The Comparative Osmoregulatory Ability of Two Water Beetle Genera Whose Species Span the Fresh-Hypersaline Gradient in Inland Waters (Coleoptera: Dytiscidae, Hydrophilidae)

Susana Pallarés; Paula Arribas; David T. Bilton; Andrés Millán; Josefa Velasco

doi:10.1371/journal.pone.0124299

Abstract

A better knowledge of the physiological basis of salinity tolerance is essential to understanding the ecology and evolutionary history of organisms that have colonized inland saline waters. Coleoptera are amongst the most diverse macroinvertebrates in inland waters, including saline habitats; however, the osmoregulatory strategies they employ to deal with osmotic stress remain unexplored. Survival and haemolymph osmotic concentration at different salinities were examined in adults of eight aquatic beetle species which inhabit different parts of the fresh—hypersaline gradient. Studied species belong to two unrelated genera which have invaded saline waters independently from freshwater ancestors; Nebrioporus (Dytiscidae) and Enochrus (Hydrophilidae). Their osmoregulatory strategy (osmoconformity or osmoregulation) was identified and osmotic capacity (the osmotic gradient between the animal’s haemolymph and the external medium) was compared between species pairs co-habiting similar salinities in nature. We show that osmoregulatory capacity, rather than osmoconformity, has evolved independently in these different lineages. All species hyperegulated their haemolymph osmotic concentration in diluted waters; those living in fresh or low-salinity waters were unable to hyporegulate and survive in hyperosmotic media (> 340 mosmol kg^-1). In contrast, the species which inhabit the hypo-hypersaline habitats were effective hyporegulators, maintaining their haemolymph osmolality within narrow limits (ca. 300 mosmol kg^-1) across a wide range of external concentrations. The hypersaline species N. ceresyi and E. jesusarribasi tolerated conductivities up to 140 and 180 mS cm^-1, respectively, and maintained osmotic gradients over 3500 mosmol kg^-1, comparable to those of the most effective insect osmoregulators known to date. Syntopic species of both genera showed similar osmotic capacities and in general, osmotic responses correlated well with upper salinity levels occupied by individual species in nature. Therefore, osmoregulatory capacity may mediate habitat segregation amongst congeners across the salinity gradient.

Citation: Pallarés S, Arribas P, Bilton DT, Millán A, Velasco J (2015) The Comparative Osmoregulatory Ability of Two Water Beetle Genera Whose Species Span the Fresh-Hypersaline Gradient in Inland Waters (Coleoptera: Dytiscidae, Hydrophilidae). PLoS ONE 10(4): e0124299. https://doi.org/10.1371/journal.pone.0124299

Academic Editor: Daniel Doucet, Natural Resources Canada, CANADA

Received: November 18, 2014; Accepted: March 11, 2015; Published: April 17, 2015

Copyright: © 2015 Pallarés et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This study was supported by funding from a pre-doctoral grant from the University of Murcia, Spain (http://www.um.es/) to SP, from a postdoctoral grant from the Royal Society UK (Newton International Program) (https://royalsociety.org/) to PA, by project CGL2010-15378 from Ministerio de Economía y Competitividad, Spain to JV and co-financed by FEDER funds (http://europa.eu/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Of all the physiological challenges organisms face in the aquatic environment, an ability to maintain the osmotic concentration of body fluids in the face of fluctuations in the external environment is essential [1, 2]. For primarily freshwater organisms such as aquatic insects, salinity is a natural stressor that can disrupt metabolism and water balance [3], and therefore species inhabiting saline waters have developed a range of physiological mechanisms to deal with ionic fluctuations, which fall into two main strategies: osmoconformity and osmoregulation. Osmoconformers concentrate organic osmolytes intracellularly and/or extracellularly in response to increasing salinity, thus avoiding the toxicity associated with salt accumulation. In osmoregulators, the internal fluid compartment is instead strictly regulated regardless of the external osmotic fluctuation mainly by the active transport of ions via specialized organs [2, 4, 5].

Osmoregulation has been widely studied in marine organisms (e.g. [6–10]), where the vast majority of taxa are osmoconformers [5, 11]. In contrast, our knowledge of the osmotic mechanisms of organisms inhabiting inland waters is severely limited, despite the fact that information on the salinity tolerance of such taxa is essential for understanding their ecology and evolutionary history (e.g. [12–14]). Inland aquatic systems encompass a wide variety of habitats across a large salinity range, from freshwaters to hypersaline water bodies (up to six times the salinity of the sea, i.e. around 200 g L^-1), which also differ in ionic composition [15, 16]. In addition, organisms inhabiting inland saline systems frequently experience large osmotic and ionic fluctuations, far exceeding those seen in most marine systems, as a result of freshwater input from rainfall, or evaporation during dry periods [16, 17]. As a consequence, tolerance of osmotic stress is one of the main constraints to colonization and survival in such ecosystems. Despite these challenges, specialization in saline waters has occurred in many primarily freshwater lineages [14, 18, 19], which offer an ideal comparative framework within which to study the physiological traits of related species adapted to different salinity optima.

In aquatic insects, osmotic patterns have been well documented in a range of larval Diptera (reviewed in [20]). However, in other orders such as Odonata, Hemiptera and Coleoptera, osmotic and ionic regulation patterns are much less well-known; most studies to date focusing on single, unrelated species or only on larval stages (e.g. [21–27]). The osmotic responses of aquatic organisms generally appear to correlate well with the salinity range occupied in nature. Strictly freshwater forms can hyperregulate in dilute waters, but die when the external osmotic concentration reaches or exceeds that of their haemolymph (e.g. [28, 29]), whilst in salinity-tolerant taxa two patterns have been found. Some species osmoregulate at concentrations below the isosmotic point and osmoconform at higher concentrations (e.g. [25, 29–32]). In nature these species are generally limited to external ion concentrations no greater than those found in sea water (about 1000 mOsm). In contrast, it is thought that all species that show tolerance to salinities above 1000 mOsm are efficient osmoregulators (e.g. [19, 33–38]). Osmoregulation therefore seems to be the most recurrent adaptation in aquatic insects inhabiting highly saline media [4], where the additional energetic costs required by osmoregulatory mechanisms may be compensated by the competitive release afforded by these habitats [39]. However, to date, this apparent association between osmotic capacity and species salinity ranges has never been explored from a comparative perspective within clades of closely related species whose members occupy different parts of the salinity gradient.

