Transcriptional responses of Biomphalaria pfeifferi and Schistosoma mansoni following exposure to niclosamide, with evidence for a synergistic effect on snails following exposure to both stressors

Background Schistosomiasis is one of the world’s most common NTDs. Successful control operations often target snail vectors with the molluscicide niclosamide. Little is known about how niclosamide affects snails, including for Biomphalaria pfeifferi, the most important vector for Schistosoma mansoni in Africa. We used Illumina technology to explore how field-derived B. pfeifferi, either uninfected or harboring cercariae–producing S. mansoni sporocysts, respond to a sublethal exposure of niclosamide. This study afforded the opportunity to determine if snails respond differently to biotic or abiotic stressors, and if they reserve unique responses for when presented with both stressors in combination. We also examined how sporocysts respond when their snail host is exposed to niclosamide. Principal Findings Cercariae-producing sporocysts within snails exposed to niclosamide express ~68% of the genes in the S. mansoni genome, as compared to 66% expressed by intramolluscan stages of S. mansoni in snails not exposed to niclosamide. Niclosamide does not disable sporocysts nor does it seem to provoke from them distinctive responses associated with detoxifying a xenobiotic. For B. pfeifferi, niclosamide treatment alone increases expression of several features not up-regulated in infected snails including particular cytochrome p450s and heat shock proteins, glutathione-S-transferases, antimicrobial factors like LBP/BPI and protease inhibitors, and also provokes strong down regulation of proteases. Exposure of infected snails to niclosamide resulted in numerous up-regulated responses associated with apoptosis along with down-regulated ribosomal and defense functions, indicative of a distinctive, compromised state not achieved with either stimulus alone. Conclusions/Significance This study helps define the transcriptomic responses of an important and under-studied schistosome vector to S. mansoni sporocysts, to niclosamide, and to both in combination. It suggests the response of S. mansoni sporocysts to niclosamide is minimal and not reflective of a distinct repertoire of genes to handle xenobiotics while in the snail host. It also offers new insights for how niclosamide affects snails. Author’S Summary Schistosomaisis control programs often employ the use of chemical molluscicides, such as niclosamide, to control the obligatory intermediate snail hosts. Despite its widespread use, we know little about how niclosamide affects snails like Biomphalaria pfeifferi, the most important vector Schistosoma mansoni in Africa. By sequencing the transcriptomes of uninfected and S. mansoni-infected B. pfeifferi exposed to niclosamide, we analyze the snail’s response to both biotic and abiotic stressors. We can also examine the response of S. mansoni to niclosamide exposure during intramolluscan development. Biomphalaria pfeifferi snails exposed only to niclosamide showed unique up-regulation of stress and defense-related transcripts not seen in snails infected with a biotic, like S. mansoni infection, alone. Schistosoma mansoni-infected B. pfeifferi exposed to niclosamide were clearly unable to regulate normal metabolic and detoxification processes. Cercariae-producing sporocysts within snails exposed to niclosamide are largely unaffected and continue to produce transcripts required for cercariae production.


INTRODUCTION
Schistosomiasis control remains elusive in many of the world's hyperendemic foci of infection in sub-Saharan Africa, jeopardizing the goals of diminishing schistosomiasis as a public health concern, or of eliminating transmission where possible by 2025 [1]. Several recent papers have called for the need to adopt more integrated control approaches instead of relying on chemotherapy alone to achieve eventual elimination [2][3], and there has been a resurgence in interest in methods to control the snails that vector human schistosomiasis [4][5]. Although the practical options available for use in snail control remain limited, molluscicides have been advocated because there are several recorded instances where their use has been associated with successful control [4,6].
Following the discovery of niclosamide's molluscicidal properties in the 1950s, it has been incorporated into the commercial preparation known as Bayluscide [7] and is the only molluscicide approved for use in schistosomiasis control by the WHO Pesticide Evaluation Scheme (WHOPES). Use of niclosamide has enjoyed a modest resurgence and its focal application in snail control is advocated by WHO [8]. It has been used widely in Egypt and China as a mainstay for control operations, and it is used in both experimental [9][10] and in new control contexts, most notably recently as part of the S. haematobium elimination program in Zanzibar [11][12].
Although some work on the effects of molluscicides on oxygen consumption and carbohydrate metabolism of snails has been undertaken [13][14], there have been relatively few studies employing modern techniques to assess the impacts of molluscicide exposure on schistosome-transmitting snails. Zhao et al. [15], working with the amphibious snail hemoglobin production was also noted. Niclosamide is able to kill schistosome miracidia and cercariae [17][18] and field experiments in China have shown that niclosamide is effective at reducing the number of viable S. japonicum cercariae in streams and downstream infection of sentinel mice [19].
