*3.5. Gene Response to NAP, PHE, FLT, and BkF Exposure*

Five genes were analyzed for adults, and all were targeted by four PAHs with the exception of *hsp70*, *COXI,* and *COXIII* (Figure 3; see also Supplementary Table S5 for the values).

**Figure 3.** Histograms show the differences in expression levels of five genes involved in stress response. *A. franciscana* adults were exposed to naphthalene, phenanthrene, fluoranthene, and benzo(k)fluoranthene at 1.45 mg/L, 1.15 mg/L, 0.81 mg/L, and 84.6 mg/L, respectively. Fold differences greater than ±1.5 (see red dotted horizontal guidelines at values of +1.5 and −1.5) were considered significant (see Supplementary Table S2 for the values). Real-time qPCR reactions were carried out in triplicate. Statistical differences were evaluated by nonparametric Mann–Whitney test. *p*-Values < 0.05 were considered significant.

In fact, *hsp70* was targeted only by PHE and FLT, whereas *COXI* and *COXIII* were not targeted only by FLT. NAP, PHE, and BkF, increased the expression levels of three genes (*hsp60*, *COXI,* and *COXIII*). Moreover, treatment with NAP also down-regulated *hsp26*; the exposure to PHE up-regulated *hsp26* and *hsp70*; FLT is able to up-regulate *hsp26* and *hsp60*, and down-regulate *hsp70*, whereas the exposure to BkF up-regulated *hsp26* and down-regulated *hsp70* (see also Supplementary Table S5).

As shown in Figure 4, among the nine genes analyzed, only one gene (*hsp70*) was not targeted by NAP, PHE, and FLT. In fact, *hsp70* was target only of BkF. Common molecular targets for four contaminants were *HAD-like*, *tcp*, *UCP2,* and *CDC48*, of which only *UCP2* was up-regulated by all treatment, whereas *tcp* and *CDC48* were up-regulated by NAP, PHE, and BkF and down-regulated by FLT; and *HAD-like* was up-regulated by PHE, FLT, and BkF and down-regulated by NAP.

**Figure 4.** Histograms show the differences in expression levels of five genes involved in stress response. *A. franciscana* nauplii were exposed to naphthalene, phenanthrene, fluoranthene, and benzo(k)fluoranthene at 0.26 mg/L, 1.15 mg/L, 0.81 mg/L, and 84.6 mg/L, respectively. Fold differences greater than ±1.5 (see red dotted horizontal guidelines at values of +1.5 and −1.5) were considered significant (see Supplementary Table S3 for the values). Real-time qPCR reactions were carried out in triplicate. Statistical differences were evaluated by nonparametric Mann–Whitney test. *p*-Values < 0.05 were considered significant.

Moreover, *hsp60* was up-regulated by all PAHs with exception of NAP; *hsp26* resulted up-regulated and down-regulated only after PHE and NAP treatment, respectively; and *COXI* and *COXIII* were down-regulated by PHE and up-regulated by BkF (see also Supplementary Table S6 for the values).

#### **4. Discussion**

Acute toxicity tests of PAHs showed similar negative impact of single four pollutants (NAP, PHE, FLT, and BkF) on both adults and nauplii. The NAP, PHE, and FLT were able to induce an increase of nauplii death already after 24 h of exposure, whereas the survival of *A. franciscana* was unaffected by exposure to BkF both after 24 h and 48 h. On the basis of lethal concentrations, the FLT (1.30 mg/L) and NAP (1.73 mg/L) appeared to be more toxic than PHE (4.44 mg/L) at 24 h. As shown in Table 2, Bellas et al. [16] showed similar results in both *C. intestinalis* and *P. lividus* embryos. In fact, exposing these two crustaceans to five PAHs for 24 h, they revealed that FLT and NAP were two and six times more toxic than NAP for *C. intestinalis* and *P. lividus*, respectively.


**Table 2.** Lethal concentration 50% (LC50) values (mg/L) of PAHs.

Instead, after 48 h of exposure, the NAP (0.40 mg/L) and FLT (0.09 mg/L) showed a toxicity 7- and 34-times higher than that established for PHE (3.07 mg/L). In comparison with PHE results from this study, the 48 h LC50 was similar to that of *C. elegans* (4.7 mg/L) but lower than those of *D. magna* (0.8 mg/L), *Chironomus tentans* (0.4 mg/L), and *Eisenia fetida* (0.1 mg/L) reported in a previous study (see also Table 2) [17]. When considering adults exposure, after 48 h, the FLT and PHE showed higher toxicity than these of NAP and BkF. Our results suggest that there is a direct relationship between toxicity and aromatic ring number of the tested compounds. Millemann et al. [2] showed the same relationship for the number of aromatic rings and their toxicity. In fact, they revealed that the PAHs with three or four aromatic rings was always more toxic than those with two aromatic rings for each of the nine exposed species (*Selenastrum capricornutum*, *Nitzschia palea*, *Physa gyrina*, *D. magna*, *Chironomus tentans*, *Gaintaurus minus*, *Pimephales promelas*, *Salmo gairdneri,* and *Micropterus salmonid*; see Table 2).