Coleoptera is one of the most specious insect orders in inland waters, including saline habitats, having colonized water at least 20 times from separate terrestrial ancestors [40]. Recent molecular phylogenetic analyses suggest that the aquatic Adephaga (which includes the familiar diving beetles and whirligigs) have entered the aquatic environment only once [40–42], whilst in some polyphagan families, as hydrophilids, multiple transitions from the terrestrial to the aquatic environment and back again have occurred [42–44]. As well as these shifts between media, the evolutionary history of beetles includes multiple independent transitions from freshwater to saline habitats (e.g. [14]) in which the evolution of specialized mechanisms to deal with salinity must have been crucially important. A number of 'true water beetle' genera (sensu [40], i.e. with both larvae and adults strictly aquatic) have occupied the full salinity gradient, including closely related species with contrasted habitat preferences, i.e. from strictly freshwater species to hypersaline specialists that are able to survive at salinity levels too toxic for any aquatic vertebrate [16]. Such taxa are therefore ideal models with which to explore the evolution and physiological diversity of osmotic stress mechanisms and their relation to habitat occupation. However, whilst a number of studies have explored osmoregulation in terrestrial beetles exposed to dehydration (e.g. [45–49]), information on the osmotic mechanisms of aquatic beetles in saline waters is almost entirely lacking, with only a few aquatic species having been studied, e.g. the freshwater Dytiscus verticalis [26, 27], larvae of freshwater Elodes [21] and a handful of saline water species such as Berosus spinosus [22] and Hygrotus salinarius [24]. A better understanding of the osmotic strategies of different lineages of water beetles should provide insights into the evolutionary processes of physiological adaptation to saline waters. In addition, information on species responses to osmotic stress may assist in the assessment of the potential for communities to deal with environmental change [50, 51]. In particular, studies on osmoregulation are important in the context of increasing aridity and salinization of inland waters, which may result in severe biodiversity losses [52, 53], especially in regions which already experience dry and Mediterranean climates [54].

Here we explore osmotic responses and survival to acute salinity exposure in adults of 8 water beetle species belonging to the genera Nebrioporus (Adephaga: Dytiscidae) and Enochrus (Polyphaga: Hydrophilidae). Within each lineage, we study species inhabiting the different parts of the fresh—hypersaline gradient (see Table 1). Our aims were to: 1) identify and describe species osmoregulatory strategies (i.e. osmoconformity or osmoregulation) to determine if the same mechanisms of dealing with salinity have evolved in these two genera which have independently colonized inland saline waters from freshwater ancestors and 2) compare species osmoregulatory strategies and osmotic capacities (i.e. the osmotic gradient between the animal’s internal medium and the external medium), checking for correlation with species salinity preferences in nature and for differences between co-habiting species of the two lineages.

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Table 1. Species´ habitat and collecting sites.

https://doi.org/10.1371/journal.pone.0124299.t001

Material and Methods

Studied species

The Mediterranean basin hosts a wide variety of inland aquatic habitats, covering the full salinity range, from freshwater to hypersaline water bodies [16, 55, 56]. Enochrus (Polyphaga: Hydrophilidae) and Nebrioporus (Adephaga: Dytiscidae) are amongst the most common and representative genera found in water bodies across the Mediterranean region [16, 57], including species occupying different parts of the salinity gradient, with both larval and adult stages being strictly aquatic.

Within each of these genera, we selected four species with different salinity occupancy ranges in the field (see Table 1), including species that commonly are found in freshwater (N. bucheti cazorlensis (Lagar, Fresneda and Hernando, 1987)), subsaline (E. salomonis (Sahlberg, 1900)), hyposaline (N. clarkii (Wollaston 1862) and E. politus (Küster, 1849)), mesosaline (N. baeticus (Schaum 1864) and E. bicolor (Fabricius, 1792)) and hypersaline waters (N. ceresyi (Aube 1838) and E. jesusarribasi Arribas and Millán, 2013).

Animal collection, maintenance and experimental design

Adults of each species were collected in different areas in Spain (Table 1), most of them located in public land not covered by any special legal protection. For those localities placed in protected areas, the collections were made with the corresponding permissions from the local authorities (Dirección General de Áreas Protegidas y Biodiversidad, Consejería de Agricultura y Medio Ambiente de Castilla La Mancha for Río Tus and Laguna de Pétrola and Dirección General de Medio Ambiente, Consejería de Agricultura y Agua de la Región de Murcia for Laguna Cotorrillo and Rambla Salada). None of the studied species is included in national or international lists of protected or endangered species. Specimens were maintained for one week in 7 L aquaria placed in an environmental chamber (SANYO MLR-351, Sanyo Electric Co., Ltd., Moriguchi City, Osaka, Japan) at 20°C and 12:12 L:D cycle. Each species was maintained at their optimum salinity (see mean conductivity of habitat in Table 1), using water from collection sites. Food was provided daily (chironomid larvae for Nebrioporus species and macrophytes for Enochrus).

Groups of 15–25 animals were exposed for 48 h to different salt concentrations that include the range that each of the species commonly occupies, and lower and upper extremes, as follow: 1, 3, 5, 10, 20, 40 and 50 mS cm^-1 for N. b. cazorlensis and E. salomonis; 1, 5, 20, 50, 75 and 100 mS cm^-1 for N. clarkii and E. politus; 1, 20, 50, 100, 140 and 180 mS cm^-1 for N. baeticus, N. ceresyi, E. bicolor and E. jesusarribasi (see equivalent osmolalities in S1 Table). Pilot trials showed that haemolymph osmolality stabilized by 2 days after transfer, as has been previously shown in other studies (e.g. [32]). Waters of different conductivity were prepared by dissolving an appropriate quantity of marine salt (Ocean Fish, Prodac, Cittadella, Padua, Italy) in distilled water. Experimental aquaria (1 L capacity) were filled with 400 mL of water at the test salinity and held in the environmental chamber at constant temperature (20°C) and 12:12 L:D cycle. Food was not supplied during this period in order to avoid variation in dietary ion intake between the species. Each treatment was replicated three times for each species. Mortality was recorded after 48 h exposure and surviving animals used for haemolymph sampling.