With respect to the effects of niclosamide on schistosome-infected snails, or on the schistosome sporocysts within them, there has been remarkably little study. Sturrock [20] investigated the effects of sublethal concentrations of niclosamide on infections of S. mansoni on Biomphalaria sudanica tanganyicensis and noted that: 1) snails exposed to molluscicide that survived were still susceptible to infection; 2) snails with prepatent infections were not initially more susceptible to molluscicide but had slightly delayed rate of parasite development and production of cercariae and did eventually exhibit higher mortality as they entered patency; and 3) survivorship of snails exposed during the patent period was less, although it takes some time for the effect to occur. Sturrock [20] commented that the combined stress of producing cercariae and exposure to molluscicide likely contributed to the higher mortality rate in patent snails. He also noted that doses sufficiently high to kill schistosome sporocysts in snails were probably above the lethal doses needed to kill the snails themselves.
In this study, building on the microarray results of Zhang et al. [16] with B. glabrata, we sought to obtain a more in-depth view of the transcriptome of molluscicide-exposed snails by using the Illumina platform to examine the responses of Biomphalaria pfeifferi to a sublethal dose (0.15 mg/L) of niclosamide. Biomphalaria pfeifferi is widely distributed in streams, ponds and impoundments in Africa and is probably responsible for transmitting more cases of Schistosoma mansoni than any other Biomphalaria species [21][22]. In addition, we examined the transcriptional responses to the same dose of molluscicide of B. pfeifferi harboring cercariae-producing S. mansoni infections. We were able to compare the responses of the above snails to both uninfected and infected B. pfeifferi not exposed to molluscicides (see companion studies [23,24]). For both the previous and present studies, we chose to examine the responses of snails recently removed from field habitats and therefore considered to be more representative of what might be expected of snails comprising natural populations actually exposed to molluscicides. The approach taken enables us to ascertain if and how the transcriptional responses of snails already coping with a massive S. mansoni infection can be further altered by simultaneous exposure to a toxic xenobiotic. For example, might snail genes up-regulated following exposure to S. mansoni trend towards down-regulation if the snail is exposed to niclosamide and required to produce increased quantities of molecules involved in detoxification?
With respect to the sporocysts of S. mansoni residing in snails exposed to niclosamide, do they exhibit any tendency to express genes that are not normally expressed during intramolluscan development, and if so, do the ensuing proteins favor survival of the sporocysts or of the stressed snail in which the sporocysts reside? Three possible scenarios for S. mansoni transcriptional response to molluscicide exposure can be considered: 1) We see an overall down-regulation of S. mansoni transcripts indicating suspension of activity; 2) Cercariae-producing S. mansoni sporocysts express unique features that are shut off in response to molluscicide exposure; and 3) Shedding S. mansoni stages exposed to molluscicide show unique transcriptional responses suggestive of a hitherto unseen ability to protect the host-parasite unit in which they reside from a xenobiotic.  [25]. Snails shedding only S. mansoni cercariae and non-shedding controls were used in this study. More details of collections and processing can be found in Buddenborg et al. [23][24].

Biomphalaria pfeifferi used in
The molluscicide niclosamide dissolved in dimethyl sulfoxide (DMSO) was purchased from Sigma. Uninfected and S. mansoni-infected B. pfeifferi were exposed to a concentration of 0.15 mg/L niclosamide with final DMSO concentrations at 1/1000 (v/v) for 24 hours at 26-28C with aeration [16]. Previous 24 h exposure of B. glabrata to varying doses of niclosamide (0.05mg/L, 0.10mg/L, and 0.15mg/L) found that the 0.15mg/L dose produced the most robust transcriptional response, as assessed by microarray analysis [16]. All snails exposed to 0.15mg/L niclosamide were alive and responding after the 24 hours dosage period. Therefore, a 0.15mg/L dose was also selected for this study as the sublethal dose administered to B. pfeifferi.
RNA extraction, library preparation, sequencing procedures, and sequencing summaries can be found in Buddenborg et al. [23,24]. Illumina RNA sequencing reads underwent extensive processing in order to separate host, parasite, and potential symbiont reads.
Normalized Schistosoma mansoni read counts acquired from RSEM were normalized using DESeq's median of ratio method [28].

