Research Article |
Corresponding author: Kokichi Aoya ( aoyak@silk.plala.or.jp ) Academic editor: Simon Vitecek
© 2023 Kokichi Aoya, Atsushi Hayakawa, Tomoya Iwata, Kazumi Tanida.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Aoya K, Hayakawa A, Iwata T, Tanida K (2023) Shrinking pupal cocoons of Rhyacophila lezeyi (Trichoptera, Rhyacophilidae) in a highly acidic stream during the summer season. Contributions to Entomology 73(2): 131-136. https://doi.org/10.3897/contrib.entomol.73.e107479
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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 (
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 (
We conducted a field survey at Shibukuro Stream, a tributary of Tamagawa River in Omonogawa River System, northern Japan. The study site (39.9388°N, 140.7059°E, elevation: 480 m, base-flow discharge = 1.4 m3 s-1) is located 4.5 km downstream from the hot spring water inputs from Tamagawa hot spring area (
We handpicked Rhyacophila lezeyi’s pupal cases containing the closed prepupal and pupal cocoons at the study site from July to September 2018, 2019 and 2021. We transported the collected samples to the laboratory alive, carefully removed sand and gravel around the cocoons and picked the cocoons within three to six hours of sampling.
In the laboratory, brownish cocoons were used for further analyses; no immature cocoons with thin membranes were examined in the present study. We checked each R. lezeyi’s pupal cocoon for pupal life (alive or dead) and morphological conditions (turgescent or shrinking). We reared all the living individuals in a dark refrigerator, in which the temperature was maintained at 1–6°C below the Shibukuro Stream water temperature. We also recorded the metamorphosis dates (pupation, breaking out from the cocoon and emergence) of each individual during rearing and prepupal and pupal mortality after rearing.
We examined the R. lezeyi cocoon membranes’ semi-permeability. First, we placed living immature pupae with undamaged turgescent cocoons in stream water and kept them overnight in a refrigerator at about 16°C. Second, we used distilled water (DW) to perform stepwise dilution in about one hour to finally transfer the pupae into the DW. Third, we transferred the cocoons to a saturated sodium chloride (NaCl) solution for 10 minutes and then directly returned them to the DW. We observed the changes in cocoon morphology in both the DW and saturated NaCl solutions.
We analysed the chemical composition of fluids inside the R. lezeyi cocoons collected at the study site on 20 July 2021. We divided the 49 cocoons containing living individuals into four types: prepupae with turgescent cocoons (n = 18), immature pupae with turgescent cocoons (n = 14), mature pupae with turgescent cocoons (n = 7) and immature pupae with shrinking cocoons (n = 10).
We pierced each cocoon membrane and, with a gentle press of the membrane, dripped the cocoon fluids on to a plastic dish. To obtain the average mass of fluids per cocoon for each cocoon type, we weighed each fluid sample. We equated 1 mg to 1 μl. Then we diluted the fluid samples, centrifuged them for 10 min at 3000 rpm and filtered them through a 0.45 µm cellulose acetate filter (Advantec Toyo Kaisha, Ltd., Tokyo, Japan).
We measured the inorganic ion concentrations in the stream water and cocoon fluids as follows: using a continuous flow auto analyser (QuAAtro2-HR, BL TEC K.K., Osaka, Japan), we measured the NH4+ concentration, we quantified the other major cations (Na+, K+, Mg2+ and Ca2+) using a microwave plasma atomic emission spectrometer (4210 MP-AES, Agilent, Santa Clara, CA, USA) and we measured all the anion species using ion chromatography (DIONEX ICS-2100, Thermo Fisher Scientific, Waltham, MA, USA). We determined the bicarbonate (HCO3-) concentration from ion balance through the difference between the total cation equivalent and total anion equivalent. We also determined the osmolarity (mOsm l-1) of the cocoon fluids and stream water from the molar concentration of each ion.