Measurements of haemolymph osmolality

Haemolymph samples were obtained in those treatments with ≤ 50% mortality. Specimens were rinsed in distilled water, dried on blotting paper and placed between two parafilm layers under the binocular microscope. A puncture was made in the pronotum and the resulting haemolymph droplet immediately collected with a 2 μl micro-syringe (Hamilton Company, Reno, Nevada, USA), transferred to cooled eppendorf tubes filled with type B immersion oil (Cargille Laboratories, Cedar Grove, New Jersey, USA) to avoid sample evaporation and melanisation, and stored at -80°C until osmolality measurements. Haemolymph samples from beetles of each treatment (i.e. 15–25 individuals) were pooled to produce the minimum volume of 2 μl required for osmolality measurements. The osmolality of the haemolymph was measured in a Wescor 5520 vapour pressure osmometer (Wescor Logan, Utah, USA) previously calibrated using Wescor standard solutions of 90, 290 and 1000 mOsmol. A special sample holder disc was used following manufacturer instructions for small sample volumes (2 μl). Haemolymph was previously separated from the immersion oil by centrifugation in a Sprout mini-centrifugue (Heathrow Scientific LLC, Vernon Hills, Illinois, USA). Samples of 10 μl of the experimental solutions were also measured with the standard sample disc to obtain external media osmolalities. A calibration curve was made to extrapolate the osmolalities of the two highest conductivities (140 and 180 mS cm^-1), which exceeded the range of the osmometer. No permits or ethical approval were required for the experimental procedures.

Data analysis

The osmotic concentration of haemolymph was plotted against external medium osmolality and compared with the isosmotic line (slope = 1) to determine if each species was an osmoconformer or osmoregulator. We also used generalized linear models (GLM) to define the relationship between haemolymph and external media osmotic concentration, assuming a gaussian error distribution and an identity link function [58]. Osmolality would scale linearly with proportional salinity in the absence of osmoregulation, and deviation from this theoretical linearity reflects the degree of osmoregulation. Therefore, linear and quadratic relationships were tested and the model that best fitted our data was selected based on lower AIC and higher deviance.

Osmotic capacity (OC) is defined as the difference between the osmotic concentration of the body fluids and that of the external medium [59]. OC represents an integrated measure of an organism’s physiological ability to compensate for the osmotic gradient that may occur between the internal and external environments [60, 61] in both hyposmotic (hyper-OC − positive values) and hyperosmotic (hypo-OC − negative values) conditions. The magnitude of this osmotic gradient across the conductivity range tested, i.e. the absolute value of osmotic capacity, was compared between Nebrioporus and Enochrus species pairs with similar salinity preferences (Table 1); i.e., N. b. cazorlensis − E. salomonis, N. clarkii − E. politus, N. baeticus − E. bicolor and N. ceresyi − E. jesusarribasi. For this, we employed two-way ANOVA with OC as the dependent variable and external medium osmolality, species and the interaction of both as factors. When the interaction of species x medium osmolality was significant, Tukey HSD post-hoc tests were used to identify the specific treatments in which OC differed amongst species. All analyses were performed with R v. 3.0.1 (R Development Core team, 2011).

Salinity tolerance limits of the species for 48 h exposure were estimated as the LC₅₀ (the osmotic concentration which resulted in the death of 50% of individuals), using Trimmed Spearman–Karber analysis (USEPA TSK Programme Version 1.5).

Results

Pattern of osmotic regulation

All studied species showed a capacity to hyperegulate in hyposmotic media (from 30 to 340 mosmol kg^-1), maintaining haemolymph osmotic concentration within a range of 280–440 mosmol kg⁻¹ (Fig 1)_. The primarily freshwater Nebrioporus b. cazorlensis, N. clarkii and E. salomonis were unable to hyporegulate in media that reach or exceed their haemolymph osmotic concentration (i.e. over 340 mosmol kg^-1), whilst the remaining saline water species (N. baeticus, N. ceresyi, E. politus, E. bicolor and E. jesusarribasi) were effective hyporegulators in hyperosmotic media. In these species, haemolymph concentration values ranged from 250 to 670 mosmol kg⁻¹, across a range of external osmolalities close to lethal levels (Table 2), i.e. until 1580 mosmol kg⁻¹ in E. politus, 2470 mosmol kg⁻¹ in N. baeticus and E. bicolor, 3550 mosmol kg⁻¹ in N. ceresyi and 4280 mosmol kg⁻¹ in E. jesusarribasi (Fig 1).

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Fig 1. Relationship between osmotic concentration of the haemolymph and the external medium.

Data are expressed as mean ± s.e. (n = 3). The isosmotic line is represented by the discontinuous line. OM: osmolality of external medium, OH: osmolality of haemolymph.

https://doi.org/10.1371/journal.pone.0124299.g001

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Table 2. LC₅₀ values (mosmol kg^-1) and 95% confidence intervals estimated by the Trimmed Spearman–Karber analysis.

https://doi.org/10.1371/journal.pone.0124299.t002

Positive and/or negative deviations of haemolymph osmotic concentration from the isosmotic line representing the theoretical osmolalitity of a strict osmoconformer (slope = 1), reflect the degree of hyper- and hyporegulation of the different species (Fig 1). Nebrioporus b. cazorlensis and N. clarkii had the lowest hyperegulation capacity, showing a gradual linear increase of haemolymph osmotic concentration as external medium concentration increased. In these species, the isosmotic point between haemolymph and the external medium was not reached at any of the salt concentrations tested. Enochrus salomonis maintained almost constant haemolymph concentration below the isosmotic point and reached this at around 300 mosmol kg^-1, being unable to hyporegulate above this concentration. Enochrus politus showed a slight linear increase of haemolymph osmolality across the experimental conductivity range, but it remained both hyper and hyposmotic to the external media. Mesosaline and hypersaline species in both genera showed the strongest deviation (mainly downside) from the isosmotic line, and the relationship between haemolymph and external osmolality was non linear (see the fitted models in Fig 1) reflecting their high osmoregulatory potential. In N. baeticus, haemolymph osmolality increased nonlinearly across the conductivity gradient (Fig 1), whilst in E. bicolor it was maintained within a narrow range (255–336 mosmol kg^-1). In the hypersaline N. ceresyi and E. jesusarribasi, haemolymph osmolality increased more markedly at the highest salinities, but in any case, hyporegulation capacity was detected until the most extreme salt concentrations tested (3550 and 4280 mosmol kg^-1, respectively).