RESULTS AND DISCUSSION
Overall B. pfeifferi and S. mansoni transcriptomic responses to molluscicide exposure Relative to uninfected and untreated control B. pfeifferi, the overall differential gene expression responses were measured for snails i) with shedding S. mansoni infections only, ii) exposed for 24 h to a sublethal dose of niclosamide only, or iii) harboring shedding S. mansoni infections and exposed to niclosamide (Fig 1A). The responses of shedding snails relative to unexposed controls have been discussed extensively by Buddenborg et al. (2017). With respect to molluscicide exposure, this is the first Illumina-based view of the transcriptomics response for any species of planorbid snail, and supplements and extends the view provided by the microarray study for uninfected B. glabrata of Zhang et al. [16].
Zhao et al. [15] undertook an Illumina-based study of the molluscicide-induced transcriptome of Oncomelania hupensis, the pomatiopsid snail host of S. japonicum. The response of B. pfeifferi to simultaneous exposure to schistosome infection and niclosamide exposure is the first glimpse we have for how snails respond transcriptionally to simultaneous exposure to these two relevant stressors.
For each of the three treatments noted, the number of upregulated snail features exceeded the number of down-regulated features. For both up-and down-regulated features, it was remarkable that over half of the transcripts proved to be distinctively represented in the combined treatment group (Fig 1B). Over 4,000 genes were distinctively up-regulated in the snails receiving the combination of stressors. This was the largest number found in any single group of either venn diagram. It was surprising to us that larger numbers of genes were not found in the cells of either venn diagram that represented two or all three of the treatments. It was also evident that although the response of niclosamide-exposed snails had features in common to those evoked by S. mansoni exposure, many genes were also uniquely differentially expressed by exposure to just niclosamide. Further inspection of the pattern in expression levels exhibited by genes uniquely expressed in the combined treatment group revealed that in comparison to genes represented in other cells, they were modest in the degree of their differential expression. The specific nature of the genes responsive to either molluscicide alone, or to molluscicides and S. mansoni are discussed further below.
The transcriptomic responses of intramolluscan stages of S. mansoni, including those from snails actively shedding cercariae are described by Buddenborg et al. [24], and are supplemented here by responses of shedding snails exposed to niclosamide (Fig 2A). S. mansoni from shedding snails expressed 18,736 transcripts whereas S. mansoni from shedding samples exposed to sublethal niclosamide expressed 23,040 transcripts ( Fig 2B), with the majority (80.6%) of these shared between the two groups. Most of the remaining transcripts were unique to S. mansoni from the niclosamide-exposed samples (19%), with only 0.4% expressed uniquely in the shedding snails. The additional genes expressed only in the presence of niclosamide raises the percentage of the S. mansoni genome of 66% shown by Buddenborg et al. [24] to be expressed in snails to 68%. S. mansoni exposed to niclosamide expressed more transcripts, but this response was variable among replicates and in general most (>90%) of these extra transcripts were expressed less than 2 log 2 normalized counts when replicate counts were averaged ( Fig 2C). Of the 80.6% of shared S. mansoni transcripts, there was little difference in overall expression levels for transcripts from samples with or without niclosamide exposure.  Specific responses of S. mansoni cercariae-producing sporocysts within B. pfeifferi exposed for 24 h exposure to sublethal niclosamide treatment: Sturrock [20] noted that the lethal dose of niclosamide for intramolluscan schistosomes must be higher than what is needed to kill the host snail. This is likely true since S. mansoni sporocysts are protected both by their syncytial tegument [29] and by being embedded in the host snail's tissues. At least with respect to a 24 h exposure to a dose of niclosamide sublethal for B. pfeifferi, we observed that sporocysts within such snails produced more transcripts relative to sporocysts from untreated shedding snails, not fewer (Fig 2). If cercariae-producing sporocysts were strongly and directly affected by niclosamide, we would have expected to see extensive and broad down-regulation or absence of numerous transcripts such as those related to nutrient uptake across the tegument, of elastases indicating a decrease or pause in cercariae production, and of transcripts associated with germ ball development and proliferation. As it was, only 0.4% of transcripts were missing as compared to untreated sporocysts.
Defense and stress responses were sustained in niclosamide-treated sporocysts relative to untreated sporocysts (Fig 3). Peroxiredoxins like glutathione peroxidase and thioredoxin peroxidase, which may be responsible for elimination of potentially lethal hydrogen peroxide produced by the snail [30][31][32], were stably maintained. Stable and consistent expression of S. mansoni planarian-like bacterial defense homologs, heat shock proteins and SODs are all indicative that protective responses were maintained following exposure to niclosamide.
