Viruses, Vol. 15, Pages 72: Aedes aegypti Strain Subjected to Long-Term Exposure to Bacillus thuringiensis svar. israelensis Larvicides Displays an Altered Transcriptional Response to Zika Virus Infection

1. IntroductionAedes aegypti can transmit several arboviruses, such as dengue, chikungunya, and Zika, which have a significant impact on global public health [1]. The control of Ae. aegypti populations is considered a global challenge [2,3], and integrated control methods are necessary to achieve a sustainable reduction of such populations [4]. The microbial larvicides based on the entomopathogenic bacteria Bacillus thuringiensis svar. israelensis (Bti) are one of the most effective tools to fight mosquito and black fly, owing to its high activity and selective action, allied with its environmental safety [5,6,7,8,9]. The mode of action of Bti is based on the presence of an insecticidal crystal, commonly formed by four major protoxins (Cry11Aa, Cry4Ba, Cry4Aa, and Cyt1Aa), that acts in the larvae midgut. Cry and Cyt are pore-forming toxins; Cry specifically binds to midgut receptors, and destroys the epithelial cells via a synergic mode of action with the Cyt toxin [10,11,12,13]. Briefly, larvae ingest the insecticidal crystals that release the protoxins that are further processed into toxins in the midgut. The Cyt1Aa toxin inserts into the membrane of epithelial midgut cells and subsequently binds to Cry toxins, promoting their oligomerization. Cry oligomers then bind with high affinity to the specific receptors on epithelial cells (e.g., aminopeptidases, alkaline phosphatases, and alpha-glucosidases), provoking cell pore formation and toxic effects that result in larval death [14,15]. The multiple protoxins found within Bti crystals and their unique mode of action on the larval midgut are the major factors that counteract the selection of resistance, and no consistent report of resistance to such crystals has been documented to date [5,16,17,18,19]. Bti crystal is among the safest larvicides available, although assessment of its long-term utilization on nontarget organisms needs to be refined, as recently reviewed [20,21]. Other aspects of mosquito biology which could potentially be affected by long-term exposure to Bti have not been widely investigated yet. This is particularly important in endemic areas for arboviruses, which require control interventions throughout the year in order to reduce the density of robust Aedes populations permanently established in urban areas [22]. To investigate the impact of continuous Bti utilization to control Ae. aegypti populations, we established a laboratory strain named RecBti. This strain has been exposed to Bti for 35 generations in order to mirror the conditions of chronic exposure to this larvicide [17]. For this purpose, third-instar larvae from every RecBti generation were treated with a Bti-based larvicide, and their susceptibility was monitored [17]. RecBti larvae from F1 to F30 were susceptible to Bti and its toxins in comparison with a reference strain [17]. The activity of the detoxifying enzymes found in the RecBti larvae was similar to that of the reference strain, suggesting that continuous Bti exposure did not induce an increase in the metabolism of xenobiotics, as opposed to the exposure to chemical insecticides, which can significantly increase the activity of such enzymes [23,24]. Another study showed that biological parameters, such as fecundity, fertility, pupal weigh, developmental time, sex ratio, and the hematophagic capacity of the RecBti (F30) individuals, did not display alterations compared with a reference strain, but females subjected to artificial oral infection with Zika virus (ZIKV) showed increased susceptibility [25]. Higher ZIKV infection rates in the RecBti females were detected at 7 and 14 days post-infection (dpi), compared with RecL. Viral dissemination after 7 dpi was two-fold higher in the RecBti females than in the control females. RecBti also showed higher infection and dissemination with DENV-2, but this was not statistically significant, unlike in the case of ZIKV [25].Together, these findings showed that the RecBti individuals displayed similar susceptibility to Bti and other biological parameters described above, compared with the reference strain, except for the increased susceptibility of females to ZIKV [25]. We hypothesized that the background of Bti exposure could be associated with the immune response to arbovirus. Therefore, we investigated the profile of expression of specific immune genes in RecBti females challenged with ZIKV, with previously confirmed heightened susceptibility to this virus, as well as the constitutive transcriptome of unchallenged females in comparison with a reference strain. 