Xanthomonadaceae, like Enterobacteriaceae, is more prominent in larvae than in other life-stages except in acetaminophen, antibiotic, and mixture containing diets, where Enterobacteriaceae dominate. Most species in Xanthomonadaceae are plant pathogens , and have been known to make use of chitin as a carbon source and utilize insects as vectors. It is possible that some of the bacteria in this family may act as symbionts with insects as they have been found in a variety of insect orders . In the NMDS plot there is distinct clustering in the microbiota by treatment. In individuals exposed to antibiotics in their diets, there is a lack of dissimilarity among their microbial communities. Insects reared on diets containing hormones had less microbial diversity in pupae. Unfortunately, we do not know if this is due to similarity in the microbial communities of third instar individuals as the statistical process of rarefication removed that particular life-stage in larvae exposed to hormones, controls, and caffeine treatments. However, the insects feeding on the mixture treatments show a distinct clustering of microbial groups in the pupal and adult stages, whereas the larval stage contains individuals with more variable microbiota. This is likely due to the early instars being exposed directly to the microbe-laden diet,macetas cuadradas de plastico while the later life-stages are only exposed to a subset of bacteria left after the gut contents were expelled at time of pupariation. The greatest dissimilarity was found in the caffeine-treated adults . The adult stage, regardless of treatment, also seems to be where the majority of variation in microbiota occurs .
Megaselia scalaris has been suggested as a model organism for bio-assays for drugs and pollutants , and our findings support this claim. However, our results also suggest that the presence of even very low concentrations of some pharmaceuticals could affect the forensic estimation of time of death based on emergence patterns of adult M. scalaris. We also caution that the pharmaceuticals used in this trial were at low concentrations found in wastewater and could be much higher in cadavers, as pharmaceuticals in humans tend to be higher than what is found in the environment. Also, due to increases in concentrations caused by water loss , pharmaceuticals could have higher toxicity in decaying matter. Perhaps most importantly, pharmaceuticals in reclaimed water are having unintended effects on the microbial community of these flies, which could lead to decreased viability of these ecologically useful detritivores. Many antibiotics and other common Contaminants of Emerging Concern , can be excreted by both humans and animals with little change in their chemical structure . Standard wastewater treatment facilities are not equipped to completely remove pharmaceuticals resulting in these compounds being found in effluent. In addition, even higher concentrations of many pharmaceuticals are released during heavy storms in the untreated wastewater overflow, which then directly contaminate the environment . Recent studies of the effects of pharmaceuticals on aquatic insects show that at concentrations found in reclaimed water these CECs can alter development of the mosquito Culex quinquefasciatus, its susceptibility to a common larvicide, and its larval microbial communities.
However, because larval forms of aquatic insects develop directly in the contaminated water, their constant exposure is likely greater than most terrestrial insects. Interestingly, many CECs, which were not designed specifically to impact microbes, have been shown to affect microbial communities. For example, caffeine, a common mental stimulant, can alter biofilm respiration, and diphenhydramine, an antihistamine, have been shown to modify the microbial community of lake biofilms. Due to such unexpected effects, accurately predicting the consequences of specific CECs, even in model insects, is not yet possible. This problem is exacerbated by a lack of information regarding effects of pharmaceuticals and other CECs on the microbial communities of any terrestrial insects. Arthropods rely on hormones to grow, develop, mate, and produce pigmentation. However, many pharmaceuticals, especially mammalian sex hormones, are structurally similar to chemicals that these organisms rely on for growth and development. These pharmaceuticals then bind to receptors and either over-express or suppress their counterparts’ natural function. This has been seen in birds, reptiles, and arthropods where endocrine disruption occurs, primary and secondary sexual characteristics are modified, and courtship behaviors are changed. While most arthropod hormones do not closely match those of mammals, their molting hormone , is very similar to the mammalian female sex hormone 17β-estradiol. In insects, 17α-ethinylestradiol, a common synthetic birth control hormone, has been shown to alter molting and lead to deformities of Chironomus riparius. In addition to these effects, pharmaceuticals have been shown to have delayed cross-generational effects . The cabbage looper is a well-studied polyphagous insect native to North America, and found throughout much of the world. This species is a pest on many agricultural crops including crucifers and a variety of other vegetables in both field and greenhouse settings .