Interestingly, there was no conspicuous evidence of greater representation of these transcripts in sporocysts from snails treated with niclosamide. Particularly noteworthy is the lack of an obvious response of the single S. mansoni cytochrome p450 gene to niclosamide presence. As noted by Ziniel et al. [33], the exact function of this gene product in S. mansoni is not known, but our results suggest it is not provoked by a xenobiotic like niclosamide. As noted by Ziniel et al. [33] and by us previously (Buddenborg et al. [24], parasitic helminths in general lack extensive cytochrome p450 repertoires, quite unlike their hosts. In contrast, B. pfeifferi does indeed deploy cytochrome p450 responses upon exposure to molluscicides (see below). Another group of S. mansoni transcripts of potential relevance to their response to niclosamide are drug efflux transporters like ABC transporters which are known to be upregulated in adult schistosomes exposed to praziquantel [34][35]. We noted several efflux transporters were expressed in cercariae-producing sporocysts of S. mansoni [24] but did not observe any conspicuous change in their expression pattern following exposure to niclosamide (Fig 4). A study on the giant liver fluke Fasciola gigantica exposed to rhodamine-labeled niclosamide did not reveal substantial changes in ABC transporter activity although the authors did not rule out the potential involvement in these proteins in drug resistance and detoxification [36].  Inspection of the transcripts produced uniquely by niclosamide-exposed sporocysts does not reveal any candidates that would seem to favor resilience to niclosamide. This coupled with the stable expression of known defense or stress response genes noted above leads us to a conclusion that sporocysts have little if any ability to mount protective responses to niclosamide and certainly do not seem to provide anything that would favor enhanced survival of their host snail in the presence of a chemical that is clearly lethal for the host. It is possible that the parasite is relying on host xenobiotic capabilities to respond to niclosamide.
S. mansoni sporocysts, and the cercariae developing within them, express a diverse array of proteases, including elastases and leishmanolysins [24,37], with likely functions in disabling snail defenses, dissolution of snail tissues to provide living space, facilitating intrasnail migration of sporocysts and for packaging in cercariae which use them both for exiting the snail host and entering the mammalian definitive host. Protease inhibitors are also produced and likely counteract proteases that the snail expresses late in infection [24,38].
The overall patterns of expression of proteases or protease-inhibitors does not differ substantially between sporocysts in untreated and niclosamide-treated snails (Fig 5). When specific proteases like elastases, leishmanolysins, and cathepsins are examined, some changes in expression were noted. Niclosamide-exposed sporocysts exhibited modest increases in expression of cercarial elastases SmCE1a, SmCE1a.2, cercarial protease, and SmCE2b ( Fig 5). The SmCE isoforms represent an expanded family of elastases unique to some Schistosoma species including S. mansoni [37]. Transcripts for leishmanolysins which are metalloproteases also known as invadolysins [39], also exhibited a modest increase in representation in niclosamide-exposed sporocysts as compared to untreated sporocysts [24].
At least some S. mansoni glucose, amino acid, and nucleoside transporter transcripts showed modestly increased representation in molluscicide-exposed sporocysts relative to untreated controls (Fig 6), as did some of the markers for germinal cell proliferation [40] such as fibroblast growth factor receptor 2, vasa, and nanos-2 noted in Buddenborg et al.
[24] (Fig 7). Very modest changes in expression were also noted (Fig 8) in neuropeptide hormones or markers of neural development important in flatworm locomotion, feeding, host location, regeneration, and development [41,42]. Shedding S. mansoni stages exposed to niclosamide had higher transcript levels for cell polarity protein, neuronal differentiation, notch, SOX transcription factor, and septate junction protein (Fig 7) and although modest, these may have important downstream effects on germinal cell proliferation or neurogenesis [43][44][45].

Shared response of two Biomphalaria species to a sublethal dose of niclosamide
Of the 30,647 probe features on the B. glabrata microarray used by Zhang et al. [16], 16,713 (55%) were homologous to a B. pfeifferi transcript (Blastn E-value <1e-10, percent identity >75%). Microarray features with homologs to B. pfeifferi transcripts and that were differentially expressed in both Zhang et al. [16] and the present study are shown in Table 1.
