*2.1.2. Horse-mussel (Modiolus capax)*

**Trial 3**. As a continuation of Trial 2, once the previous experiment (21 days) was completed, a pathogen challenge was performed at CIBNOR with those treated juvenile scallops. About 30 juveniles were randomly selected from each previous treatment (in duplicate) and challenged with a pathogenic strain of *Vibrio alginolyticus* (CAIM57: www.ciad.mx). An initial single dose (1 × 10*<sup>7</sup>* CFU ml−1) was provided as based on the mean lethal dose (LD50) determined by the Probit method and based on the dose–response model described by Finney [19]. To obtain greater clarity in the results, in addition to the groups previously treated PaV-PhA (T1), PaV-SiT (T2), antibiotic AMP (T3) and ET (T4), two new groups that did not receive any previous treatment were included. First, a new group was infected with CAIM57 and defined as positive control (CTRL **+**), and another new group was not infected and defined as negative control (CTRL -). Survival (%) of juveniles was evaluated at 0, 24, 48, 72 and 120 h after infection, and the activity of SOD before infection at 48, 72 and 96 h post-infection was determined. For each treatment, soft tissues (100 mg) from six juveniles were weighted and 500 μl phosphate buffer (pH 7.5) were added. The tissues were homogenised and centrifuged at 9327 × *g* for 10 min at 4°C, recovering the supernatant and storing it at −20°C until further analysis. SOD activity was determined with a commercial kit (SOD Assay Kit #19160, Sigma-Aldrich). Results were expressed as an indirect measure of SOD activity as a per cent of the water-soluble tetrazolium salt formazan complex inhibition. During the challenge, no water changes were made. All juvenile scallops not treated but challenged (CTRL +) died at 72 h while untreated and unchallenged scallops attained the highest survival (95%). The HOM-treated scallops also survived the challenge; T1 scallops attained 85 *versus* 40% survival in those treated with antibiotic (T3). Finally, the SOD activity increased significantly with respect to the other treat-

72 Aquaculture - Plants and Invertebrates

ments and controls in the juveniles of the HOM T1 (81%), 72 h post-infection.

**Trial 4.** To assess the effects of HOM treatments on the microbial communities of the gastrointestinal tract (GIT) of juvenile *A. ventricosus* an experimental design was applied at CIBNOR during nursery management with five homoeopathic treatments and three controls, four replicates each. Juveniles were placed in recirculating upwelling nursery units (40 l) each one with four PVC upwelling cylinders for 21 days; the following HOM treatments and controls were applied: ViP-ViA/a (T1), ViP-ViA/b (T2), AcF-MsS (T3) PhA-SiT (T4), ViT (T5), ET (T6), and NT (T7). At the end of the experiment, eight scallops were randomly taken from each replica and washed, removed fouling organisms of external sides of shell and sprayed with ethanol and dried. Immediately one of the shells was removed and soft tissues dissected to isolate the gastrointestinal tract (GIT) of each scallop, which were fixed in RNAlater® (Thermo Fisher Scientific, Waltham, MA, USA) and preserved at −20°C. The technique of massive DNA sequencing was applied, which is widely used in the study of microbial communities associated with biological systems. The bacterial 16S rDNA was extracted according to Garcia-Bernal *et al*. [3], amplified for sequencing in the Illumina MiSeq Platform (Illumina, San Diego, CA, USA) in a certified Genomic Services Laboratory (www.langebio.cinvestav.mx; Irapuato, Guanajuato, México). Afterward, a bioinformatic and statistical analysis of the generated database was carried out. Initially, significant differences were detected (*p* < 0.05) in growth rate of shell length (μm d−1) (*p* < 0.05), with the best results (140 μm d−1) in the HOM T2. The groups that received T3 and T5 showed a significantly higher survival rate (*p* < 0.05) than the other groups. Moreover, the dominant phylum was *Proteobacteria*, followed by *Actinobacteria*, *Firmicutes*, and *Bacteroidetes*. The prevalent genera in GIT were *Microbacterium*, *Bacillus*, The Horse mussel *Modiolus capax* is a native species from the Gulf of California with aquaculture potential, but scientific knowledge must be generated to achieve a sustainable production [20]. A study was developed at CIBNOR to assess the effect of HOM treatments on gonadal, physiological and transcriptomic maturation in *M. capax.* Microalga and wheat meal were used as food for broodstock mussels. The experiment was designed in triplicate (482 adult mussels; 60 days) and applied to evaluate three HOM treatments: SiT-CaS-HeS (T1), PhA-FeP-ZiP (T2), ViP-ViA-ViT (T3), ethanol as positive control (T4), and NT as negative control (T5). A sequential sampling in time (days) was made (t0 , t30 and t60). The histological analyses showed that mussels treated with T1 and T3 attained (t**30**) the best results (*p* < 0.05) in total weight increase, gonadal maturation of 100% females (**Figure 2** left), highest frequency of vitellogenic and postvitellogenic oocytes and highest oocyte quality according to the total area, theoretical diameter and % of ovoplasm. Also, mussels treated with T3 attained the best reproductive condition of the females (gonadal coverage area, ovarian maturity index, gonadal development index and reproductive potential); however, those receiving T2, increased oogonia proliferation and bioenergetic quality of the oocytes (amount of lipids and neutral carbohydrates). Histochemical and biochemical analyses revealed that HOM treatments (T1, T2 and T3) contributed to increase the overall energy reserves (lipids, carbohydrates and proteins) in the ovary, digestive gland and adductor muscle. López-Carvallo *et al.* [20] using wheat enriched di-algal diet, barely reaching gonad to maturity ~ 25% of the *M. capax* broodstock. Thus, we considered that better results in reproductive condition and oocyte quality of the species were attained with HOM treatments with respect to control.

