Shrinking pupal cocoons of Rhyacophila lezeyi were often found during summer in Shibukuro Stream, a highly acidic mountain stream in northern Japan (pH = 2.82 on average). We performed both field surveys and laboratory rearing experiments to clarify the mechanisms of R. lezeyi cocoon shrinkage. The R. lezeyi cocoon shrinkage proportion increased in years with high stream water temperatures and was related to water temperatures before and after pupation at the study site. Approximately 90% of the prepupae and pupae inside the shrinking cocoons died during the rearing experiment, implying that cocoon shrinkage caused by high water temperature strongly influenced R. lezeyi pupal survival. Laboratory experiments showed that R. lezeyi’s pupal cocoon membranes were semi-permeable and that the cocoon fluids were always hyperosmotic, indicating that water molecules can continuously enter the cocoon fluids from the stream water until the turgor of the cocoon wall is reached. However, the shrinking cocoons showed lower fluid volume and higher osmolarity than the normal turgescent cocoons. The reduction of osmotic gradient across the membrane during decreased stream flow due to less precipitation and/or the damage to the cocoon membrane and pupal body from high and fluctuating water temperatures and low pH are possible mechanisms for R. lezeyi pupal cocoon shrinkage.

cocoon shrinkage, membrane semi-permeability, osmolarity, pH, water temperature

The eastern and northern parts of Japan have many acidic mountain streams because of the high volcanic activity in the region (Goto 1982). Rhyacophila lezeyi, Navás, 1933, often inhabits such extreme environments (Tsutsumi and Yokoyama 2020). One of these is Shibukuro Stream, where stream water is a mixture of a nearby hot-acidic spring water with high temperature (97°C) and low pH (pH 1.1) and streams diluting those conditions. In the middle reaches of Shibukuro Stream, R. lezeyi larvae and pupae occur in high densities, predominating the faunal communities’ biomass (Aoya 2018). Rhyacophila lezeyi is distributed in the south of Honshu Island and is one of the most common species of the genus Rhyacophila in Japan, except the northern region (Hattori 2005). The larvae and pupae of R. lezeyi are confined to cool stenothermic running waters. The larvae feed mainly on nemourid stonefly nymphs in Shibukuro Stream, often exerting strong predation pressure on the lower trophic levels (Aoya 2018). R. lezeyi has an asynchronous univoltine life cycle and its larvae pupate and emerge continuously from May to October, peaking in June, in the stream (Aoya and Tanida 2023). The final instar larva of R. lezeyi normally constructs a turgescent pupal cocoon (Fig. 1A) inside the pupal case and the pharate adult breaks out from the cocoon for emergence (Aoya 2020; Aoya 2022). However, shrinking cocoons (Fig. 1B) are frequently found in Shibukuro Stream in summer and the cocoon shrinkage mechanisms have yet to be elucidated.

Figure 1. 

Rhyacophila lezeyi pupae inside cocoons collected in Shibukuro Stream. 1A: Normal (turgescent) cocoon (29 Aug 2018). 1B: Shrinking cocoon (19 Aug 2019). The scale is 5 mm.

We performed both field surveys and laboratory experiments to identify plausible mechanisms that cause the R. lezeyi summer cocoon shrinkage in Shibukuro Stream. We focused specifically on the osmotic gradient of the R. lezeyi pupal cocoon membrane between stream water and cocoon fluids because osmosis can supply cocoon fluids with oxygenated stream water across a semi-permeable cocoon membrane (Wichard 1991; Wichard et al. 1997), as has been reported for Rhyacophila fasciata Hagen, 1859 (Wichard et al. 1993). We hypothesise that high solute concentrations in stream water during summer reduce the osmotic gradient across the cocoon membrane, thereby causing the cocoon shrinkage and prepupal and pupal mortality of R. lezeyi.

In the highly acidic Shibukuro Stream, the increase of Rhyacophila lezeyi shrinking cocoons was associated with higher and fluctuating water temperatures and a lower pH (Table 1). Such changes in the physicochemical environment are probably related to increased hot spring water contributions resulting from the decreased stream flow due to less precipitation. In fact, the mean summer precipitation (July-September) was the lowest and the third lowest for 2019 and 2021, respectively, during the 1979–2022 period, while the precipitation level was the 13^th highest for 2018 during that period. Larvae and pupae exposed to higher water temperatures more frequently experienced collapsed cocoons (Fig. 2). Cocoon shrinkage also was correlated with severe mortality in R. lezeyi (Table 2). Therefore, increased water temperature may influence R. lezeyi cocoon morphology and pupa physiology, which may eventually suppress their development.