These features represent a conservative view of genes characteristic of Biomphalaria's response to sublethal niclosamide exposure. The entire differential expression analysis of B.
pfeifferi's response to niclosamide showed 895 transcripts up-regulated and 604 downregulated when compared to uninfected control B. pfeifferi.  [16] and B. pfeifferi that were significantly differentially expressed after exposed to 0.15mg/L niclosamide. As a lipophilic xenobiotic, niclosamide would likely be eliminated in animals by increasing its hydrophilicty (phase 1 reaction), conjugating the phase I product with a charged chemical group (phase 2 reaction), and then removing it with the aid of a transmembrane transporter (phase 3 reaction) [46]. A key enzyme superfamily of heme-thiolate proteins responsible for initial phase I detoxification are the cytochrome p450s (CYPs). CYPs are found in all kingdoms of life and most commonly perform monooxygenase reactions adding one oxygen atom to the xenobiotic with the other oxygen atom reduced to water [46]. Zhang et al. [16] found that 9 of the features that were up-regulated ≥ 2-fold change following exposure to 0.15mg/L of niclosamide were CYPs. The B. glabrata genome has about 99 genes encoding heme-thiolate detoxification enzymes with tissue-specific expression patterns suggesting that CYPs serve specific biological processes [47].

B. pfeifferi
CYPs are also up-regulated in B. pfeifferi in response to niclosamide exposure, including two in common with B. glabrata (Table 1) and 8 more as noted in Fig 9A,   Glutathione-S-transferase 7-like (GST) was up-regulated 5-fold following niclosamide exposure in B. glabrata [16]. In addition to CYPs, GST has also been shown to be upregulated following niclosamide-based molluscicide exposure in Oncomelania hupensis [15].
GST was also represented in the B. pfeifferi Illumina DE transcripts with up-regulation of GST omega-1-like, and microsomal GST-1 and 3-like (Fig 9).
Transmembrane transporters complement the detoxification and conjugation reactions of phases 1 and 2 by eliminating the xenobiotic or toxin present in an organism [46]. ATPbinding cassette (ABC) transporters, particularly ABC efflux transporters, play an important role in eliminating toxic compounds from cells. For instance, ABCG2, a non-specific, multixenobiotic transporter is known to be expressed at high levels in the gills and hemocytes of Mytilus edulis [49]. One family of ABC efflux transporters, the multidrug resistance proteins (MRPs) act to eliminate drugs and toxic chemicals transporting anionic compounds detoxified in phases 1 and 2. One MRP-1 is expressed 2.8-fold higher than controls in B.
glabrata [16] and 10 MRP-1 transcripts were up-regulated in B. pfeifferi suggesting these transporters are removing toxic waste products produced directly by niclosamide or indirectly through cell death or tissue necrosis ( Table 2). Heat shock proteins show increased expressed to a variety of stressors including elevated temperature, hypoxia, ischemia, heavy metals, radiation, calcium increase, glucose deprivation, various pollutants, drugs, and infections [50]. Up-regulation of HSPs has been associated with susceptibility of B. glabrata to S. mansoni [51][52]. HSPs have also been identified in other molluscs as indicative of environmental stress. The disk abalone Haliotis discus discus up-regulates HSP 20 when exposed to extreme temperatures, changing salinity, hssseavy metals, and microbial infection [53]. The marine bivalve, Mytilus galloprovincialis up-regulates HSPs 24.1, 70, 90, and sequestosome-1 following toxic metal exposure [54]. Biomphalaria glabrata mounts a multifaceted HSP response to niclosamide by up-regulating HSPs 12, 40, and 70 [16] and the selective autophagosome cargo protein sequestosome-1. We also saw up-regulation of these specific HSPs but the more comprehensive sequencing available from the Illumina study revealed mixed responses of isoforms of HSP 12.2 and down-regulation of HSP 30 (Fig 10). al. [16].
In response to exposure to S. mansoni infection, B. pfeifferi shows a more complex transcriptional expression of HSPs, cytochrome p450s, and glutathione-S-transferases than it does to molluscicide with no general up-or down-regulation of any group of these transcripts [23]. Biotic stressors such as parasites with intimate and prolonged contact with host tissues may induce a more complex stress response with up-and down-regulation of various HSPs in comparison to a general up-regulated of CYPs, glutathione-S-transferases, small and large molecular weight HSPs, and sequestosome noted in the response of several molluscs to abiotic stressors.

Additional responses of Biomphalaria to sublethal molluscicide exposure detected with Illumina RNA-Seq
Because of the unbiased sequencing available in Illumina RNA-Seq, we were able to acquire additional information on the transcriptomic responses of B. pfeifferi to niclosamide beyond that allowed by the B. glabrata microarray study. Transcripts involved in protection from oxidative damage, generalized pathogen defense and innate immunity, protease inhibitors and feeding behavior were all noted.