On the other hand, a *de novo* transcriptome characterisation of the ovarian tissue treated with homoeopathy was performed using RNAseq. *In silico* analysis of differential gene expression revealed that mussels treated with T3 showed the highest number of differentially expressed transcripts (**Figure 2** right), and some of them were related to genes that encoded oestrogen

**Figure 2.** Frequency of ovarian developmental stages (left) determined by histology and number of genes (right) with significant differential expression (*p* < 0.01) in the ovary transcriptome of broodstock mussels (*Modiolus capax*) conditioned with homoeopathy for gonad maturation. Stage S0 undifferentiated; stage SI Previtellogenesis; stage SII Vitellogenesis; stage SIII Posvitellogenic; stage SIV partial spawning; stage SV post spawning; *n* = 482 mussels. Overexpressed genes = black bars; under-expressed genes = Grey bars.

receptors ER and ERR2- like (*p* < 0.01). Estradiol levels have been reported to have a direct influence on gonadic development and oocyte quality in marine bivalves [21] that allowed explaining the high reproductive condition found in the mussels that received T3.

oocytes were observed. To evaluate the quality of the oocytes matured under different treatments, the Sudan Black histochemical technique was used staining the lipid components of the cells. The triglyceride lipid index (TLI) was calculated as described by Rodríguez-Jaramillo *et al*. [22], and significant differences were found in oocytes from different treatments and sampling times. Lipid content (TLI) was significantly higher (*p* < 0.0001) in oysters receiving HOM T2 in

lower TLI values, including Veterinary HOM T3 and T4. A difference between HOM treatments lies in the fact that some of them seem to trigger a constant production of new generations of oocytes, which may be useful to hatchery purposes because it could be associated to the possibility of several partial spawning events that could derive into several larval batches and more

**Trial 2**. A study was conducted at CIBNOR to analyse the transcriptomic response to five HOM treatments in broodstock oyster *C. virginica* in order to understand the response mechanisms that are activated by these nanomedicines, thus, helping to characterise their mode-of-action. About 25 groups of 25 adult oysters (120 g; 10 ± 12 cm), each from a homogeneous population, were conditioned in 80-l plastic boxes provided with continuous microalgal food at COTET for 45 days. An experimental design with seven different treatments, each one with three replicates, was developed to determine the transcriptomic effect of an *Actinomycetes* strain (1 × 106 CFU ml−1 = RL8) and four homoeopathic drug complexes, alone and in combination: RL8 (T1), ViP-ViA (T2) PhA-SiT (T3), ViP-ViA + RL8 (T4), PhA-SiT+RL8 (T5) and two control groups: ethanol as a positive control (T6) and NT negative control (T7). Five oysters of each

oysters were collected and dissected at the end of the experiment (t45). Several tissues, including mantle, gills, gonad, muscle and digestive gland (DG), were separately placed on snap-frozen tubes in RNA later® (Thermo Fisher Scientific, Waltham, MA, U.S.A.) and stored at −80°C. The rest of tissue portion were fixed in Davidson solution for histological examination. DG tissues fixed in RNA later® (Thermo Fisher Scientific, Waltham, MA, U.S.A.) from five individuals of each experimental group were subjected to RNA extraction, for the transcriptomic analysis, using Illumina Hiseq 2000 platform (Illumina, San Diego, CA, U.S.A.). The comparison of the transcriptome data with the KEGG database indicated that the treatments influenced associated metabolic pathways in gonadal development and maturation including "Developmental process involved in reproduction", "Meiotic cell cycle process", "Steroid metabolic process", "Response to oestrogen "and" Regulation of reproductive process ". These pathways were assigned to the KEGG categories of "Developmental process involved in reproduction" which was among the most important category, indicating the significance of signal transduction

systems and endocrine regulation of gonad development and function in *C. virginica*.

The transcriptome of T2 and T3 had 998 and 881 genes with a significantly increased level of expression, respectively, compared to the control (*p* < 0.05). In T4 and T5, there were 748 and 789 genes with an increased level of expression, respectively, compared to a separate control (*p* < 0.05). No genes were significantly differentially expressed under T1 compared to a separate T2, T3, T4, T5 and controls (*p* < 0.05). Genes with increased expression following T2 and T3 were associated with biological processes, including metabolic pathways, ribosomal biogenesis, and transport of nitrogen compounds and anions. Following T4 and T5, genes with increased expression were involved in metabolism processes related with response to

). The rest of the treatments and replicates recorded significantly

Aquacultural Homoeopathy: A Focus on Marine Species http://dx.doi.org/10.5772/intechopen.78030 75

) collected and dissected. The rest of the

a short conditioning time (S1

opportunities for seed production.

homoeopathic and control treatments were initially (t0

This work has provided a *de novo* transcriptome characterisation for *M. capax* for the first time and together with the evaluation of physiological variables, it constitutes the first research about the beneficial effect of homoeopathy in gonadal maturation of the species, which has a clear technological applicability during broodstock gonadic conditioning for seed spawning and production of the species. It will likely be applicable to other bivalve molluscs with commercial interest.