4. Discussion Ae. aegypti RecBti females that displayed an increased susceptibility to ZIKV in previously performed laboratory infection experiments [25] herein showed an altered expression of some genes potentially involved in the arbovirus response. Previous studies have found that these genes are crucial for the defense against ZIKV [27,28,29,30,31]; however, only a few have assessed them in mosquitoes challenged with larvicides [27,41,42], particularly under the condition of long-term exposure, as investigated in our study. We provided evidence that a reduced expression of genes from the JAK-STAT and Toll immune pathways was associated with the increased viral susceptibility found for the RecBti strain subjected to Bti exposure for 29 generations. The data are consistent with the potential activation of the Ae. aegypti JAK-STAT pathway in response to viral infection, as reported in previous studies [27,42], including response to ZIKV [28]. The downstream component domeless plays an essential role in the regulation of antiviral signaling through the JAK-STAT pathway [43]. In our study, transcripts of the domeless and hop genes from that pathway showed to be upregulated in the ZIKV-infected reference strain, while they were downregulated in the RecBti strain. Downregulation of these immune genes in ZIKV-infected RecBti individuals was confirmed using a comparison between strains. The Toll pathway plays an important role in Ae. aegypti antiviral immune responses [29,30,31,44]. Activation of this pathway in Ae. aegypti in response to ZIKV was shown by RNA-seq, and the depletion of cactus, a negative regulator from Toll, resulted in significantly lower intensity of ZIKV infection [28]. Herein, we observed that relish 1, which stimulates the viral defense, showed significant downregulation in ZIKV-infected RecBti females, while this was not found for cactus. Thus, the expression pattern of this target, which is an antagonist in the Toll pathway, suggests the involvement of this pathway for arbovirus defense in the RecBti strain. The activation of five Toll-like receptors from this pathway in Ae. aegypti resistant to permethrin, after ZIKV infection, was also recorded [41]. Antimicrobial peptides can be produced by induction of the immune signaling pathway in order to neutralize viral pathogens [45]. The Toll and/or IMD pathways, which can be activated in response to viral infection, can trigger the transcriptional activation of defensins and cecropins [40], while gambicin can be induced by the IMD, Toll, and JAK-STAT pathways via combinatorial regulation [46]. In our study, the defensin A and the cecropin G genes were downregulated in the ZIKV-infected RecL strain. A comparison of uninfected females between strains showed that RecBti females had reduced expression of those AMPs, compared with RecL females, and this difference was significant for defensin A. The relative expression of the defensin A and cecropin G genes between infected strains could not be compared, owing to the undetectable transcription levels in RecBti individuals. Therefore, the data suggest that, in both strains, the expression of these AMPs was downregulated, and in RecBti females, their expression was so low that they were not detected by RT-qPCR. Previous studies have demonstrated the specific defensin A action against ZIKV infection in Ae. aegypti [30]. For gambicin, a comparison between infected strains showed reduced expression in RecBti females after viral infection. Therefore, the AMPs investigated herein may be relevant to understand that the immune system can be altered as a function of long-term exposure to Bti and, thus, can trigger a change in the viral susceptibility of these individuals. Together, the quantitative expression of these genes in a pool of females that showed increased ZIKV susceptibility demonstrated a profile of repression. The lack of detection of some AMPs transcripts in RecBti-infected samples is likely to reinforce the repressed transcription profile found for this strain. In this study, the altered expression of immune genes in ZIKV-infected females was investigated in heads with attached salivary glands, which is the last escape barrier for the virus development in mosquito. It is important to consider that other tissues can be assessed, such as the midgut which is the first escape barrier for the virus and also the site of action of Bti toxins. It should be considered that although the gene expression can display variations according to the tissue analyzed [29,47,48,49,50], our data demonstrated a trend of repression of some transcripts involved in arbovirus response from IMD, Toll, and JAK-STAT pathways in the head of infected females, which corroborates their increased susceptibility to ZIKV. However, it is important to note that the antiviral response in mosquitoes can be also regulated by other pathways, whose related genes were not assessed in this study [51].An important aspect that remains unknown is Bti’s ability to impact larvae and/or adult microbiota. Tetreau et al. [52] showed