Osmotic capacity

There were significant differences in osmotic capacity (OC) between the four species pairs compared, except in the case of hyposaline species (Table 3). There was also a significant species x external medium osmolality interaction, showing that species differed in their specific response patterns of OC across the range of osmotic concentrations tested.

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Table 3. Effect of osmotality of external medium (OM), species (Sp) and their interaction on osmotic capacity (OC).

https://doi.org/10.1371/journal.pone.0124299.t003

Species occupying fresh-subsaline waters (N. b. cazorlensis and E. salomonis), showed similar salinity tolerances (see LC₅₀ values in Table 2) and similar mean hyper-OCs in media up to 90 mosmol kg^-1. This was followed by a significant reduction in OC as haemolymph osmolality was closer to the isosmotic point with the external medium, at 180 and 340 mosmol kg^-1. OC was significantly lower in E. salomonis at these osmotic concentrations (P < 0.01) (Fig 2A).

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Fig 2. Osmotic capacities of Nebrioporus and Enochrus species pairs.

Data are expressed as mean ± s.e. (n = 3). Asterisks indicate statistically significant differences between species (P ≤ 0.01) at each conductivity treatment. OC: osmotic capacity.

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In the hyposaline species (N. clarkii and E. politus), hyper-OC showed the same decreasing tendency across an osmolality range of 30–340 mosmol kg^-1, which was within the tolerance limits of both species (Table 2). The main differences between these species were due to the fact that E. politus was also able to osmoregulate in hyperosmotic conditions, i.e. at 1000 and 1580 mosmol kg^-1, which were over the lethal limit of N. clarkii (Fig 2B, Table 2).

Mesosaline species displayed similar mean values of hyper-OC at 30 and 340 mosmol kg^-1. From this concentration, hypo-OC increased with increasing osmotic stress in both species, and was significantly higher in E. bicolor than N. ceresyi at 2470 mosmol kg^-1 (Fig 2C).

Hypersaline species showed an identical pattern of hyper-OC to mesosaline taxa in media below the isosmotic point. Above this osmolality, hypo-OC progressively increased, being significantly higher in E. jesusarribasi than in N. ceresyi across all hyperosmotic treatments (Fig 2D). In addition, this species could still osmoregulate at the highest experimental concentration (4280 mosmol kg^-1), which was above the lethal limit for N. ceresyi (Table 2).

Discussion

We have, for the first time, characterized the osmoregulatory strategies of adult aquatic beetles with different salinity tolerances in nature. Our study shows that species from two genera that have independently invaded saline waters are able to osmoregulate in chloride-rich waters, with no osmoconformity being observed in any of the species studied.

In media below 340 mosmol kg⁻¹ (i.e. 20 mS cm^-1), all species showed a similar pattern of hyperegulation, maintaining haemolymph concentration within a narrow range close to the typical osmolality of insect haemolymph (300 mosmol kg⁻¹) [62]. Hyperegulation is a universal adaptation for life in freshwater [18], and involves the production of dilute urine to compensate for water that enters the body osmotically coupled with the replacement of lost salts by dietary intake [63] and active ion uptake [5, 20]. In most insects, Malphigian tubules and the rectum are responsible for urine formation [64, 65], and some species posses anal papillae for active ion uptake from the external environment (e.g. [21, 66–69]). In terms of the ecological implications of the osmotic patterns found here in hyposmotic media, the high survival and osmoregulatory capacity of the studied saline species in these conditions demonstrate that, at least during the adult stage, they can potentially survive in freshwaters, despite rarely being found in fresh or-low conductivity habitats in nature. This is in agreement with another recent study on saline beetle species that tolerate a wide range of salinities (including freshwater) under experimental conditions, but are restricted to waters with salinities close to their upper tolerance limits in nature [70], something also observed in saline water corixids (e.g. [23, 71]). Therefore, restriction to saline habitats may be driven by other factors such as interspecific competition and/or larval requirements (see below).

In media above the isosmotic point, only the hyposaline E. politus and the meso and hypersaline species studied were able to regulate their haemolymph osmotic concentrations. Hyporegulation capacity has previously been reported in other water beetles, as adults of the dytiscid Hygrotus salinarius [24], and larvae of the hydrophilid Berosus spinosus [22]. However, the very wide osmotic gradients that the hypersaline species here studied were able to maintain have never been demonstrated in any beetle species before. For example, the Iberian endemic species E. jesusarribasi was able to maintain its haemolymph at approximately 3500 mosmol kg⁻¹ below that of the media, displaying a hyporegulation capacity comparable to those reported for some of the most effective insect osmoregulators known to date, such as the larvae of Ephydra brine flies [37, 38], the dolichopodid Hydrophorus plumbeus [72], the soldier fly Odontomyia cincta (Stratiomyidae) [73] or larvae and adults of some species of corixid bugs as Trichocorixa verticalis interiores [23] or Sigara stagnalis [74].

A diversity of mechanisms could be behind the extraordinary hyporegulation capacity showed by the species studied here. In general, insect adaptations to live in saline waters are designed to a) limit the entry of ions into the body and the loss of water to the external medium by osmosis, and b) actively excrete excess ions and retain water via specialized organs and tissues. The cuticle of insects represents a relatively impermeable boundary with their environment, with epicuticular lipids, and especially hydrocarbons, serving as a barrier to water loss and a waterproofing layer. However, aquatic insects seem to be in general more permeable to water than their terrestrial counterparts [5,75–77], and water loss through the cuticle has been recorded in some saline Enochrus species when subject to aerial desiccation (J. Velasco, unpublished data). Data on epicuticular hydrocarbons are available for a few freshwater beetle species [78, 79], but nothing is known to date regarding these in saline aquatic taxa. Despite the adaptations to minimize fluxes of water through the body wall, ion entry by drinking the external medium and feeding on food rich in salts (aquatic macrophytes and biofilms in Enochrus species and macroinvertebrates in Nebrioporus) is likely to represent an important salt input for the saline species studied here. Since access to freshwater or to food with low osmotic concentration is not available in inland saline habitats, reducing drinking rates could be a complementary behavioural adjustment to minimize ionic input [29, 80]. Such adaptations, coupled with mechanisms for active ion excretion and water conservation in specialized excretory organs likely account for the osmoregulatory capacities observed here. Insect excretory adaptations typically involve the Malpighian tubules for primary urine formation and the hindgut—the rectum in particular—as the major site of water conservation [18, 19, 81]. Osmotic and water homeostasis are often under complex hormonal control (e.g. oxytocin- and vasopressin-like peptides have been related to osmoregulatory functions in several invertebrate species [82–84]) and the specific mechanisms at morphological, biochemical and cellular levels are widely diverse between different insect groups (see 69 for an extensively review) or even between related taxa with different ecological requirements. For example, some saline-tolerant dipteran larvae possess a two-part rectum, with the posterior segment serving as a salt gland [81, 85], whilst closely related freshwater species do not show such morphological differentiation [19]. The osmotic regulation patterns reported here provide an ideal basis for further comparative studies on the specific osmoregulatory mechanisms in saline and freshwater beetles, which are so far unknown for this group of aquatic insects.