We observed high expression of several glutathione peroxidase transcripts, presumably associated with enhanced conversion of hydrogen peroxide to water. In cancerous colon cells, niclosamide increased cell death when used with a therapeutic drug through hydrogen peroxide production [55], therefore, it is not inconceivable that niclosamide in snails is directly or indirectly involved in increasing hydrogen peroxide levels although there is thus far no direct evidence for this in exposed snails. Glutathione peroxidase has been shown to increase the general tolerance of cells to oxidative stress resulting from exposure to xenobiotics [56].
Glutathione reductase, a critical oxidoreductase enzyme that catalyzes the reduction of glutathione disulfide to glutathione, surprisingly was down-regulated. As noted above, glutathione is a key ingredient needed in phase II conjugation mediated by the enzyme glutathione-S-transferase, which is up-regulated in B. pfeifferi following molluscicide exposure. An impaired ability to regenerate glutathione because of down-regulated glutathione reductase activity could then impair both the detoxification process and interfere with maintenance of redox balance by allowing hydrogen peroxide to accumulate.
One of the more striking responses of B. pfeifferi exposed to niclosamide was the high up-regulation of transcripts for several protease inhibitors including antitrypsin-like and serpins (serine protease inhibitors) and the down-regulation of metallo, cysteine, and serine proteases. In contrast, only one serine protease (chymotrypsin-like elastase family member 1) and a single aminopeptidase N-like transcript were up-regulated. Caspases are cysteinedependent proteases that play essential roles in programmed cell death [57] and isoforms of caspase-2 and 3 were down-regulated in niclosamide-exposed B. pfeifferi. The downregulation of protease activity may be part of a compensatory stress response made by the snail to minimize metabolic changes associated with niclosamide exposure that if left unchecked would lead to apoptosis and protein degradation.
Responses typically classified as innate immune responses because they occur following exposure to parasites like S. mansoni were also noted in B. pfeifferi exposed only to niclosamide. One such transcript was homologous to CD109 antigen-like, a thioestercontaining protein, which is highly enriched in plasma from both resistant and susceptible strains of B. glabrata containing miracidia transforming into mother sporocysts [58]. We also noted up-regulation of a transcript identified as complement C1q-like protein that we have reported to be up-regulated in early S. mansoni-infected B. pfeifferi [23]. Fibrinogen-related proteins (FREPs) 1 and 2 were both up-regulated after niclosamide exposure; FREP2 was also up-regulated in S. mansoni-shedding B. pfeifferi [23]. Dermatopontin, a parasiteresponsive gene frequently noted in studies of both B. glabrata and B. pfeifferi, was also upregulated following niclosamide exposure.
A conspicuous response was the high up-regulation of over 100 diverse transcripts identified as LBP/BPI1 (lipopolysaccharide binding protein/bacterial permeability-increasing protein 1) in B. pfeifferi after exposure to niclosamide. LBP/BPI1 is an antimicrobial molecule found in the albumen gland of B. glabrata and egg masses [59]. Silencing of LBP/BPI1 expression in B. glabrata resulted in significant reduction of egg-laying, and death of eggs attributable to oomycete infections, providing evidence that LBP/BPI is involved in parental immune protection of offspring [60].
Transcripts homologous to B. glabrata tyrosinases (Tyr) 1, 2, and 3, are also upregulated in response to niclosamide. In early-stage pre-patent S. mansoni infections Tyr-1 is up-regulated, and Tyr-3 is down-regulated in B. pfeifferi harboring cercariae-producing sporocysts [24]. Tyrosinases are involved in melanin synthesis and additionally might mark an early phase in initiation of castration by diverting tyrosine towards the production of melanin instead of dopamine in S. mansoni-infected B. pfeifferi [23]. Like LBP/BPI1, tyrosinase has also been isolated from B. glabrata egg masses and is presumed to provide an immunoprotective effect for developing embryos by contributing to the melanization of the egg membrane [59,61]. The additional considerable effort by the snail to make two egg mass-associated proteins in response to niclosamide is baffling, but might represent a lastditch attempt to produce offspring before death. Alternatively, perhaps this is best viewed as an example of relatively non-specific innate immune responses that can be invoked by exposure to an unusual stressor, even if it is of an abiotic nature. Another consideration is that it represents a response to the presence of bacteria in the snail that might appear due to impaired hemocyte function or possibly due to failure to contain the gut microbiome in its usual compartment.