One possible cocoon shrinkage mechanism was at least partially due to the change in the osmotic gradient over the cocoon membrane (Fig. 5). Rhyacophila lezeyi’s pupal cocoon membrane was found to be semi-permeable (Fig. 3), as was R. fasciata’s in Fulda River in Germany (Rudolph and Wichard 1993; Wichard et al. 1993). As the observed cocoon fluids’ osmolarity was always higher than that of stream water, water molecules were generally allowed to permeate from stream water into the cocoons. Wichard et al. (1993) suggested that dissolved organic matter, in addition to inorganic components in the cocoon fluids of R. fasciata, had an osmotic effect. Even if we consider the osmolarity of only the organic components present in the cocoon fluids of R. lezeyi, the water influx direction does not change. In fact, for normal turgescent cocoons, cocoon fluids’ osmolarity and cocoon volume decreased and increased with pupal development, respectively (Fig. 4), implying that the cocoon fluids were diluted by stream water permeation. However, the shrinking cocoons showed lower fluid volume and higher hyperosmotic conditions than did the normal cocoons (Fig. 4). Thus, water removal from the cocoons via the membranes due to events of higher osmolarity (> 40–90 mOsm l^-1) of the Shibukuro Stream water might be mechanisms of pupal cocoon shrinkage and high osmolarity.

Figure 5. 

Plausible pathways for water and solutes in cocoon fluids, body fluids and stream water. 5A: Under normal conditions, stream water is forced into the cocoon fluids due to the large osmotic gradient across the semi-permeable membrane. As the prepupae moult, exuvial fluids rich in ions are released and the cocoon fluids are gradually diluted. The pupae might excrete excess water from their bodies into the cocoon fluids. 5B: In high-temperature water, the rate of water influx into the cocoon fluids may decrease because the reduced flow is associated with less precipitation to enrich the stream water’s solute concentration. High and fluctuating water temperatures and low pH might also influence the physical and physiological properties of the cocoon and pupa, resulting in cocoon shrinkage.

In fact, higher ion concentrations, as revealed by higher conductivity values (EC range = 2,317–2,570 μS cm^-1), were often observed during the summer months compared with the day of osmotic pressure measurement (20 July 2021, EC = 1,971 μS cm^-1). In addition, ion concentrations of stream water are likely further enriched during periods of decreased stream flow with less precipitation. Therefore, we argue that the removal of cocoon fluids via osmoregulation is responsible for the shrinkage of the pupal cocoon under the influence of the temporary-occurring hyper-osmotic water medium in the acidic stream.

Other plausible factors for the cocoon shrinkage are high water temperature and low pH, which may cause physical and physiological stress. The increase in water temperature results in the decrease in dissolved oxygen concentration, which might affect pupal respiration (Wiggins and Wichard 1989; Wichard et al. 2002). Strong acidic conditions may weaken the cocoon threads that support morphological cocoon turgescence. The inhibition of water excretion from pupal body fluids might also be possible due to the physiological stress of high and fluctuating water temperature and a low pH (Fig. 5).

The present study revealed that Rhyacophila lezeyi cocoon shrinkage observed in the highly acidic Shibukuro Stream was associated with relatively high water temperatures and a low pH, both of which were resulted from the decreased stream flow due to low precipitation during summer. Moreover, the cocoon shrinkage caused severe mortality in R. lezeyi, suggesting that temporal and seasonal rainfall patterns may influence the persistence of R. lezeyi population via the alteration of flow regime and physicochemical environments in the stream. The present study also showed that the summer cocoon shrinkage of R. lezeyi may be caused by reduced osmotic gradient across the membrane during decreased flow and/or physical and physiological stresses due to high water temperature and low pH in that period. Further long-term studies are necessary to identify the detailed mechanisms of pupal cocoon shrinkage and its resultant effects on the R. lezeyi population dynamics.

The authors thank Prof. Emer. J. C. Morse of Clemson University, USA, for helpful information; Prof. W. Wichard, University of Köln, Germany, for his kind and detailed advice on the physiology of rhyacophilid pupal cocoons; and the staff of the Natural Science Research Office, Inc., Daisen City, Akita Prefecture, Japan, for their support of the field survey. This work was partially supported by MEXT KAKENHI Grant Numbers 19K22449 and 21H02561.