Many antibiotics and other common contaminants of emerging concern can be excreted by both humans and animals with little change in their chemical structure . Not surprisingly, pharmaceuticals have been appearing in wastewater, surface waters, and in some cases tap water, over the past few years . 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 . These pharmaceuticals have been found at biologically active concentrations in surface waters around the world . There is also an increasing effort to use reclaimed wastewater in drought-affected areas, such as Southern California . In agriculture/livestock operations, pharmaceuticals are found in manure that is used as fertilizer, effectively compounding the pharmaceutical concentrations . Current research shows these chemicals tend to be both pseudopersistent in soil and detrimental to soil and rhizosphere microbes . Our 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 . Watts et al. showed 17α- ethinylestradiol, a common birth control agent, and Bisphenol-A, a common plasticizer, can cause deformities in the midge Chironomus riparius. 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,hydroponic dutch buckets has 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, such as insects and crustaceans, rely on hormones to grow, develop, mate, and produce pigmentation . However, many pharmaceuticals, especially mammalian sexhormones, 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 . Although most arthropod hormones do not closely match those of mammals, their molting hormone is very similar in structure to the mammalian female sex hormone 17β-estradiol. In crustaceans, mammalian hormones have been known to cause both increased molting events and inhibition of chitobiase, the enzyme responsible for digestion of the cuticle during insect molting . In insects, 17α-ethinylestradiol, a common synthetic birth control hormone, has been shown to alter molting and lead to deformities of C. 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 is found throughout much of the world . T. ni are yellow-green to green in color and can complete their life cycle in as little as 21 d depending on temperature . This species is a pest on many agricultural crops including crucifers and a variety of other vegetables in both field and greenhouse settings .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. Many herbivores can be exposed to these contaminants after the CECs enter surface waters, soil, and plants from wastewater reuse and unintended discharge. To investigate the function of the gut microbes in insects, several studies have used antibiotics applied at high doses . There is also no information regarding effects of CECs when translocated through plants to terrestrial insects.
To test the hypothesis that common pharmaceuticals affect mortality, development, and microbial communities of T. ni, we conducted a series of bio-assays in artificial diet and on a key host plant utilizing surface water concentrations of common important pharmaceuticals. We used a culture-independent approach by performing a 16S rRNA gene survey on both diet and whole-body insects. Any effects would have potentially important implications from agricultural perspectives. Also, as there is currently no information on effects of CECs on terrestrial insects acquired through a plant matrix, our findings would have possible interest for integrated pest management research.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-containing 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. Ciprofloxacin is a quinolone topoisomerase IV and DNA gyrase inhibitor that acts by stabilizing the DNA-topoisomerase IV and DNA-girase so that it is no longer reversible . This blocks DNA replication and eventually causes cell death of bacteria. However, unlike bacteria, when higher-level organisms evolved, the A and B subunits of the topoisomerases fused, creating homodimers that cannot be targets of ciprofloxacin , and thus damage to the ribosomes of insects is not a possible mechanism of toxicity. Interestingly, we did not see the increased time to adulthood in T. ni reared on plants 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 or by photodegradation. However, recent studies have shown pharmaceutical concentrations in surface waters, which appear to remain constant over the course of several years . More studies would be needed to determine how CECs at concentrations found in reclaimed water for agriculture would interact with current IPM strategies , and how soil matrices would affect the chemical acquisition and translocation by plants. Many insects rely on microbial communities and endosymbionts to grow and develop; however, it has been shown that Lepidoptera species do not have a vertically transmitted microbial community .
In addition, because the effects of microbial communities on T. ni survival and development have not been documented, we present these data only to show that microbial communities change when exposed to CECs, and not as a proven factor influencing survival. 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 is one family, Lactobacillaceae, which appears in all treatments and life stages in high proportions, except for adults. They are fairly common in insects and can be responsible for at least 70% of the bacterial community . Lactobacillaceae is responsible for ∼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 ; however, its function in T. ni is still unknown. Alcaligenaceae has been shown to be present in other moths ,bato bucket but Lepidopterans are not thought to have a functional microbiome . 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. The microbial community becomes predominately Lactobacillaceae for sixth instars and pupae. Once the insects reach the adult stage, their most predominant family is Pseudomonadaceae. This pattern holds in the acetaminophenand caffeine treatment groups as well. Interestingly, the other treatment groups do not share this pattern. For antibiotic- and hormone-treated T. ni, Lactobacillaceae is the predominant microbial family in the immature stages, but at the adult stage microbial community reverts to predominantly Pseudomonadaceae. We suspect that this is because, once the larvae undergo metamorphosis and shed their gut contents in preparation for pupation, they are no longer exposed to the pressures exerted by the CECs on the microbial community. Fig. 3 provides a visual indication of the changes in the bacterial communities over time. The increase in β diversity after eclosion could be due to the larvae no longer being exposed to CECs or diet-borne bacteria after being moved to sterile containers. Also, when bacteria are lost as larvae digest their gut contents during pupation, the microbial β diversity could change. Interestingly, the hormone-treated T. ni follow a similar pattern to those exposed to antibiotics, but their ellipses are always much smaller, suggesting the entire insect population is showing a uniform response within their microbial communities. However, in the mixture-treated insects, larvae displayed a greater average diversity in their microbial community structure than either pupae or adults. This finding has not been shown in any single category of treatment, and we suspect the microbes exposed to mixtures could be experiencing potential interactive effects among chemicals . Such interactions should be the focus of future studies along with investigations of plant rhizosphere bacteria, particularly since we found a difference in the Bradyrhizobiaceae family for all treatments. These results show that a terrestrial insect pest of commercial crops can be affected by CECs found in reclaimed wastewater for agricultural use. Our results suggest that CECs found in wastewater can impact T. ni growth and development, survivorship, and alter their microbial communities. Because T. ni is a common agricultural pest found around the world, feeds on a wide variety of plants, and has a history of developing pesticide resistance, its ability to deal with toxins is likely higher than many other insects. In addition, the responses we observed to CECs could have interesting implications for IPM practices on plants such as lowering the amount of pesticides needed or increasing susceptibility to insect pathogens, as has been shown in mosquitoes . These potential effects may be understated because some insects cannot detect the presence of the pharmaceuticals . However, we do not recommend purposefully exposing crops to CECs specifically for the control of insects because our study documented that these pharmaceuticals are translocated into crops and we do not yet know their possible effects on humans if consumed . We specifically want to note that ingestion of these compounds through uptake and translocation by a plant is not the only way T. ni or any other insect would be exposed to these compounds. Overhead sprinkler irrigation could cause contact absorption by the plants or insects, and simply drinking water on leaves at contaminated sites could expose insects to higher concentrations than were found in plant tissues. In fact, the ciprofloxacin concentration used was less than one-third of the highest rate . We urge caution in extrapolating to plants growing in soil, because variation in soil type and potential soil bacterial degradation could affect persistence [although soil bacteria are often negatively impacted by CECs ]. However, CEC exposures are considered pseudopersistent because they are reapplied with each irrigation. Thus, the effects reported here are likely to be conservative. Additional studies with other insects, particularly those with other feeding strategies, will be necessary before any patterns can be discerned.The growth of the human population places an ever-increasing demand on freshwater resources and food supply. The nexus of water and food is now well recognized. One promising strategy to sustain food production in the face of competing water demands is to increase the reuse of treated human wastewater. Municipal wastewater reuse for food production has been successfully adopted in some regions of the world. For example, Israel uses ~84% treated wastewater in agriculture production .