Field sampling results of Weishan Lake
From 2004 to 2016 the snail population was continually monitored. Each year, 1/10 of the volume of the soil in breeding cages was sampled, and live snails were counted to estimate the total population. Fluctuating snail populations, ranging from 1590 to 14550, were observed (Fig. 3A). In the first year, the population rapidly increased more than 3 times the initial population (from 2000 to 8230) from 2005 to 2006, and then it slowly dropped down to 1580 from 2007 to 2009. Between 2010 and 2013, the population dramatically increased to more than 7 times the initial population to 14550; however, a sharp drop was again observed again from 2014 to 2016, and the population decreased to 1590 in 2016.
Snail eggs were also collected and counted to estimate the productivity of the snails.The number of eggs laid per female snail was 27.73 in 2005, 6.29 in 2006, 1.6 in 2015 and 0.7 in 2016 (Fig. 3B).
Shell morphology of O. hupensis collected from Yangtze River and Weishan Lake
In 2004, 2005, 2006, 2015 and 2016 snails were collected both from Yangtze River and Weishan Lake to compare their morphological features. The shell length (L), shell width (W), aperture length (AL), aperture width (AW) and longitudinal ridge number (LRN) of the collected snails were measured.
The average shell length of snails in the Yangtze River and Weishan Lake ranged from 5.87 ± 1.27 mm to 7.33 ± 0.63 mm (Fig. 4A). The average shell width of snails in the Yangtze River ranged from 2.50 ± 0.48 mm to 3.43 ± 0.27 mm (Fig. 4B). The average aperture length of snails in the Yangtze River and Weishan Lake ranged from 1.58 ± 0.21 mm to 2.52 ± 0.22 mm (Fig. 4C). The average aperture width of snails in the Yangtze River and Weishan Lake ranged from 1.57 ± 0.34 mm to 2.33 ± 0.33 mm (Fig. 4D). The average longitudinal ridge number of snails in Yangtze River and Weishan Lake ranged from 12.41 ± 2.61 to 13.75 ± 1.24 (Fig. 4E).
No significant difference in any of the 5 features of snail morphology was observed between different times and locations, although decreases in shell length (L), shell width (W) and aperture width (AW) were observed after 2005 in the snails of Weishan Lake when compared to the ones from the Yangtze River.
Gonad ultrastructure of O. hupensis from Yangtze River and Weishan Lake
In sections of male gonads of snails from Yangtze River, sperm cells could be divided into head, neck, middle piece and principal piece of tail (Fig. 5A). In the head region, the acrosome is separated from helical chromatin and composed of electron opaque material, similar to the nucleus. The nucleus is long and helical, while the plasma membrane surrounding the nucleus is rough, with space between it and the nuclear membrane. The neck region is defined as the area connecting the head and middle piece, composed of nuclear fossa (NF) and nuclear invagination (NI). The NF is a lumen connecting to middle piece, and NI is formed by chromatin projected on the neck (Fig. 5A). Observation of a cross-section of NF demonstrates electron lucent material in the center which forms the axoneme (Fig. 5B). A cross-section shows that the middle piece (MP) of the tail consists of the axoneme coarse fiber (ACF) complex in the center, surrounded by mitochondrial sheath (MS). The axoneme is composed of nine peripheral microtubular doublets and a central doublet, 9 + 2 (Fig. 5C). The mitochondrial sheath is composed of 9 regularly arranged mitochondria bunches; each bunch contains 5 mitochondria (Fig. 5C). The mitochondrial sheath is absent in the principal piece (PP) of the tail. A cross-section shows the 9 + 2 structure surrounded by the fibrous sheath (FS), which forms a ninogan-shaped lumen. Outermost is the loosely surrounding plasma membrane (PM). ACF is surrounded by electron lucent material (Fig. 5D).
In sections of male gonads of snails from Weishan Lake, the main features are the same as those in the snails from the Yangtze River. However, some changes in detailed features were observed. In the head region, the nucleus had lower nuclear electron density than in those from the Yangtze River; in the neck region, axoneme is missing from the NF (Fig. 6B), and in the middle piece and principle piece, the 9 + 2 ACF structure is absent (Fig. 6C and D).
For female gonads of snails from the Yangtze River, oocytes are observed in the primary follicles of O. hupensis. Abundant endoplasmic reticulum (ER) and lipidosomes (L) with electron-opaque interiors are distributed in the cytoplasm. Pale vesicles (PV) are visible in extracellular areas. The nuclear membrane (NM) is irregular in shape and attached to a large amount of ER. The nucleolus is round in shape with high electron density; abundant chromatin (Cr) and a small amount of heterochromatin (HCr) are distributed inside the nucleus (Fig. 7).
For oocytes of snails from Weishan Lake, ER, PV and L are much reduced in the cytoplasm when comparied to those from the Yangtze River. The nucleus is relatively larger in size, the NM is smooth and round in shape, without visible ER attached, and the nucleolus and Cr are not visible in the nucleus (Fig. 8).