Ovigerous females of H. inermis Leach, 1815, were collected in Lacco Ameno d’Ischia (Gulf of Naples, Italy) at depths of 3–15 m. A P. oceanica meadow extends continuously from 1 to about 33 m depth in this area (Mazzella and Buia 1989), and previous studies on the population dynamics and the sex reversal of H. inermis were accomplished on individuals collected in the same area (Zupo 1994). The collections were performed at noon, by towing a plankton net (400 mm diameter; mesh size 100 µm) horizontally across a Posidonia meadow, from a boat at a speed of about 3.7 km h−1. Each collection consists of a 3–5 min tow, to avoid clogging the net with seagrass leaves that could hurt the collected individuals. The first sorting was performed visually, and shrimps were stored in a container (300 × 400 mm) filled with 30 mm of seawater (38 psu). In the laboratory, after species identification, each H. inermis female was individually transferred in a conical flask filled with 1.5 L of filtered seawater, aerated by means of an air pump connected to a plastic tube. Nauplii of Artemia cf. franciscana (Coppens® premium cysts, 3 individuals/mL) were added to the seawater (38 psu) and the conical flasks were kept in a thermostatic chamber (18 °C) until the release of H. inermis larvae. Culture vessels were checked every morning for the presence of larvae, indicated also by the occurrence of adult female exuviae (Zupo and Messina 2007), since the release of larvae corresponds with exuviation of ovigerous females. When larvae were found, they were sieved through a 60 µm filter, counted and transferred to their rearing vessels.
Collected larvae were cultured under laboratory conditions until they reached the juvenile stage, which occurred within 18–22 days under our experimental conditions (Zupo 2000). These involve culturing larvae in 1 L conical flasks, each containing 800 mL of culture medium. The larval phase consists of eight stages and has variable development time depending on culture conditions (Lebour 1931; Le Roux 1963; Zupo and Buttino 2001). The size of the zoea ranges from 1 to 1.6 mm (first zoel stage) to 3.0–4.0 mm (last zoel stage, Zupo and Buttino 2001). Larvae were fed ad libitum on nauplii of Artemia franciscana (four nauplii of freshly hatched Coppens® premium cysts per mL) along with the rotiferid Brachionus plicatilis (4 ind/mL) for the first 7 days. From the 8th day onwards, enriched A. franciscana nauplii were used instead of freshly hatched nauplii, at the same density. For enrichment, 24 h old nauplii were kept in enrichment media (AlgaMac-2000, Aquafauna Bio-Marine, Inc) for at least 12 h. After 24 h of enrichment, the nauplii were harvested. Enriched nauplii were added at the same rate during the whole larval growth, whilst the number of Brachionus was reduced to 4, 3 and 2 ind/mL on the 9th, 10th and 11th day, respectively. Brachionus administration was stopped from the 12th day onwards.
Rearing of juveniles
Larvae that changed into juveniles were transferred into deep-walled Petri dishes of 500 mL at a density of 1 juvenile/16 mL of filtered and sterilised seawater (25 juveniles in 400 mL of seawater 38 psu) and were cultured until they reached sexual maturation, under the same conditions as mentioned above. A maximum of 5 days was needed to complete the stocking of juvenile vessels with 25 individuals each.
Juveniles were fed on A. franciscana at a density of three freshly hatched nauplii per mL, 2 A. franciscana /mL and 1 A. franciscana /mL for the first 3 days, respectively, along with artificial food (below). Administration of A. franciscana was ended after the 3rd day. To obtain females derived from the diatom-induced sex change described above, two groups of juveniles were allotted, based on the artificial food type given. The food types given were: (1) composed dry food (CTRL−) and (2) composed dry food added with the benthic diatom Cocconeis scutellum parva (CTRL+). The food CTRL− was composed of three dry ingredients mixed in equal quantities (Zupo et al. 2007), i.e., dry A. franciscana, dry Spirulina sp. and “Microperle” (micro-encapsulated supplementary feed for marine invertebrates); they were all provided by SHG (Ovada, Italy). In addition to the ingredients of CTRL−, CTRL+ contained 50% in weight of the diatom Cocconeis scutellum parva, known for causing regression of androgenic glands in young juveniles (Zupo and Messina 2007; Zupo et al. 2014). This diatom was used to trigger the production of young females.