We found that species in both genera show parallel osmotic strategies in relation to the salinity ranges they occupy in nature, i.e. the species living in fresh-subsaline waters possess hyperregulation but not hyporegulation capacity, whilst species found in more highly saline waters are euryhaline osmoregulators. Osmotic capacities were similar between species of the two lineages occupying habitat with similar salinity, differing only significantly at the most elevated osmotic stress levels. Likewise, individual species were able to osmoregulate within a specific range of osmotic concentrations, which correlate with the upper salinity levels they commonly occupy in nature. This is clear, for example, in N. clarkii and E. politus. Although both species can be found in hyposaline waters, N. clarkii occupies a narrower range of salinities (Table 1) and accordingly its osmotic response and LC₅₀ were similar to that of the freshwater-subsaline species, whilst E. politus, which lives within a broader salinity range, showed a similar hyper- and hyporegulation pattern to the mesosaline species E. bicolor and N. baeticus. These results together sustain the idea that within each genus, the differing osmotic capacities of the species may mediate their differential tolerances to salinity and consequently their habitat segregation across the salinity gradient [56, 70, 86].

On the other hand, despite the general concordance between field salinity and osmotic response ranges observed, our experimental data show that the saline-tolerant species studied could osmoregulate and survive at salinities that exceed both the upper and lower limits they commonly occupy in nature. The balance between the metabolic costs of osmoregulation and interspecific competition may play an important role in constraining habitat occupancy in saline waters. The osmotic stress posed by inland saline environments limits the number of species that are able to colonize them, resulting in a significant reduction in interspecific interactions, such as competition or predation in such habitats [39, 87]. The high energy demands required for homeostatic adjustment in the face of osmotic stress may result in trade-offs with other biological traits, resulting in a negative correlation between tolerance to salt and competitive ability [39, 88]. This may at least partly explain the absence of euryhaline hyper and hyporegulator species (e.g. N. baeticus, N. ceresyi, E. bicolor and E. jesusarribasi) in physiologically suitable habitats with higher species richness, such as freshwaters. In addition, however, our experiments may overestimate the true osmotic capacity of these species at salinities exceeding their natural ranges, since their regulatory mechanisms might not be maintained in the long term at these conditions [62]. Also, although larval and adult stages are truly aquatic [40] and apparently coexist during their entire life at similar salinities in nature, experimental data on salinity tolerance of larvae are lacking and it is unknown if their osmotic capacities differ to that found in adults (e.g. [24]). This could also be behind the absence of saline water species in freshwater habitats. Further studies on the osmoregulatory mechanisms and capacities of larvae would be welcome, as they may aid our understanding of the ecological and evolutionary implications of salinity tolerance in water beetles. Unfortunately such studies are hampered by a number of factors, including short larval lifespan in most species, and the taxonomic intractability of the majority of relevant larval stages.

Our understanding of the evolutionary history of colonization of saline waters by beetles is limited, but it is clear that salinity tolerance has arisen independently in a number of different aquatic lineages; for example, independent and direct transitions from freshwater to saline habitats have been reported in Enochrus species of the subgenus Lumetus [14]. Our results and previous work on osmoregulation in beetles [21, 22, 24] suggest that hyporegulation capacity has arisen in independent lineages to deal with salinity. Salt tolerance has also apparently arisen independently in larvae of many genera of mosquitoes [4, 20, 89], but in this case instead a diversity of osmotic strategies has evolved: from strictly freshwater hyperegulators, to osmoconformers and true euryhaline osmoregulators [72]. To date, the osmoconformist strategy seems to be absent amongst aquatic beetles, and generally in those lineages of insects that have successfully colonized highly saline waters [19]. Therefore, despite the fact that osmoconformity is less energetically costly [5, 90] and the most common osmotic strategy amongst marine invertebrates, osmoregulation appears as the most effective and successful adaptation to osmotic stress in insects in inland waters.

In a recent study on the evolution of salinity tolerance in Enochrus, [14] found evidence of multiple direct transitions to saline waters, apparently associated with periods of global aridification, as well as strong concordance between the position of species on habitat salinity and aridity gradients. The authors therefore hypothesised that the mechanisms behind salinity and desiccation tolerance might have co-evolved in this lineage. Our discovery of a generalised osmoregulation mechanism in saline water beetles is consistent with the idea of correlated evolution of such tolerances, since the physiological basis of osmoregulation has multiple commonalities with mechanisms underlying desiccation resistance [91–94]. In fact, examples of the correlation between good osmoregulatory ability and tolerance to arid conditions are abundant amongst a variety of terrestrial xeric beetles, e.g. desert tenebrionids [46, 47, 95, 96] or the meloid Cysteodemus armatus [97]. In the case of aquatic Coleoptera, the development of drought tolerance in lineages subjected to strong seasonal aridity may, therefore, have provided the genetic and physiological basis behind hyporegulation capacity, making colonisation and diversification in saline waters possible.

In conclusion, our findings suggest that osmoregulation could be a generalized strategy to deal with osmotic stress among adult aquatic beetles, and reveal that the evolution of enhanced hyporegulation capacities might have played a key role in the colonization of saline waters by some lineages.