that Ae. aegypti larvae exposed to Bti for 25 h experienced significant changes in the midgut microbiota. Another study showed that variations in the microbiota of larvae can have an impact on the microbiota of adults and their susceptibility to DENV-2 [53]. The role of the microbiota and its impact in mosquito susceptibility to arbovirus has been demonstrated by several studies [54,55,56,57,58]. Therefore, the exposure of mosquitoes to microbial larvicides should be investigated to elucidate their role in the microbiota and the respective impact on the susceptibility to pathogens. It is important to notice that the potential role played by the genes in the respective immune pathways was based on the literature only, as their role was not directly assessed in our study.In another step of our study, the transcriptome of RecBti females was assessed to offer a broader view of the expression status of genes that could be differentially expressed by RecBti F35 unchallenged females, in this case, not induced by ZIKV infection. Before this analysis, the susceptibility of F35 larvae to Bti was confirmed, corroborating previous studies which assessed this strain until F30 [17,25] and other investigations which showed that long-term exposure to Bti did not evolve to resistance to the Bti crystal [5,16,19]. The transcriptome of RecBti F35 females, after 34 generations had been exposed to Bti-based larvicides, but not submitted to ZIKV infection, did not show a marked profile of changes compared with the reference females. RecBti females had a similar profile to the reference strain, as only a limited set of DEGs were detected considering the cutoff used in this study. Genes encoding for Cry toxin receptors, endopeptidases involved in Bti protoxin processing, detoxifying enzymes, immunity, metabolism of xenobiotics, and chitin/cuticle metabolism that showed differential expression through RNA-seq analysis in other Ae. aegypti strains exposed to Bti [59,60,61], or in Ae. aegypti-resistant strains to individuals Bti toxins [60,62,63], in general, were not found to be differentially expressed in RecBti. It is worth noting that none of the seven altered immune genes found in F30 females challenged with ZIKV were found to be differentially expressed in the transcriptome of unchallenged F35 females, which is likely to be related to this condition. The transcriptomic profile is in agreement with previous studies which showed that RecBti and the reference strains displayed several similar phenotypes investigated previously; for example, the susceptibility to Bti, susceptibility to chemical insecticides, activity of detoxifying enzymes, and some life traits [17,25]. A completely different scenario was found, for instance, in the transcriptome of a Cx. quinquefasciatus strain, which displays a high level of resistance to the Binary (Bin) toxin (RR50 > 5000) from Lysinibacillus sphaericus larvicides. The Bin-resistant larvae, also unchallenged with this toxin, showed a remarkable constitutive profile of differential expression characterized by 1300 DEGs, including the repression of several genes with LFC values greater than 3, including the gene coding the receptor of the Bin toxin, which is responsible for resistance [64]. Although the status of susceptibility to Bti was unaltered for the RecBti strain, which is consistent with the transcriptomic profile found for genes involved in the action of Bti, the increased susceptibility to ZIKV was associated with only two repressed DEGs related to arbovirus response; namely, “poor Imd response” and “leucine-rich immune protein,” and the repressed status of the former was not validated by RT-qPCR. On the contrary, the upregulation of vitellogenin-related genes in RecBti females should be noted, as vitellogenin can play an important role not only in reproduction, but also in immunity, including antibacterial action against Bacillus thuringiensis [65,66,67,68]. A comparison of Cx. quinquefasciatus populations from Florida, with different susceptibility to West Nile virus (WNV), showed that the most susceptible individuals had greater expression of vitellogenin and bigger ovary size after blood feeding. Such a finding suggests a correlation between ovary development and competence to WNV [69]. It should be noted that such variations in arbovirus susceptibility of mosquito populations have been documented [70,71,72,73,74], but the factors playing a role in such variations remain unknown. In our study on the RecBti strain kept under long-term exposure to Bti, there was an association between the increased susceptibility to ZIKV and the altered transcription profile of genes involved in the immune response to arbovirus. Nevertheless, it should be highlighted that continuous exposure of this Ae. aegypti strain to Bti did not impact its susceptibility to this larvicide.

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