Formulated Feed for Strombus pugilis (Mollusca, Gastropoda) Allowed Effective Gonad Maturity

Fighting conch Strombus pugilis is one of six conch species distributed throughout the Caribbean Sea on sandy bottoms in inshore waters [1]. Along with the conches S. gigas and S. costatus, S. pugilis is a marine resource of ecological and economical importance [2]. Until recently, S. gigas meat was a popular staple food among human populations in the Caribbean region but is now used mostly as an ingredient in tourist restaurants. S. pugilis is still widely consumed by people in the Caribbean, and its shell is used in jewelry making. This conch species is also now sought after for use in aquariology, with prices ranging from $6 to $30 USD per animal in markets as varied as Southeast Florida, Brazil and the West Indies. Finally, conch species are grazers, and provide the important environmental service of keeping sea grass and algae in balance.


Introduction
Fighting conch Strombus pugilis is one of six conch species distributed throughout the Caribbean Sea on sandy bottoms in inshore waters [1]. Along with the conches S. gigas and S. costatus, S. pugilis is a marine resource of ecological and economical importance [2]. Until recently, S. gigas meat was a popular staple food among human populations in the Caribbean region but is now used mostly as an ingredient in tourist restaurants. S. pugilis is still widely consumed by people in the Caribbean, and its shell is used in jewelry making. This conch species is also now sought after for use in aquariology, with prices ranging from $6 to $30 USD per animal in markets as varied as Southeast Florida, Brazil and the West Indies. Finally, conch species are grazers, and provide the important environmental service of keeping sea grass and algae in balance.
Extraction of wild conch has compromised some populations to the point that protective measures have been implemented. For example, queen conch S. gigas is considered to be commercially threatened in some Caribbean countries and is consequently protected by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES); indeed, in many countries a total ban is in place protecting organisms and egg masses. Culture of conch species is a promising alternative for producing animals for consumption and the aquarium trade, without harvesting wild individuals, thus ensuring the conservation of natural populations. Culture of S. gigas has been successful in terms of hatchery spat production, but still depends on wild egg masses, and spat growth still depends on the use of large areas of natural environment [3]. Dependence on wild egg masses is one of the main hindrances to completely autonomous conch culture. Two of the bottlenecks in intensive conch culture are lack of formulated feed adequate for producing a good growth rate, and attaining gonad maturity at an equal or greater rate than in wild populations. In a natural environment, conches feed on a complex diet of macroalgae, microbenthic organisms and biofilm ingested with sediment [4][5][6]. No data are available on the diet nutrient profile (i.e. energy level, protein and micronutrients) required by S. pugilis for proper gonad maturity [7], however, macroalgae is probably a primary component. The red algae Halymenia floressi is abundant in the waters of the Yucatan Peninsula, mainly on sublittoral rocky substrates [8]. The present study objective was to compare the progress of gonad maturity in adult S. pugilis between wild individuals and cultured individuals fed one of two isoprotein and isoenergetic diets enriched with different percentages of Halymenia floressi.

System and experimental animals
Reproductive performance in adult S. pugilis was evaluated in an experimental aquaculture system at the Center for Research and Advanced Studies (Centro de Investigación y de Estudios Avanzados -CINVESTAV) in Merida, Yucatan, Mexico. The experimental system was composed of twelve 20 L glass aquaria (40 × 20 × 25 cm). Each aquaria contained filtered (25 µm) sea water continuously oxygenated Abstract Fighting conch Strombus pugilis is one of six Strombidae species distributed throughout the Caribbean. It is used as food, as an aquarium organism and its shell are popular in jewelry production. Conch aquaculture has been done traditionally by extracting egg masses from wild adults. This is an issue for several conch species protected by CITES. Intensive conch culture requires good growth rates and gonad maturity under laboratory conditions using formulated feed. An evaluation was done of the effect of inclusion of the red algae Halymenia and Spirulina on gonad maturity in S. pugilis using two experimental diets containing low and high concentrations of these algae (2% and 8% of each). Each diet was fed to six groups of conch kept in 20 L aquaria at 27.5°C. They were fed twice daily at 0.1 g feed/conch for 105 days. Gonad development and digestive gland structure were analyzed with histological techniques. Analysis of gonad development and vitellus granule diameter were analyzed for the two treatments and a control (wild conch). Wild conch females exhibited a reproductive cycle with 100% maturity at the beginning of this study, followed immediately by spawning (in two peaks: 50% and 34%) and initiation of a new oogenesis cycle. Females fed the 8% H. floresii and 8% Spirulina diet exhibited two spawning peaks (75% and 100%) spaced a month apart, and larger yolk granules than those in the control and the 2% H. floresii and 2% Spirulina diet. Proteoglycan granule abundance in the digestive cells did not differ between treatments. H. floressi and Spirulina may function as a feeding stimulant, enhancing feed intake and promoting gonadal maturity in S. pugilis broodstock under laboratory conditions. using an air pump, and kept at a temperature of 27.5°C. Photoperiod was 12 h light/12 h dark throughout the experimental period. Adult individuals were collected in the Ria Celestún Biosphere Reserve (20°52'13.96"N; 90°24'00"W). One hundred eight animals were randomly distributed at a density of nine per aquarium.
Two isoprotein and isocaloric diets were formulated with different proportions of algae: Diet 1 (D1) contained 8% Spirulina and 8% H. floressi; Diet 2 (D2) contained 2% of each algae; and the control (WC) consisted of wild conch. Diets were tested simultaneously, with six replicates per diet. The animals were fed twice daily at a rate of 0.1 g per conch -1 for 105 days. Uneaten feed was removed each day.