Another unexpected response was the high up-regulation of myomodulin-like neuropeptide in niclosamide-treated B. pfeifferi. Myomodulins are neurotransmitters involved in regulating feeding behavior by controlling radula protractor muscles used for feeding [62] in Lymnaea stagnalis [63] and Aplysia californica [64]. Myomodulin is down-regulated in prepatent S. mansoni-infected B. glabrata and this was implicated as possibly diminishing feeding efficiency in infected snails [65]. Down-regulation of a B. pfeifferi feeding circuitry peptide was seen in early and patent S. mansoni infections [23]. The up-regulated myomodulin activity noted provides evidence that basic physiological activities such as feeding are altered after niclosamide exposure. The mussel Mytilus edulis shows a decreased rate of feeding after exposure to hydrophobic organic chemicals, organochlorine compounds, organophosphate and carbamate pesticides, and pyrethroids [66][67].
With respect to features down-regulated following niclosamide exposure, it would seem pomatia and Cepaea nemoralis [68]. Although we did not observe down-regulation of the hemoglobin-encoding gene noted by Zhang et al. [16] following exposure of B. glabrata to niclosamide, down-regulation of neuroglobin in niclosamide-exposed B. pfeifferi was observed. This could be associated with reduced availability of oxygen, at least for neural cells.
Significant down-regulation of a Cu-Zn SOD (-9.3 log 2 FC) in B. pfeifferi indicates that SODs have a more complex response to niclosamide than previously thought from the microarray study by Zhang et al. [16]. High expression of certain alleles of Cu-Zn SOD have been implicated in resistance of B. glabrata strain 13-16-R1 to S. mansoni [69][70][71] so it is not unlikely that different Cu-Zn SODs show distinctive responses to other stressors like niclosamide. Calmodulins, ubiquitous calcium-dependent signaling proteins responsible for regulating the uptake, transport, and secretion of calcium in gastropod shell formation [72][73], are expressed by B. glabrata in response to gram (-) and gram (+) bacteria, yeast [74], and in B. glabrata snail plasma containing larval S. mansoni. Here, we saw down-regulation of calmodulin in B. pfeifferi exposed to the niclosamide, raising the possibility that calmodulin expression is more responsive to biotic challenges. Transcripts related to cell adhesion like spondins that are expressed in Biomphalaria hemocytes [75] are also down-regulated.

Responses of B. pfeifferi with cercariae-producing S. mansoni infections to sublethal niclosamide treatment
As previously noted, snails exposed to the combined effects of the biological stressor S.
mansoni and the abiotic stressor niclosamide were surprisingly responsive (Fig 1), exhibiting large numbers of uniquely up-and down-regulated features, with many of these only modest in the degree of their differential expression. Among the more notable responses were several features associated with managing cell death in damaged tissues ( Table 3). The transmembrane transporter ABCA3 is associated with resistance to xenobiotics and engulfment during apoptosis [76]. The enzymes glutaredoxin-2-like and catalase-like are both involved in reduction of hydrogen peroxide that may be released during niclosamideinduced apoptosis. An increase in apoptosis could account for the up-regulation of lysosomal endopeptidases such as cathepsin-L-like. Two mitochondria-associated transcripts that also play a role in gluconeogenesis, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glycerol-3-phosphate dehydrogenase-like (GPDH) were also up-regulated. GAPDH accumulates in mitochondria during apoptosis and induces proapoptotic mitochondrial membrane permeability [77]. Niclosamide has been screened as a potential promoter of mitochondrial fragmentation by disrupting membrane potential, reducing ATP levels, and inducing apoptosis by caspase-3-activation in HeLa cells [78]. As with snails exposed to niclosamide alone, once again a down-regulation of transcripts for ribosomal proteins was noted. Reduction in ribosome production can be considered a stress response because it is a rapid and effective response against misfolded proteins [80] but may simply be an indication of a downgrading of general condition. Other downregulated transcripts show diverse functional activity. Several annexins, intracellular Ca 2+ and phospholipid binding proteins are down-regulated showing the possible disruption of regulation of membrane organization, trafficking, and the regulation of Ca 2+ concentrations within cells [81].
Unlike the general up-regulation of CYPs in B. pfeifferi exposed only to niclosamide, B.
pfeifferi with dual stressors highly down-regulate several CYPs (microsomal CYPs 2J1-like, 2B4-like, 3A29-like, 26A1-like, and mitochondrial CYP12A2-like). Mitochondrial CYP12A2like is known to metabolize a variety of insecticides and xenobiotics [82]. We cannot discount that contribution to the down-regulation of this particular CYP is a result of mitochondrial degradation caused, in part, by niclosamide as noted previously as well as the additional stress of a patent S. mansoni infection.