The food CTRL− was administered to produce a higher abundance of males, according to the physiology of this species (Zupo 2000) while CTRL+ treated postlarvae supposed to produce a larger abundance of females. Food was offered at the rate of 3.50 mg/day per 400 mL of seawater (38 psu) for the first 15 days. The quantity of food given was increased up to 7.0 mg/day in the next days, to guarantee it was provided ad libitum. The survivors were counted daily and then transferred into new culture solution in clean vessels, using a Pasteur pipette. Exuviae were collected regularly and observed using a microscope (Leica DMLB) for any sign of maturation, i.e., presence/absence of A.M. and A.I.. Juveniles were fixed in 70% alcohol when they reached the size of 7–8 mm and were assumed to be sexually mature. Two individuals were sampled from each (CTRL− and CTRL+) treated juvenile culture vessel at 5-days interval (0, 5, 10, 15, 20, 25 days, respectively) and fixed in 70% alcohol to investigate ontogenetic changes of gonopores.
Analysis of sex
In total, 50 individuals (10 mature, 40 immature) were analysed for the presence/absence of A.M. and their gonopores were observed as well. Twelve additional individuals were taken for analysis of ontogenetic changes of gonopores. Their total body length was measured under a complanar apochromatic macroscope (Leica Z16-APO) using a millimetric paper. Individuals fixed in 70% alcohol were stained in 2% methylene blue solution and immediately observed for the location and shape of gonopores. Males were characterised by the presence of gonopores at the far proximal end of the coxa of the fifth pair of pereiopods, whilst the gonopores of females were located at the far proximal end of the third pair of pereiopods (Bauer 2004). In addition, mature individuals were also observed and their second pleopods were collected to detect the absence/presence of A.M. using bright-field light microscopy (Leica DMLB). Presence of an A.M. on the pleopod II corresponds to males, while its absence characterises females.
The lengths of exopodite, endopodite, basipodite and A.I. of each of the 40 immature individuals as well as their body size were measured. These individuals belong to the same age group and they have similar sizes. All measurements were made on images of the shrimps obtained by a Leica Z16-APO macroscope equipped with a computerised system of image analysis.
The ratio number of females/total number of mature individuals (F/Mat) was calculated for each group. The ration number (F/Mat) between CRTL+ and CTRL− were compared by means of the z test on proportions. The ratio females/total number of mature individuals was chosen to make the experiment independent from the number of immature. Large values for F/mat ratios indicate apoptogenic activity.
Regression curves were obtained for each couple of biometric parameters and, in particular, the lengths of A.I., basipodites, endopodites and exopodites were compared to the total lengths of males and females. Slopes and correlation coefficients were computed to test the relationships between the size of shrimps and the lengths of basipodite, exopodite, endopodite and A.I.. Morphometric data were separately computed for males and females, to allow a comparison of the two sexes. We compared the regression slopes of males and females to test the null hypothesis (Ho):
where Bf is the slope for females, and Bm is the slope for males.
To perform this analysis, we used t test and analysis of covariance (ANCOVA). It compares the independent variable X (here, total length) and the dependent variable Y (basipodite, exopodite, endopodite and A.I.) between two groups. The purpose of ANCOVA, in our case, is to compare two or more linear regression lines. It is a way of comparing the Y variable among groups, while statistically controlling for variation in Y influenced by the values assumed by the X variable. Two null hypotheses are tested in ANCOVA. The first is that the slopes of the regression lines are all the same. If this hypothesis is not rejected, the second null hypothesis is tested, that the Y-intercepts of the regression lines are all the same. We performed a power analysis on the sample size needed for ANCOVA using the method proposed by Borm et al. (2007).