Our study also provides new information on the large-scale genotoxicity information of PAHs on *A. franciscana* adults and embryos. Firstly, when considering the genotoxicity on adults, all five genes were molecular targets of four pollutants, with only the exceptions of *hsp70*, *COXI,* and *COXIII* (Figure 3). When considering the real-time qPCR experiments on nauplii, all nine genes were molecular targets of four pollutants, with the only exception of *hsp26*, *hsp60*, *hsp70*, *COXI,* and *COXIII* (Figure 4). These data suggest that the nauplii

treated with NAP, PHE, FLT, and BkF were very similar, as also shown in the heatmap reported in Figure 5.

**Figure 5.** Heatmaps showing the expression profiles and hierarchical clustering of nine genes analyzed through real-time qPCR in nauplii treated with naphthalene (NAP), phenanthrene (PHE), fluoranthene (FLT), and benzo(k)fluoranthene (BkF). Color code: red, up-regulated genes with respect to the control; green, down-regulated genes with respect to the control; black, genes for which there was no variation in expression with respect to the control.

All together, these molecular results revealed that the majority of affected genes in *A. franciscana* were involved in the development processes. In fact, all genes belonging to these classes were affected by the four toxicants. *HAD-like*, *tcp*, *UCP2,* and *CDC48* in *A. franciscana* are involved into molecular mechanisms underlying post-diapause, a common event in diverse taxa from plants to animals [49,52–54]. These data could indicate that PAHs affect some common molecular pathways by changing the normal biological mechanisms, which, in turn, generate death in nauplii and adults. Interestingly, several genes followed by RT-qPCR in the present study were previously found to be functionally interconnected [35,49,52]. In particular, Varó et al. [35] showed that nanoparticles (PS NPs) altered the expression of all genes belonging to the network except for *tcp*, whose relative expression was not significant (Supplementary Figure S1). It is important to underline how the evaluation of the changes in gene expression induced by these toxicants has given the opportunity to uncover some key results that are not easily noticed through observations (i.e., mortality). In fact, the BkF was not able to impact the survival of both nauplii and adults but contemporarily altered the expression level of all tested genes.

It has been widely demonstrated that PAHs are mutagenic, carcinogenic, and teratogenic compounds with long-term effect, especially on human health [55,56]. For these reasons, embryos and larvae of marine invertebrates can be considered as suitable indicators in understanding the toxicological response induced by organic compounds since they are also capable to accumulate high levels of them in their tissues [9,16,19]. Moreover, invertebrate species have a key trophic position in benthic food web, playing the role of intermediate consumers [57]. As a result, the toxicological risk is addressed not only to marine species but also to human beings, which could be exposed to such contamination through the food chain [58,59]. Thus, there is the need to develop early warning systems on consolidated biological models supporting sensitive sub-lethal endpoints. An increase of knowledge on changes of *A. franciscana* genes expression can provide great added values in toxicity assessment. In fact, despite its widespread past use, few studies have been

conducted on the change of gene expression of *A. franciscana* in response to environmental contamination. The identification of molecular pathways in which the targeted genes were involved represents a key step in understanding how crustacean *A. franciscana* protects itself from the stress caused by toxic substances.

In conclusion, genotoxicity may be considered as a possible new biomarker to detect the presence and effects of key environmental pollutants impacting the survival of marine invertebrates. The great simplicity of handling *A. franciscana* in laboratory conditions together with the high sensitiveness of the molecular endpoints could support future applications of this model organism.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w14101594/s1, Figure S1: Gene network; Table S1: Solubility of PAHs [60–62]; Table S2: Tukey's test on nauplii survival data after 24 h and 48 h of PAHs exposure; Table S3: Tukey's test on adults survival data after 24 h and 48 h of PAHs exposure; Table S4: LC50 and 95% confidence intervals calculated after 24 h and 48 h of PAHs exposure; Table S5: Data of gene expression levels in adults; Table S6: Data of gene expression levels in nauplii.

**Author Contributions:** Conceptualization, L.A. and G.L.; methodology, L.A. and S.S.; software, L.A. and V.Z.; validation, L.A., G.L., M.C. and M.G.; formal analysis, L.A. and M.T. (Maria Toscanesi); investigation, L.A. and S.S.; resources, G.L., M.G. and M.C.; data curation, L.A.; writing—original draft preparation, L.A. and G.L.; writing—review and editing, G.L., D.A.L.V., M.T. (Marco Trifuoggi) and M.G.; visualization, D.A.L.V.; supervision, G.L. and M.G.; project administration, L.A. and G.L.; funding acquisition, G.L. and M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Luisa Albarano was supported by a Ph.D. (Ph.D. in Biology, University of Naples Federico II) fellowship co-funded by the Stazione Zoologica Anton Dohrn and University of Naples Federico II.

**Conflicts of Interest:** The authors declare no conflict of interest.