The monthly average values (mean ± SD) for stream water temperature, pH and EC at the study site were 11.8 ± 5.0°C, 2.82 ± 0.24 and 1,572 ± 661 µS cm-1, respectively. The temperature and EC values were about 4°C higher and about 20–40 times higher, respectively, than those in nearby mountain streams receiving no hot spring water. We compared annual variations in temperature and pH of the stream from July to September. Both the daily water temperature and its daily range were significantly increased and the pH was significantly decreased in 2019 and 2021 compared to those in 2018 (Table
Stream water temperature, pH and electrical conductivity (EC) at the study site of Shibukuro Stream and precipitation at a nearby weather station from July to September (2018, 2019 and 2021).
2018 | 2019 | 2021 | Significance test | |
---|---|---|---|---|
Daily stream water temperature (°C) | 17.6b ± 2.02 | 20.1a ± 1.88 | 20.0a ± 1.78 | p < 0.001AN |
Diurnal range of stream water temperature (°C) | 2.26b ± 1.02 | 2.86a ± 1.07 | 2.77a ± 1.09 | p < 0.001AN |
pH | 2.93b ± 0.16 | 2.68a ± 0.16 | 2.68a ± 0.09 | p < 0.01KW |
Electrical conductivity (EC) (µS cm-1) | ― | ― | 2191 ± 357 | |
Daily precipitation (mm)* | 8.02c ± 17.3 | 2.88b ± 6.83 | 3.96a ± 8.22 | p < 0.01KW |
a×b: p < 0.05WR |
In the summers of 2019 and 2021 with high water temperatures, Rhyacophila lezeyi cocoon shrinkage occurred frequently and they experienced high pupal mortality in the study stream. The average proportion of shrinking cocoons in the collected sample year was approximately 4–6 times higher in 2019 (54.1%, n = 74) and 2021 (31.5%, n = 197) than in 2018 (8.9%, n = 124). We also found a significant positive correlation between the mean water temperature for 20 days before collection and the proportion of shrinking cocoons (Fig.
The relationship between the mean stream water temperatures for 20 days prior to collection days and the proportion of shrinking Rhyacophila lezeyi pupal cocoons from July to September (2018, 2019 and 2021). The horizontal bars show the range of the daily mean stream temperatures. The numbers above the symbols show the total number of individuals collected.
The laboratory rearing experiment also confirmed the extremely high mortality of prepupae and pupae in shrinking cocoons (> 90%), although the mortality of those in turgescent cocoons was relatively low (< 10%, Table
Mortality of prepupae and pupae of Rhyacophila lezeyi during the laboratory rearing experiment.
before pupation | before breaking out from the cocoon | Total | |||||||
---|---|---|---|---|---|---|---|---|---|
prepupae | pupated prepupae | pupae | |||||||
Normal cocoon (Turgescent) | 2018 | 2.4 % | (42) | 7.1 % | (42) | 9.9 % | (71) | 9.7 % | (113) |
2019 | 0.0 % | (8) | 25.0 % | (8) | 0.0 % | (26) | 5.9 % | (34) | |
Shrinking cocoon (Collapsed) | 2018 | 75.0 % | (4) | 25.0 % | (4) | 85.7 % | (7) | 90.9 % | (11) |
2019 | 73.3 % | (15) | 26.7 % | (15) | 92.0 % | (25) | 95.0 % | (40) |
The experiment to confirm the cocoon membrane semi-permeability revealed that the R. lezeyi pupal cocoons lost fluid immediately after the transfer to the saturated NaCl solution, resulting in significant cocoon shrinkage (Fig.
Under natural conditions, the mean volume of prepupal cocoon fluids (12 µl) increased to 16 µl for immature pupae and 18 µl for mature pupae. However, the immature pupae inside shrinking cocoons had less fluid volume (10 µl) than normal. Chemical analyses revealed that the cocoon fluids’ ion concentrations were generally higher than those of stream water and that half the ion species (Na+, NH4+, NO3-, HPO42- and HCO3-) were only present in the cocoon fluids, indicating an internal source of such chemical substances for the fluids (Fig.
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
One possible cocoon shrinkage mechanism was at least partially due to the change in the osmotic gradient over the cocoon membrane (Fig.
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 (
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.