Potential agricultural losses are exacerbated by a history of pesticide resistance development . Currently there is little to no information regarding pharmaceutical effects at the concentrations found in reclaimed water on the growth or microbial community composition of any terrestrial herbivore. To investigate the function of the gut microbes in insects, several studies have used antibiotics applied at high doses. To test the hypothesis that common pharmaceuticals affect mortality, development, and microbial communities of Trichoplusia ni, we conducted a series of bio-assays in artificial diet and on a key host plant. Tomatoes were grown from seeds in 10.16 cm pots in UC soil mix No. 3 and fertilized with Miracle Gro nutrient solution at labeled rate. At approximately 10 cm, tomato plants’ roots were washed with water and transplanted to sand culture as in Hladun et al. . Transplants were treated with CECs in hydroponic growth media with concentrations described in Table 1. Plants were watered every 2 hr from 6 am- 6 pm and every 4 hr thereafter. Hydroponic solutions were kept in 120 L containers. Each container included one of five CEC treatments or an untreated control hydroponic solution. Plants grew 4-6 wk before cabbage loopers were bagged onto whole leaves with white mesh organza bags . If T. ni devoured the entire leaf they were immediately moved to a leaf of similar location on the plant. Data regarding growth and development and mortality were collected daily. Growth index data were calculated as in Zhang et al. . Data were then analyzed in R. Plants were separated into parts and frozen at -62 ± 2°C until analyzed.For the first three life-stages in all treatments, the majority of microbes belong to the family Lactobacillaceae . Lactobacillaceae’s proportional abundance increases in third and sixth instar; remain high, but decrease in the pupae; and then decrease further in adult insects where the majority of microbes were Pseudomonadaceae. Alcaligenaceae, Pseudomonadaceae, and Enterobacteriaceae, the next three families with the highest average percentages of rarefied OTU counts,maceta 10 litros have similar patterns of high percentages in third instars followed by a decline in sixth instars and pupae, and then a spike in the adult life-stage. This same pattern was seen in both Chitinophagaceae and Sinobacteraceae, as well. For the control, acetaminophen, caffeine, and mixture treatment groups containing all life-stages , the trends of average percentages follow the same patterns. The families with highest average percentage of rarefied OTU counts were Lactobacillaceae, Pseudomonadaceae, Alcaligenaceae, and Enterobacteriaceae, respectively. Interestingly, this pattern changes for antibiotic, hormone, and mixture treatment groups. For insects fed diets containing antibiotics and hormones, the average most proportionate families were Lactobacillaceae, Pseudomonadaceae, Alcaligenaceae, and Enterococcaceae.
When examining the differential abundance of the individual OTUs by life-stage in each treatment , an interesting pattern appears. There were significant differences in controls between third instars and all other life-stages, between acetaminophen fed third instars and sixth instars, and between caffeine-fed third instars and sixth instars and pupae. However, in these three treatment groups, there were no significant differences between sixth instar and pupae versus their adult life-stages. In the T. ni treated with antibiotics and the mixture treatment, microbes were significantly different between any early life-stage and adults. For insects fed a hormone-contaminated diet, microbes were only different for third instars when compared to adults. There were similar trends when individual OTUs were examined for each life stage comparing differences in treatments . For third instar insects, controls had significant differences in all other treatment groups; acetaminophen was significantly different when compared to antibiotic, hormone, and mixture treatments, but not the caffeine treatment; caffeine-fed insect microbes followed a similar trend to acetaminophen. For third instar, there were no significant differences between antibiotic, hormone, and mixture treatments. Between all of the multiple treatment comparisons with OTUs, there were no significant differences in the sixth instar insects. For pupae, acetaminophen and caffeine-fed insects’ microbes were different compared to antibiotic treatments. The microbial communities in the antibiotic treatment were significantly different from hormone and mixture treatments. Adults had significant differences in microbe composition in all treatments versus the mixture treatment. Principle Component Analyses visualize these findings .In our study, CECs at concentrations found in reclaimed wastewater were shown to increase mortality of T. ni, especially on artificial diets contaminated with antibiotics, hormones, and a mixture of the chemicals. The mortality effect was also evident when T. ni were reared on plants grown in antibiotic-contaminated hydroponic growth media. Because plants grown in the hydroponic system contained quantifiable levels of ciprofloxacin in the leaf tissue, and the antibiotic treatments significantly changed the microbial community of the insect, we think this is possibly a cause of the mortality but we cannot exclude direct effects of the CECs on the insects or indirect effects through the plants. Interestingly we did not see the increased time to adulthood in T. ni reared on plants as compared with those reared on contaminated artificial diet. We postulate the discrepancy is possibly due to a number of factors such as dilution of CECs, as they were acquired from the water by the plants or there was bio-degradation of the chemicals occurring in the plant 200 or by photodegradation. More studies would be needed to determine how CECs at concentrations found in reclaimed water for agriculture would interact with current integrated pest management strategies , and how soil matrices would effect the chemical acquisition and translocation by plants. Many insects rely on microbial communities and endosymbionts to grow and develop. While Adonis does not have a post hoc test for direct pairwise comparisons, we can evaluate changes in the bacterial communities based on adjusted p-values and PCA ellipses. We found significant shifts in the microbial community in the various life-stages examined within the control treatments notably from third instar to subsequent life-stages. A similar result has been reported for mosquitoes and other insects. However, there were no significant differences in any of the later life-stages. This suggests that T. ni may require certain microbes to advance to later instar, but more information is needed to confirm this. Not surprisingly, insects that undergo complete metamorphosis and also rely on a different food source as adults would require a different bacterial community throughout the life stages; however there is one family, Lactobacillaceae, which appears in all treatments and life-stages in high proportions, except for adults. Species in this family are Gram positive Firmicutes that are known to produce lactate, formate, and succinate through fermentation . They are fairly common in insects and can be responsible for at least 70% of the bacterial community. Lactobacillaceae is responsible for approximately 42% of the bacteria in all life-stages, followed by Pseudomonadaceae, Alcaligenaceae, and Enterobacteriaceae. Lactobacillaceae have been shown to act as beneficial bacteria in Drosophila and aid systemic growth when larvae are reared on nutrient-poor diet . Pseudomonadaceae and Enterobacteriaceae families contain known symbionts in insects. Alcaligenaceae has been shown to be present in other moths but at a much lower proportionality than we found. These microbes could be commensal or mutualistic but more research would be needed to determine this. There are clear patterns regarding the changes in microbial community proportionality according to the heat map . In controls, third instar microbial communities are relatively evenly spaced by family.