CONCLUDING REMARKS
This study provides a distinctive and detailed view of the nature of the response of fieldderived B. pfeifferi to relevant stressors likely to be encountered in its environments, including infections with S. mansoni, just one of several digenetic trematodes known to commonly infect this snail in Africa [83], and exposure to the commonly used molluscicide, niclosamide. It is important to gain additional detailed information regarding the effects of niclosamide on snails, particularly those that harbor schistosome infections. For example, do infected snails succumb more readily to treatment and if so, why? This particular aspect of molluscicide use has not been widely investigated.
In general, exposure to niclosamide alone resulted in the fewest responsive features in B. pfeifferi (1,711) followed by infection with S. mansoni (2,271) and then by the combination of niclosamide and S. mansoni (7,683). Snails in these three groups all responded in very distinct ways, but in each case with more features up-than down-regulated. Sublethal exposure to a single xenobiotic provoked about 67% as large a transcriptomic response as was noted for snails shedding S. mansoni cercariae, snails that had probably been infected with the parasite for at least a month and harbored large numbers of daughter sporocysts.
The fact that snails that received the combination of infection and niclosamide responded so much more vigorously with so many distinctive features suggests that they were under greater duress and that their responses in some sense preempted the responses of snails in the other two groups.
Exposure to niclosamide alone provoked up-regulation of several features associated with response to xenobiotics including cytochrome p450s, heat shock proteins, multidrug resistant transporters and glutathione-S-transferases, confirming many of the observations made by Zhang et al [16] in a microarray study of B. glabrata exposed to sublethal doses of niclosamide. Several additional unique aspects of the response to niclosamide were also noted given the increased resolution provided by Illumina sequencing. We note that one of the effects of niclosamide on B. pfeifferi may be to contribute to redox imbalance because glutathione is being used by glutathione-S-transferases to conjugate xenobiotics but may not be sufficiently regenerated because of down-regulated activity of glutathione reductase.
Exposure of infected snails to niclosamide was noteworthy in revealing the involvement of several features not found to be responsive to either stressor alone. Although many of the uniquely expressed features did not respond dramatically, the ones that did were indicative of responses associated with apoptosis, reduced protein synthesis, reduced production of some CYPs and thus diminished detoxification ability, and diminished innate immune function. Accordingly, we hypothesize that the combination of stressors was likely overcoming the snail's ability to maintain homeostasis. The snail mounts a considerable transcriptomic response to the presence of cercariae-producing sporocysts [23] and it is not hard to imagine that the energy demand placed on infected snails by continual production of cercariae takes an additional toll. The mortality rate of B. pfeifferi infected with S. mansoni is significantly higher than that noted for unexposed control snails [25]. The molluscicideexposed infected snails selected for sequencing were alive when sampled, but the transcriptional profiles suggested they were not thriving. This is broadly in agreement with observations made to indicate that B. sudanica with S. mansoni infections succumb to sublethal niclosamide treatment at a higher rate than do uninfected controls [20]. In other words, the combination of stressors used here exposed the limits of what these snails can do to maintain homeostasis.
Even though S. mansoni sporocysts within snails exposed to niclosamide expressed more transcripts than in untreated snails, there was little about the response to suggest they possessed any distinctive or large-scale ability to respond to a xenobiotic like niclosamide.
Furthermore, the sporocyst response did not appear to be as indicative of a failure to maintain homeostasis as we noted for snails. This is in keeping with the general observation that the lethal dose of niclosamide for sporocysts is probably much higher than for snails [20]. Although it is clear that both miracidia and cercariae are vulnerable to niclosamide [17][18], this may be a reflection of their more aerobic metabolism and that they would be more fully exposed to the action of niclosamide as compared to sporocysts nested within the tissues of an infected snail. Although we did not observe a strong negative effect of molluscicide exposure on the transcriptomics responses of sporocysts, given the relatively unhealthy state of the snail that we detected, it would inevitably follow that the condition of the sporocysts would degenerate.
In conclusion, we noted remarkably distinctive transcriptomics responses for B. pfeifferi depending on the nature of the stressor treatment they received, and that the combination of niclosamide and S. mansoni infection imposed a level of stress on the snails that resulted in a massive response comprised of many features we had not observed previously. This study contributes to the growing list of molecular participants that may govern the outcomes of the intimate interrelationships between snails and schistosomes, and that may help us understand how snail host biology might be targeted for disruption by molluscicidal chemicals.