*2.1.1. Catarina scallop (Argopecten ventricosus)*

The production of mollusc bivalves around the world is still challenged every year by the propagation and emergence of new diseases. Scallops are especially susceptible to epizootic pathogenic bacteria in the hatchery, mainly those related to *Vibrio* spp. and *Aeromonas* spp. [14]. Four experiments described below were performed with *A. ventricosus*.

**Trial 2**. To compare between homoeopathy and antibiotic efficiency in *A. ventricosus* juveniles, an experimental design was performed by triplicate (6 treatments and 18 replicates, 120 seeds each replicate) at CIBNOR for 21 days. Juveniles (4.14 ± 0.06 mm; 13.33 ± 0.03) received PaV-Pha (T1), PaV-Sit (T2) as HOM treatments, ampicillin AMP (T3) as antibiotic treatment, and ethanol ET (T4) and NT (T5) as control treatment. Liquid treatments were applied (100 μl l

**Table 1.** Haemocyte count and growth rate in *Argopecten ventricosus* spat treated with homoeopathic medicines for 21 days.

Numbers show mean ± standard error. Identical lowercase letters denote lack of significant differences between

**Haemocyte count Growth rate (μm day−1)**

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

for homoeopathy and 10 ppm for antibiotic) after seawater exchange every 48 h and before feeding scallops. Evaluations were performed for growth in height (mm), total wet weight of the shell (mg), biochemical flesh composition (mg g−1) and SOD activity. Thirty juveniles were taken at random from each replica at 7, 14 and 21 days to measure size and total wet weight

Juveniles grew significantly more in size with HOM T1 (6.22 ± 0.11 mm; 0.05 mm d−1) and T2 (6.99 ± 0.09 mm; 0.08 mm d−1) compared with NM (T5) (5 ± 0.02 mm; 0.02 mm d−1). A significant increase in total wet weight was recorded with HOM T2 (41.16 ± 0.35mg; 1.3 mg d−1) compared to NT group T5 (24.33 ± 0.10 mg; 0.5 mg d−1). Survival was 100% in all treatments and their

**Figure 1.** Growth in size (μm day−1) (left) and weight (mg day−1) (right) of juvenile Catarina scallop *Argopecten ventricosus*

and to determine absolute growth in height and weight (mm, mg) for each treatment.

replicates (**Figure 1**).

treated with homoeopathic medicines.

treatments at *p* < 0.05.

Initial 19 ± 0.38 **<sup>e</sup>**

T1 **44 ± 0.54 <sup>a</sup> 117 ± 1.32 <sup>a</sup>** T2 41 ± 0.57 **<sup>b</sup> 108 ± 0.97 <sup>a</sup>** T3 34 ± 0.75 **<sup>c</sup>** 53 ± 6.31 **<sup>c</sup>** T4 24 ± 0.40 **<sup>d</sup>** 94 ± 1.38 **<sup>b</sup>** T5 31 ± 0.32 **<sup>c</sup>** 36 ± 5.83 **<sup>d</sup>** T6 21 ± 0.87 **<sup>e</sup>** 14 ± 0.40 **<sup>e</sup>** T7 22 ± 1.13 **de** 34 ± 0.06 **<sup>d</sup>** T8 16 ± 0.60 **<sup>f</sup>** 20 ± 0.14 **<sup>e</sup>**

−1

71

**Trial 1**. To assess the effects of homoeopathic treatments (HOM treatments) in the immune system of Catarina scallop *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. Continuous aeration and a microalgal food mix 1:1 (*Isochrysis galbana* - *Chetoceros calcitrans*; 150,000 cel ml−1) were provided. The following HOM treatments and controls were applied: ViP (T1), ViA (T2), PhA Metasilicate (T3), PhA-SiT (T4), ViT (T5), dynamised ET (T6), diluted ET (T7) and NT nor ethanol added (T8).

At the end of the assay, mainly nosodes (ViP, ViA) formulated from pathogenic bacterial compounds, stimulated a significant (*p* < 0.05) growth rate and increased haemocyte count which were counted using three images at 100X from scallops tissue were processed by Image Pro Plus 6.0 to count the number of haemocytes in a tissue area (0.21 mm2 ). Haemocytes count was 1–3 times higher than controls (**Table 1**). These results suggested immune system enhancement by the action of HOM treatments because it is known they stimulate enzymatic activity of superoxide dismutase (SOD) and catalase (CAT) related with antioxidant responses of the organisms during oxidative stress [15]. The proliferation of haemocytes is related to activation of immune response because they are the primary cells responsible to protect organisms against infections [16]. Antioxidant activity increases have also been linked to improving survival when organisms are challenged against stressful conditions [16–18]. These findings make it clear that HOM treatments can activate a quantifiable biological response on the immune and antioxidant system in juvenile scallop *A. ventricosus*.


Similia® (PaV); Cyme-Heel, Gal-Heel, Hepa-Heel, Mucs-Heel and Chol-Heel of Rubiopharma*®* (INM); Endecto (END) and Infecçoes (INF) of Arenales Homoeopathy®; *Phosphoric acid* (PhA), *Phosphoric acid* (AcF), *Silicea terra* (SiT), *Sodium methasilicate* (MsS), Scorpion toxin Vidatox® (ViT); *Calcium sulphuricum* (CaS); *Hepar sulphuris* (HeS), *Ferrum phosphoricum* (FeP); *Zincum phosphoricum* (ZiP); *Magnesium phosphoricum* (MaP), *Mercurius solubilis* (MeS). Also, other nosode-type homoeopathic medicines from *Vibrio* compounds (ViP, ViA) were applied. These and other nosode type HOM-products have been designed by CIBNOR that is processing the respective trademark and industrial property titles (Office for industrial protection and technology transfer; OTT-CEPAT/CIBNOR; www.cibor.gob.mx). Ethanol (ET) and no-HOM nor ethanol (NT) were used as control treatments. Homoeopathic medicines (HOM) were

sprinkled on balanced food or inert sugar pills or added directly to culture seawater.