Analytical methods
Formulated diets were analyzed for crude protein content (total nitrogen × 6.25 [9]), carbon and calories in triplicate using CN Flash EA (Thermo Quest Ltd. Milan, Italy). Crude lipid concentrations were determined by petroleum ether extraction using a micro Foss Soxtec Avanti 2050 Automatic System. Ash content was obtained by incinerating samples in a muffle at 600°C for 3 h. Nitrogen-free extract with fiber was calculated by difference [100% − (protein % + lipid% + ash %)]. The same procedure was used to measure muscle nitrogen and carbon content in each treatment and the control at 5, 15, 30, 45, 60, 75, 90 and 105 days.

Histological analysis
Histological analyses were done by first cutting the organ mass through the mid-section containing the digestive gland and gonad. Tissue samples were fixed in alcoholic Bouin fluid, and processed using standard histological techniques [10]. After dehydration in an ethanol series and clearing with Histosol Clearing Agent, the sections were embedded in Paraplast wax. Tissue sections (6 µm thick) were stained with a trichrome stain [10], which included Alcian blue (8GX Sigma-Aldrich) at pH 2.5 to differentiate proteoglycans (blue granules). Gonad and digestive gland examination were done using a Leica DM2700 microscope. Images were taken with a Leica MC17 digital camera mounted to the microscope, and corrected for contrast and color (Adobe Photoshop CS6 software).
Effect of the diets on the analyzed individuals was determined based on histological features of gonadal maturity and digestive gland structure. Two microscope slides with five histological sections each were prepared for each individual. Gonad maturity stages were identified considering the amount of connective tissue between the ovigerous tubules, ovigerous tubule diameter, oocyte length and width, and yolk granule diameter. Testicular tissue maturity stage was based on the number of seminiferous tubules and their diameter. Yolk granule diameter was quantified for each oogenesis stage by measuring 100 yolk granules on three sections from three individuals (Toup View software by Toup Tek). Average values and standard deviations were calculated for each trait for each of the two treatments and wild conch (i.e. D1, D2 and WC). Glycoprotein granule frequency [11] was measured by counting the total number of granules observed in three fields of the five sections on each slide under 40x magnification and calculating the mean and standard deviation for each diet.
For each treatment and wild conch, the structure of digestive glands was evaluated using the feed index established by Aranda and Frenkiel [12]. Average values and standard deviations were calculated for each trait for each of the two treatments and the control (i.e. D1, D2 and WC).

Statistical methods
Significant differences (P<0.05) between diet feed index values per date were identified with a non-parametric Tukey test [13]. A one-way ANOVA [13] was applied to identify significant differences (P <0.05) between diets in yolk granule and ovocyte diameter; tubule diameter at various spermatogenesis stages; digestive cells; glycoprotein granules; and feed index per diet.

Diet composition
Calorie content (Kcal kg -1 ) of formulated diets was 3943.5 in D1 and 3824.4 in D2, providing the same amount of energy (Table 1). Tissue wet weight and proximate biochemical composition for the two treatments (D1, D2) and wild conch (WC) were quantified. At the end of the experimental period, average tissue wet weight was 13.7 ± 3.3 for D1, 10.6 ± 1.5 for D2 and 17.9 ± 3.6g for WC. Initial organism protein content was 535 g Kg -1 , whereas final content was 541 g Kg -1 for WC, 627 g Kg -1 for D1 and 610g Kg -1 for and D2 (Table 2).

