally not possible. Similarly, it may be possible to determine some origins via comparison with the water in rearing pans over time, although there was no DNA found in water at the start of the experiments. However, our focus was not on the origin of bacterial species in these mosquitoes,maceta cuadrada plastico and we do not have these data. Analyses or the microbial community in such pans would be an interesting follow-up study. Interestingly, Enterobacteriaceae, which includes the genus Buchnera and other common endosymbionts, is the predominant family of the third instars in the control treatment. For example, the gut symbiont of the plataspid stinkbug is phylogenetically similar to Buchnera species . In potato psyllids various genera of the family Enterobacteriaceae have been reported in the life stages and faeces accounting for at least 21% of the microbiome. Enterobacteriaceae is one of, if not the most important family of endosymbionts in the pea aphid , and is commonly used in research regarding the effects of antibiotics on insect-symbiont interactions.
Similarly, Chouaia et. al described a slowing of larval development in Anopheles mosquitoes when they removed Asaia bacteria from the family Acetobacteraceae. However this family has one of the lowest proportionalities in all of the mosquitoes including control treated. This suggests it is not an endosymbiont of this Culex mosquito specie. However, reports of the effects of other PPCPs on insect-symbiont interactions are rare. It is interesting to note that the hormones found in wastewater from treatment plants are all mammalian female sex hormones and would not be expected to affect bacteria. We would not expect an effect of these hormones on bacteria, as there is no endocrine system; nonetheless, substantial changes in the microbiome occurred in response to exposure to these hormones . Similarly, caffeine and an antihistamine, would not be expected to effect biofilms, but were shown to repress respiration in stream biofilms 51. We think there may be some influence the hormones have on bacterial gene expression however that is not in the scope of this paper and thus, specific effects for each PPCP or combination of contaminants will need to be determined from more experimental data. The increased bacterial diversity during mosquito ontogeny could result from either bacterial replication during development or by acquisition through ingestion. In the hormone and control treatment groups, we are unsure if bacteria are lost during development, or if the change in bacterial diversity was caused by differential growth among taxa. Interestingly, in pairwise comparisons of the hormone treated mosquitoes , the majority of the significant differences were between second and fourth instar larvae. The hormone treated mosquitoes also had the most families that were correlated to the first principal component at a minimum of 85%. Combined this suggests that the mosquitoes exposed to hormones had the most diverse microbial communities and that this diversity increases over time. Mammalian hormones change the microbiome of C. quinquefasciatus mosquitoes and it is possible they are responsible for the increased richness and diversity seen in the mixture treatments compared to antibiotics alone; however more studies will need to be conducted to confirm this conclusion. Regardless, our results indicate that reclaimed wastewater has the potential to impact mosquito ecology. Considerably more research will be required to discern how mixtures of PPCPs could affect bacterial microbiomes for important medical pests. If similar results are found for agriculturally important insects exposed to these emerging contaminants, additional research documenting the effects of increasing use of reclaimed water and associated changes to the insect microbiome will become even more important. Similarly, because insects are a critical food source for higher trophic level organisms in terrestrial surface waters, releases of PPCPs in aquatic environments have the potential to modify the ecology of these ecosystems.Pharmaceuticals have been increasingly prescribed for the past 30 years, and prescription rates have almost tripled in the past 14 years. In food-producing animals alone, there were 9.1 million kg of medically important antibiotics used in 2013. Of those 9.1 million kg used, 73.6% was used for the purpose of increasing production of the animals, and this use continues to increase. Many antibiotics and other common Contaminants of Emerging Concern , are excreted by both humans and animals with little change in their chemical structure . It is no surprise pharmaceuticals have been appearing in wastewater, and in some cases tap water, over the past few years .Many pharmaceuticals are released during heavy storms in the untreated wastewater, due to overflow, which then flows directly to the environment 46. These pharmaceuticals are now found at biologically active concentrations in surface waters around the world . In addition to runoff, there is an increasing effort to use reclaimed wastewater in drought affected areas, such as Southern California. In agriculture/livestock operations, pharmaceuticals are also found in manure that is then used as fertilizer, effectively compounding the pharmaceutical concentrations. Current research shows these chemicals tend to be both long lived in soil and detrimental to soil microbes . Recent studies on the effects of pharmaceuticals on aquatic insects show that at environmentally relevant concentrations they can alter development of the mosquito Culex quinquefaciatus, its susceptibility to a common larvicide, and its larval microbial communities. Watts et al. 11 showed alterations and deformities in the midge Chironomus riparius after treatment with a common birth control agent, 17α- ethinylestradiol, and a common plasticizer, Bisphenol-A.