Supporting Information

S1 Table. Equivalent osmolalities of the experimental conductivities.

https://doi.org/10.1371/journal.pone.0124299.s001

(DOC)

Acknowledgments

We thank F. Picazo, S. Guareschi, J.A Carbonell, P. Abellán, A. Gómez and C. Andújar for their useful field assistance, V. Céspedes for helping with haemolymph extraction and research technicians of the SAI (Servicio de Apoyo a la Investigación de la Universidad de Murcia) for providing access to the osmometer.

Author Contributions

Conceived and designed the experiments: SP PA DTB AM JV. Performed the experiments: SP. Analyzed the data: SP PA. Contributed reagents/materials/analysis tools: SP JV AM. Wrote the paper: SP PA DTB AM JV.

References

1. Barton-Browne LB (1964) Water Regulation in Insects. Annu Rev Entomol 9: 63–82.
- View Article
- Google Scholar
2. Evans DH (2008) Osmotic and ionic regulation: cells and animals. Boca Raton: CRC Press.
3. Kefford BJ, Papas PJ, Crowther M, Nugegoda D (2002) Are salts toxicants? Australas J Ecotoxicol 8: 63–68.
- View Article
- Google Scholar
4. Bradley TJ (2008) Saline-water Insects: Ecology, Physiology and Evolution. In: Lancaster J, Briers R, editors. Aquatic Insects: Challenges to Populations. Wallingford: CAB International. pp. 20–35.
5. Bradley TJ (2009) Animal osmoregulation. New York: Oxford University Press.
6. Deaton LE (1981) Ion regulation in freshwater and brackish water bivalve molluscs. Physiol Zool 54: 109–121.
- View Article
- Google Scholar
7. Evans DH (1981) Osmotic and ionic regulation by freshwater and marine fish. In: Ali MA, editor. Environmental Physiology of Fishes. New York: Plenum Press. pp 93–122.
8. Sang HM, Fotedar R (2004) Growth, survival, haemolymph osmolality and organosomatic indices of the western king prawn (Penaeus latisulcatus Kishinouye, 1896) reared at different salinities. Aquaculture 234: 601–614.
- View Article
- Google Scholar
9. Serrano X, Serafy J, Grosell M (2011) Osmoregulatory capabilities of the gray snapper, Lutjanus griseus: salinity challenges and field observations. Mar Freshw Behav Physiol 44: 185–196.
- View Article
- Google Scholar
10. Torres G, Giménez L, Anger K (2011) Growth, tolerance to low salinity, and osmoregulation in decapod crustacean larvae. Aquat Biol 12: 249–260.
- View Article
- Google Scholar
11. Willmer P, Stone G, Johnston I (2005) Environmental Physiology of Animals. Malden: Blackwell Publishing.
12. Muñoz J, Gómez A, Green AJ, Figuerola J, Amat F, Rico C (2008) Phylogeography and local endemism of the native Mediterranean brine shrimp Artemia salina (Branchiopoda: Anostraca). Mol Ecol 17: 3160–3177. pmid:18510585
- View Article
- PubMed/NCBI
- Google Scholar
13. Pinceel T, Brendonck L, Larmuseau MHD, Vanhove MPM, Timms BV, Vanschoenwinkel B (2013) Environmental change as a driver of diversification in temporary aquatic habitats: does the genetic structure of extant fairy shrimp populations reflect historic aridification? Freshw Biol 58: 1556–1572. pmid:23871383
- View Article
- PubMed/NCBI
- Google Scholar
14. Arribas P, Andújar C, Abellán P, Velasco J, Millán A, Ribera I (2014) Tempo and mode of the multiple origins of salinity tolerance in a water beetle lineage. Mol Ecol 23: 360–373.
- View Article
- Google Scholar
15. Bayly I (1972) Salinity tolerance and osmotic behavior of animals in athalassic saline and marine hypersaline waters. Annu Rev Ecol Syst 3: 233–268.
- View Article
- Google Scholar
16. Millán A, Velasco J, Gutiérrez-Cánovas C, Arribas P, Picazo F, Sánchez-Fernández D, et al. (2011) Mediterranean saline streams in southeast Spain: What do we know? J Arid Environ 75: 1352–1359.
- View Article
- Google Scholar
17. Velasco J, Millán A, Hernández J, Gutiérrez-Cánovas C, Abellán P, Sánchez-Fernández D, et al. (2006) Response of biotic communities to salinity changes in a Mediterranean hypersaline stream. Saline Syst 12: 1–15.
- View Article
- Google Scholar
18. Bradley TJ, Briscoe AD, Brady SG, Contreras HL, Danforth BN, Dudley R, et al. (2009) Episodes in insect evolution. Integr Comp Biol 49: 590–606. pmid:21665843
- View Article
- PubMed/NCBI
- Google Scholar
19. Albers MA, Bradley TJ (2011) On the evolution of saline tolerance in the larvae of mosquitoes in the genus Ochlerotatus. Physiol Biochem Zool 84: 258–267. pmid:21527816
- View Article
- PubMed/NCBI
- Google Scholar
20. Bradley TJ (1987) Physiology of osmoregulation in mosquitoes. Annu Rev Entomol 32: 439–462. pmid:2880552
- View Article
- PubMed/NCBI
- Google Scholar
21. Treherne JE (1954) Osmoregulation in the larvae of Helodes (Coleoptera: Helodidae). Trans R Ent Soc Lond 105: 117–130.
- View Article
- Google Scholar
22. Nemenz H (1969) Physiologische und ökologische untersuchungen an einem hydrophiliden aus hyperhalinen gewässern (Berosus spinosus Stephen) (Coleoptera). Vie Milieu 20: 171–230.
- View Article
- Google Scholar
23. Tones PI, Hammer UT (1975) Osmoregulation in Trichocorixa verticalis interiores Sailer (Hemiptera.Corixidae)-an inhabitant of Saskatchewan saline lakes, Canada. Can J Zool 53: 1207–1212.
- View Article
- Google Scholar
24. Tones PI (1977) Osmoregulation in adults and larvae of Hygrotus salinarius Wallis (Coleoptera, Dytiscidae). Comp Biochem Physiol A Comp Physiol 60: 247–250.
- View Article
- Google Scholar