Control (WC):
In the wild conch, the digestive gland exhibited an array of adenomers ( Figures 1A and 1B). All these secreting structures are connected to small ducts, which join larger ducts attached to the stomach. Two cell types make up the functional glandular structure: digestive and vacuolated. Digestive cells in the wild conch animals had an average length of 24.8 ± 25.7 µm and an average width of 8.3 ± 2.0 µm; they contained large granules up to 6.2 ± 1.7 µm in diameter. These  cells alternated with vacuolated cells, which were always occupied by brown inclusions. These inclusions were sporozoa-like microorganisms belonging to the Apicomplexa group.

Laboratory-reared adults:
In treatments D1 and D2, the digestive gland exhibited digestive cells like those in the wild conch, but with fewer and smaller blue granules ( Figures 1C and 1D). The sporozoalike microorganisms were also present. Blue granule diameters were 6.2 ± 1.7 µm for WC, 5.6 ± 1.5 µm for D1 and 5.3 ± 1.5 µm for D2. Granule frequency was highest in the wild conch (22.7 ± 9.0), which was very similar to that of D2 (21.2 ± 9.8) ( Table 3 and Figure 1).
Average feed index in the wild conch treatment was 5.5 ± 3.3, with a maximum value of 11.2. In D1, the average feed index value was 3.2 ± 1.8 and in D2 it was 3.5 ± 1.6 ( Figure 2). The Kruskal-Wallis test identified significant differences (P<0.005) in median feed index values between treatments on days 45, 60 and 105.

Reproductive stages
Reproductive stages did not vary between the control and the two treatments. The gonadal maturation process could be divided into four maturity stages, as described and characterized below ( Figures 3A-3H).

Females:
(a) Early oogenesis: This stage exhibited initial yolk formation with extensive connective tissue between tubules. The ovigerous ducts measured 94.2 ± 19 µm, smaller oocytes measured 41.9 ± 13.2 µm in length and 19.9 ± 7.9 µm width, and exhibited no yolk granules in the cytoplasm (previtellogenic oocytes). The nucleus had a very large nucleolus, with loose chromatin ( Figure 3A and Table 4).
(b) Mid oogenesis: Connective tissue between the ovigerous tubules occurred in smaller amounts, and the ovigerous ducts were higher (203.7 ± 35.3 µm) than in early oogenesis. Some oocytes had a yolk while others did not. In large oocytes (108.8 ± 28.2 µm), yolk was present and the nucleolus was larger. Yolk granules measured 5.6 ± 1.0 µm ( Figure 3B and Tables 4 and 5).
(c) End oogenesis (maturity): Connective tissue was almost nonexistent and ovigerous tubule walls were very thin. Most eggs were mature and completely occupied the tubule lumen. Eggs measured 213

Males
(a) Early spermatogenesis: Abundant connective tissue structures were observed between the sperm tubules, which measured 82.2 ± 29.3 µm in diameter ( Figure 3E and Table 6).
(b) Mid spermatogenesis: Less connective tissue was present between the tubules, and tubule diameter increased to 151 ± 38.1 µm (Table 6). Numerous primary spermatocytes and some sperm packet formation were present. Large apyrenic sperm were frequent in the tubule lumen ( Figure 3F).    (d) Spawning: Connective tissue between ovigerous tubules was abundant, and tubule diameter decreased. No oocytes were observed in the tubules, and only yolk remnants from eggs expelled during spawning were present ( Figure 3D). The female gonad structures at different oogenesis stages demonstrated that tubule diameter and previtellogenic and vitellogenic oocyte size increased during oogenesis process. Tubule, oocyte and yolk granule diameters in the D1 treatment were larger than D2 and wild conch (Tables 4 and 5). The one-way ANOVA showed mean yolk granule diameter to differ (P<0.0001) between treatments.  maturity. Minimal conjunctive tissue was present between the seminiferous tubules. The tubules contained a higher amount of eupyrenic sperm packages, whereas apyrenic sperm and feeder cells were observed ( Figure 3G and Table 6).