The production of mollusc bivalves around the world is still challenged every year by the propagation and emergence of new diseases. Scallops are especially susceptible to epizootic pathogenic bacteria in the hatchery, mainly those related to *Vibrio* spp. and *Aeromonas* spp.

**Trial 1**. To assess the effects of homoeopathic treatments (HOM treatments) in the immune system of Catarina scallop *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. Continuous aeration and a microalgal food mix 1:1 (*Isochrysis galbana* - *Chetoceros calcitrans*; 150,000 cel ml−1) were provided. The following HOM treatments and controls were applied: ViP (T1), ViA (T2), PhA Metasilicate (T3), PhA-SiT (T4), ViT (T5), dynamised ET (T6), diluted ET (T7) and NT nor ethanol added (T8). At the end of the assay, mainly nosodes (ViP, ViA) formulated from pathogenic bacterial compounds, stimulated a significant (*p* < 0.05) growth rate and increased haemocyte count which were counted using three images at 100X from scallops tissue were processed by Image Pro

1–3 times higher than controls (**Table 1**). These results suggested immune system enhancement by the action of HOM treatments because it is known they stimulate enzymatic activity of superoxide dismutase (SOD) and catalase (CAT) related with antioxidant responses of the organisms during oxidative stress [15]. The proliferation of haemocytes is related to activation of immune response because they are the primary cells responsible to protect organisms against infections [16]. Antioxidant activity increases have also been linked to improving survival when organisms are challenged against stressful conditions [16–18]. These findings make it clear that HOM treatments can activate a quantifiable biological response on the

). Haemocytes count was

[14]. Four experiments described below were performed with *A. ventricosus*.

Plus 6.0 to count the number of haemocytes in a tissue area (0.21 mm2

immune and antioxidant system in juvenile scallop *A. ventricosus*.

**2. Effects of homoeopathy in marine species**

*2.1.1. Catarina scallop (Argopecten ventricosus)*

**2.1. Mollusc**

70 Aquaculture - Plants and Invertebrates

Numbers show mean ± standard error. Identical lowercase letters denote lack of significant differences between treatments at *p* < 0.05.

**Table 1.** Haemocyte count and growth rate in *Argopecten ventricosus* spat treated with homoeopathic medicines for 21 days.

**Trial 2**. To compare between homoeopathy and antibiotic efficiency in *A. ventricosus* juveniles, an experimental design was performed by triplicate (6 treatments and 18 replicates, 120 seeds each replicate) at CIBNOR for 21 days. Juveniles (4.14 ± 0.06 mm; 13.33 ± 0.03) received PaV-Pha (T1), PaV-Sit (T2) as HOM treatments, ampicillin AMP (T3) as antibiotic treatment, and ethanol ET (T4) and NT (T5) as control treatment. Liquid treatments were applied (100 μl l −1 for homoeopathy and 10 ppm for antibiotic) after seawater exchange every 48 h and before feeding scallops. Evaluations were performed for growth in height (mm), total wet weight of the shell (mg), biochemical flesh composition (mg g−1) and SOD activity. Thirty juveniles were taken at random from each replica at 7, 14 and 21 days to measure size and total wet weight and to determine absolute growth in height and weight (mm, mg) for each treatment.

Juveniles grew significantly more in size with HOM T1 (6.22 ± 0.11 mm; 0.05 mm d−1) and T2 (6.99 ± 0.09 mm; 0.08 mm d−1) compared with NM (T5) (5 ± 0.02 mm; 0.02 mm d−1). A significant increase in total wet weight was recorded with HOM T2 (41.16 ± 0.35mg; 1.3 mg d−1) compared to NT group T5 (24.33 ± 0.10 mg; 0.5 mg d−1). Survival was 100% in all treatments and their replicates (**Figure 1**).

**Figure 1.** Growth in size (μm day−1) (left) and weight (mg day−1) (right) of juvenile Catarina scallop *Argopecten ventricosus* treated with homoeopathic medicines.

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

*Symbiobacterium* and *Burkholderia*. In general terms, phyla *Proteobacteria* and *Actinobacteria* play an essential role in immunity and nutrition of invertebrates, and both were dominant in

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

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

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. Over-

, t30 and t60). The histological analyses showed

Aquacultural Homoeopathy: A Focus on Marine Species http://dx.doi.org/10.5772/intechopen.78030 73

juvenile scallop *A. ventricosus* in all groups treated with HOM.

species were attained with HOM treatments with respect to control.

expressed genes = black bars; under-expressed genes = Grey bars.

*2.1.2. Horse-mussel (Modiolus capax)*

sequential sampling in time (days) was made (t0

**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*, *Symbiobacterium* and *Burkholderia*. In general terms, phyla *Proteobacteria* and *Actinobacteria* play an essential role in immunity and nutrition of invertebrates, and both were dominant in juvenile scallop *A. ventricosus* in all groups treated with HOM.