25. Nicholls SP (1983) Ionic and osmotic regulation of the haemolymph of the dragonfly, Libellula quadrimaculata (Odonata: Libellulidae). J Insect Physiol 29: 541–546.
- View Article
- Google Scholar
26. Frisbie MP, Dunson WA (1988) Seasonal aspects of sodium, potassium, and water balance in the predaceous diving beetle Dytiscus verticalis. Can J Zool 66: 1553–1561.
- View Article
- Google Scholar
27. Frisbie MP, Dunson WA (1988) Sodium and water balance in larvae of the predaceous diving beetle, Dytiscus verticalis: An air-breather resistant to acid-induced sodium loss. Comp Biochem Physiol A 89: 409–414.
- View Article
- Google Scholar
28. Wigglesworth VB (1938) The regulation of osmotic pressure and chloride concentration in the haemolymph of mosquito larvae. J Exp Biol 15: 235–247.
- View Article
- Google Scholar
29. Patrick ML, Bradley TJ (2000) The physiology of salinity tolerance in larvae of two species of Culex mosquitoes: the role of compatible solutes. J Exp Biol 203: 821–830. pmid:10648224
- View Article
- PubMed/NCBI
- Google Scholar
30. Garrett MA, Bradley TJ (1984) The pattern of osmotic regulation in larvae of the mosquito Culiseta inornata. J Exp Biol 113: 133–141. pmid:6151963
- View Article
- PubMed/NCBI
- Google Scholar
31. Garrett MA, Bradley TJ (1987) Extracellular accumulation of proline, serine and trehalose in the haemolymph of osmoconforming brackish-water mosquitoes. J Exp Biol 129: 231–238. pmid:3108441
- View Article
- PubMed/NCBI
- Google Scholar
32. Havird JC, Santos SR, Henry RP (2014) Osmoregulation in the Hawaiian anchialine shrimp Halocaridina rubra (Crustacea: Atyidae): expression of ion transporters, mitochondria-rich cell proliferation, and hemolymph osmolality during salinity transfers. J Exp Biol https://doi.org/10.1242/jeb.103051
33. Nayar JK, Sauerman DM (1974) Osmoregulation in larvae of the salt-marsh mosquito, Aedes taeniorhynchus. Entomol Exp Appl 17: 367–380.
- View Article
- Google Scholar
34. Bradley TJ, Phillips JE (1975) The secretion of hyperosmotic fluid by the rectum of a saline-water mosquito larva, Aedes taeniorhynchus. J Exp Biol 63: 331–342. pmid:1202126
- View Article
- PubMed/NCBI
- Google Scholar
35. Neumann D (1976) Adaptations of Chironomids to Intertidal Environments. Annu Rev Entomol 21: 387–414.
- View Article
- Google Scholar
36. Kokkinn MJ (1986) Osmoregulation, salinity tolerance and the site of ion excretion in the halobiont chironomid, Tanytarsus barbitarsis Freeman. Aust J Mar Fresh Res 37: 243–250.
- View Article
- Google Scholar
37. Herbst DB, Conte FP, Brookes VJ (1988) Osmoregulation in an alkaline salt lake insect, Ephydra (Hydropyrus) hians Say (Diptera: Ephydridae) in relation to water chemistry. J Insect Physiol 34: 903–909.
- View Article
- Google Scholar
38. Herbst DB (1999) Biogeography and physiological adaptations of the brine fly genus Ephydra (Diptera: Ephydridae) in saline waters of the Great Basin. Gt Basin Nat 59: 127–135.
- View Article
- Google Scholar
39. Herbst DB (2001) Gradients of salinity stress, environmental stability and water chemistry as a templet for defining habitat types and physiological strategies in inland salt waters. Hydrobiologia 466: 209–219.
- View Article
- Google Scholar
40. Jäch MA, Balke M (2008) Global diversity of water beeltes (Coleoptera) in freshwater. Hydrobiologia 595: 419–442.
- View Article
- Google Scholar
41. Shull VL, Vogler AP, Baker MD, Maddison DR, Hammond PM (2001) Sequence alignment of 18S ribosomal RNA and the basal relationships of adephagan beetles: evidence for monophyly of aquatic families and the placement of Trachypachidae. Syst Biol 50: 945–969. pmid:12116642
- View Article
- PubMed/NCBI
- Google Scholar
42. Hunt T, Bergsten J, Levkanicova Z, Papadopoulou A, John OS, Wild R, et al. (2007) A comprehensive phylogeny of beetles reveals the evolutionary origins of a superradiation. Science 318: 1913–1916. pmid:18096805
- View Article
- PubMed/NCBI
- Google Scholar
43. Bernhard D, Schmidt C, Korte A, Fritzsch G, Beutel RG (2006). From terrestrial to aquatic habitats and back again—molecular insights into the evolution and phylogeny of Hydrophiloidea (Coleoptera) using multigene analyses. Zool. Scripta 35, 597–606.
- View Article
- Google Scholar
44. Short AEZ, Fikáček M (2006) Molecular phylogeny, evolution and classification of the Hydrophilidae (Coleoptera). Syst Entomol 38: 723–752.
- View Article
- Google Scholar
45. Coutchie PA, Crowe JH (1979) Transport of water vapor by tenebrionid beetles. II. Regulation of the osmolality and composition of the haemolymph. Physiol Zool 52: 88–100.
- View Article
- Google Scholar
46. Nicolson SW (1980) Water balance and osmoregulation in Onymacris plana, a tenebrionid beetle from the Namib Desert. J Insect Physiol 26: 315–320.
- View Article
- Google Scholar
47. Riddle W (1986) Hemolymph osmoregulation in three species of beetles. Comp Biochem Physiol A 83: 619–626.
- View Article
- Google Scholar
48. Naidu S (2001) Water balance and osmoregulation in Stenocara gracilipes, a wax-blooming tenebrionid beetle from the Namib Desert. J Insect Physiol 47: 1429–1440. pmid:12770149
- View Article
- PubMed/NCBI
- Google Scholar
49. Naidu S (2006) Haemolymph amino acid, sugar and glycerol levels in the Namib Desert tenebrionid Physadesmia globosa (Coleoptera: Tenebrionidae) during dehydration. Eur J Entomol 103: 305–310.