(c) End spermatogenesis: This stage corresponds to testicle
(d) Spent: Very little connective tissue was present between the seminiferous tubules in this stage. No spermatocytes were observed, and spermatogonia were present in smaller numbers ( Figure 3H).
The tubules in the male gonads increased in size during spermatogenesis. Tubule diameter was similar between D1 and WC, but less so when compared to D2 (Table 6).
(e) Rest: The gonads were composed of connective tissue only or connective tissue with tubule remains. No differences were present between male and female structures.

Reproductive cycle (a) Females
Reproductive cycle in the wild conch during the study period began with 100% of females in end oogenesis (maturity), followed by a spawning stage and then immediately thereafter the beginning of a new oogenesis stage ( Figure 4A). Spawning peaks were observed at 15 days (50%) and 75 days (34%). The early oogenesis process in the wild conch was very fast and therefore almost imperceptible in the sampling dates. In the D1 treatment, the reproductive cycle began with a period of maturity (end oogenesis) from days 0 to 45, followed by spawning peaks at day 60 (75%) and day 90 (100%) ( Figure 4B). This one month period between spawning peaks was about half the time required (two months) in the wild conch. The overall reproductive cycle in D2 was similar to D1, although end oogenesis lasted from days 0 to 30, and only minor spawning peaks (25% at day 60; 50% at day 75) were observed ( Figure 4C).

(b) Males
In the wild conch reproductive cycle, 50% of males were in the mid-spermatogenesis stage and 50% in the spent stage ( Figure 4D). The latter exhibited two peaks: one at day 15 (50% to 100%) and a second at day 60 (32%). Two peaks were also present in the spent stage in the D1 treatment, although these were shorter and less intense than in the wild conch: the first was at day 15 (67%) and the second at day 90 (25%) ( Figure 4E). Early spermatogenesis was broad and intense. Two peaks in the spent stage in the D2 treatment were very similar to those in D1, and the resting stage was long, from day 5 to day 75 (30% to 50%) ( Figure 4F).
The Kruskal-Wallis test identified no differences between treatments (D1, D2 and WC) in the median number of females and males in the different reproductive stages: early/mid gametogenesis (Figures 5A and  5E); end gametogenesis ( Figures 5B and 5F); spawning/spent stages ( Figures 5C and 5G); and rest stage -females only ( Figure 5D). The one exception was for the rest stage in males (H=4.69; p<0.011; Figure 5H).

Discussion and Conclusion
The cultivation of fresh and saltwater fish, shellfish and algae is an important and growing source of food production [14]. It has also helped to improve the condition of harvestable stocks of some marine resources. Sustainable aquaculture of Strombidae species requires harvest of broodstock raised using appropriate diets. A study of the natural diet of S. gigas in the Bahamas using I3C to identify components showed the principal food source to be macroalgae, particularly Laurencia spp. and Batophora oerstedi [4]. In another study of the natural diet of S. gigas juveniles and adults on San Pedro Bank, Belize, a total of 22 items were identified in the stomach contents. The most diverse phylum was Rodophyta, followed by Cyanophyta and Protozoa [6].
In mollusks, energy reserves are stored in the muscle [15,16] and the digestive gland [16][17][18]. Proteoglycan granules have been identified inside digestive cells [19]. Based on the amount of proteoglycan granules in the digestive gland structure of S. gigas, a feed index has been proposed to assess its nutritional status [12]. Under moderate stress, energy distribution in invertebrates maintains basal metabolism, but under total stress, the organism suppresses energy storage and reproductive functions [20,21]. The digestive gland was used as an energy storage indicator in the present study. Feed index values were best in the wild conch, followed by conch fed D1 and D2, which did not differ between them.
Formulated diets most commonly use fishmeal to supply proteins, fatty acids, minerals and vitamins, and to make feed palatable [22]. Fatty acid levels affect growth and gametogenesis in mollusks [23][24][25]. The fatty acids profile of H. floresii includes high values of palmitic acid (28.36% to 64.67%), oleic acid (6.62% to 13.92%), linoleic acid (1.03% to 4.65%) and arachidonic acid (4n6, 1.2% to 6.9%) [26]. Addition of feeding stimulants can enhance feed intake. Alginate and carrageenan   function as feeding stimulants in diets for abalone [25]. Carrageenans are compounds extracted from certain red seaweeds (Rhodophyceae), and are widely used as gelling agents and stabilizers in aqueous mixtures and emulsions [27]. Among the algae known to contain carrageenans is H. floresii [8,28]. These compounds are vital to retaining watersoluble nutrients in feed, and maintaining feed particle integrity once in the water. This benefits slow feeders, and is crucial to the success of any formulated diet [29]. Bromophenols identified in the alga Eisenia bicyclis were found to be a chemical defense against herbivore attack [30]. Total bromophenol content varies between algae species, and is known to be lower in H. floresii than in Ulva species [31].
Adding algae to mollusk diets has produced positive results. In a feeding experiment using red abalone H. rufescens, the highest growth was observed with Porphyra columbiana, followed by a mixture of P. columbiana with a formulated diet [32]. It was found that growth of juvenile H. rufescens was highest with the macroalgae diet and the high macroalgae supplementation diet (76.1% macroalgae: 23.9% formulated feed) [33]. Supplementation of a formulated feed (catfish chow) with the alga Agardhiella sp. in juvenile S. gigas resulted in better growth (0.23 mm. d -1 ) than the formulated feed alone (0.11 mm.d -1 ) [5].
Spirulina has also been used to optimize artificial diets for growth performance in mollusks. It is a rich source of protein (70% dry weight), carotenoids (4000 mg/kg), omega-3 and omega-6 polyunsaturated fatty acids, gamma linolenic acid (GLA), sulfolipids, glycolipids, polysaccharides, vitamins (A, E and B) and minerals [34]. The effect of five protein-rich ingredients Spirulina, casein, fishmeal, soya oil and torula yeast were tested in diets for the abalone H. midae. Fish meal and Spirulina diets produced higher increases in length and specific growth rate compared to the diets containing soya oil, torula yeast, and casein alone [35]. When fed formulated diets containing a combination of Spirulina, fish meal and shrimp meal the abalone H. asinine exhibited better growth rates than those fed diets containing only vegetable source protein sources [36]. In a study in which juvenile H. iris were fed one of nine diets containing different protein sources (white and red fishmeal, blood meal, meat and bone meal, casein, soybean concentrate, wheat gluten, maize gluten, and Spirulina ) the Spirulina diet produced growth similar to that of the fishmeal, soybean, and casein protein diets [37].
Feed availability and quality are critical factors in the induction of final maturation and spawning in invertebrate species [38]. In a study of the scallop Aequipecten irradians, nutrient reserves from ingested feed were apparently utilized during gonad growth and gametogenesis [17]. This process involved their transfer from the digestive gland to the gonad for use by the developing gametes for synthesis of various biochemical constituents. Testing of three diets (red seaweed Gracilariopsis bailinae only, formulated feed only, and a combination of G. bailinae and feed) in abalone H. asinina broodstock produced better reproductive performance (i.e. mean instantaneous fecundity and hatching rate percentages) with the combined seaweed/ feed and feed only treatments [23]. In other study, three diets (fresh green seaweed Ulva armoricana only, formulated feed only and a combination of U. armoricana at 20% and feed) were tested in the sea urchin Tripneustes gratilla, observing a higher gonad production with the combined seaweed/feed treatment [39].
The effects of diet on reproductive performance in conch species have only been addressed in two previous studies. The feasibility of a captive breeding program for S. gigas, S. raninus, S. alatus and S. costatus was tested using a prepared feed (36% Mazuri Koi pellets, 16% Ulva sp.), and all four species were observed to produce egg   H G masses [40]. Another study on the effects of two diets (koi chow and catfish chow) on reproductive output in S. alatus found no significant difference between the two treatments in the number of egg masses laid [41].
The relationship between dietary protein content and fecundity has received very little attention in mollusk aquaculture [42]. Fatty acids such as arachidonic acid are known to be precursors of the prostaglandins involved in reproductive processes in mollusks [43,44]. High arachidonic levels may not be required for muscle growth, but are clearly necessary for oogenesis and embryogenesis [45]. Carotenoids are known to modulate reproductive performance and enhance fertility in sea urchin (Loxechinus albus) [46]. In a similar manner, use of Spirulina as a carotenoid source in diets for the fish Pseudotropheus acei was found to increase egg laying rates compared to treatments using only fish meal [47].
The present results indicate that Diet 1, containing the highest H. floresii and Spirulina levels, exhibited the best reproductive performance and the highest energy reserves in the digestive gland. These results suggest that the carrageenan present in H. floressi may function as a feeding stimulant, and the arachidonic acid may promote gonadal maturity. In addition, the carotenoids in the Spirulina very probably promoted gonadal ripening in the tested S. pugilis broodstock under laboratory conditions.