- View Article
- Google Scholar
50. Hofmann GE, Todgham AE (2010) Living in the now: physiological mechanisms to tolerate a rapidly changing environment. Annu Rev Physiol 72: 127–145. pmid:20148670
- View Article
- PubMed/NCBI
- Google Scholar
51. Somero GN (2011) Comparative physiology: a “crystal ball” for predicting consequences of global change. Am J Physiol Regul Integr Comp Physiol 301: R1–R14. pmid:21430078
- View Article
- PubMed/NCBI
- Google Scholar
52. Kefford BJ, Papas PJ, Metzeling L, Nugegoda D (2004) Do laboratory salinity tolerances of freshwater animals correspond with their field salinity? Environ Pollut 129: 355–362. pmid:15016457
- View Article
- PubMed/NCBI
- Google Scholar
53. Cañedo-Argüelles M, Kefford BJ, Piscart C, Prat N, Schäfer RB, Schulz CJ (2013) Salinisation of rivers: an urgent ecological issue. Environ Pollut 173: 157–167. pmid:23202646
- View Article
- PubMed/NCBI
- Google Scholar
54. Sala OE, Chapin FS, Armesto JJ, Berlow R, Bloomfield J, Dirzo R, et al. (2000) Global biodiversity scenarios for the year 2100. Science 287: 1770–1774. pmid:10710299
- View Article
- PubMed/NCBI
- Google Scholar
55. Montes C, Martino P (1987) Las lagunas salinas españolas. In: Bases científicas para la protección de los humedales españoles. Madrid: Real Academia de Ciencias Exactas, Físicas y Naturales de Madrid. pp. 95–145. pmid:3270297
56. Arribas P, Gutiérrez-Cánovas C, Abellán P, Sánchez-Fernández D, Picazo F, Velasco J, et al. (2009) Tipificación de los ríos salinos ibéricos. Ecosistemas 18: 1–13.
- View Article
- Google Scholar
57. Millán A, Abellán P, Ribera I, Sánchez-Fernández D, Velasco J (2006) The Hydradephaga of the Segura basin (SE Spain): twenty five years studying water beetles. Monograph on Hydroadephaga. Memorie Soc Entom Ital 85: 137–158.
- View Article
- Google Scholar
58. Quinn GP, Keough MJ (2002) Experimental design and data analysis for biologists. Cambridge: Cambridge University Press.
59. Charmantier G, Charmantier-Daures M, Aiken DE (1984) Variation des capacités osmoreégulatrices des larves et postlarves de Homarus americanus Milnes-Edwards, 1837 (Crustacea, Decapoda). C R Acad Sci Paris 299: 863–866.
- View Article
- Google Scholar
60. Lignot JH, Spanings-Pierrot C, Charmantier G (2000) Osmoregulatory capacity as a tool in monitoring the physiological condition and the effect of stress in crustaceans. Aquaculture 191: 209–245.
- View Article
- Google Scholar
61. Calosi P, Ugolini A, Morritt D (2005) Physiological responses to hyposmotic stress in the supralittoral amphipod Talitrus saltator (Crustacea: Amphipoda). Comp Biochem Physiol A Mol Integr Physiol 142: 267–275. pmid:16183309
- View Article
- PubMed/NCBI
- Google Scholar
62. Chown SL, Nicolson S (2004) Insect Physiological Ecology: Mechanisms and patterns. Oxford: Oxford University Press.
63. Frisbie MP, Dunson WA (1988) The effect of food consumption on sodium and water balance in the predaceous diving beetle, Dytiscus verticalis. J Comp Physiol B 158: 91–98.
- View Article
- Google Scholar
64. Pannabecker T (1995) Physiology of the Malpighian tubule. Annu Rev Entomol 40: 493–510.
- View Article
- Google Scholar
65. O’Donnell MJ (1997) Mechanisms of excretion and ion transport in invertebrates. In: Dantzler WH, editor. Handbook of Physiology. Vol. 2. Oxford: Oxford University Press. pp. 1207–1289.
66. Wichard W, Komnick H (1974) Fine structure and function of the rectal chloride epithelia of damselfly larvae. J Insect Physiol 20: 1611–1621. pmid:4852050
- View Article
- PubMed/NCBI
- Google Scholar
67. Donini A, O’Donnell MJ (2005) Analysis of Na⁺, Cl^-, K⁺, H⁺ and NH4⁺ concentration gradients adjacent to the surface of anal papillae of the mosquito Aedes aegypti: application of self-referencing ion-selective microelectrodes. J Exp Biol 208: 603–610. pmid:15695753
- View Article
- PubMed/NCBI
- Google Scholar
68. Patrick ML, Aimanova K, Sanders HR, Gill SS (2006) P-type Na⁺/K⁺-ATPase and V-type H⁺-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti. J Exp Biol 209: 4638–4651. pmid:17114398
- View Article
- PubMed/NCBI
- Google Scholar
69. Larsen EH, Deaton LE, Onken H, O´Donnell M, Grosell M, Dantzler WH, et al. (2014) Osmoregulation and Excretion. Compr Physio 4:405–573.
- View Article
- Google Scholar
70. Céspedes V, Pallarés S, Arribas P, Millán A, Velasco J (2013) Water beetle tolerance to salinity and anionic composition and its relationship to habitat occupancy. J Insect Physiol 59: 1076–1084. pmid:23973816
- View Article
- PubMed/NCBI
- Google Scholar
71. Carbonell JA, Millán A, Velasco J (2012) Concordance between realised and fundamental niches in three Iberian Sigara species (Hemiptera: Corixidae) along a gradient of salinity and anionic composition. Freshw Biol 57: 2580–2590.
- View Article
- Google Scholar
72. Herbst DB, Bradley TJ (1988) Osmoregulation in dolichopodid larvae (Hydrophorus plumbeus) from a saline lake. J Insect Physiol 34: 369–372.
- View Article
- Google Scholar
73. Gainey LF (1984) Osmoregulation in the larvae of Odontomyia cincta (Diptera: Stratiomyidae). Physiol Zool 57: 663–672.
- View Article
- Google Scholar
74. Scudder GE (1976) Water-boatmen of saline waters (Hemiptera: Corixidae). In: Cheng L, editor. Marine Insects. New York: Elsevier. pp. 265–290
75. Holdgate MW (1956) Transpiration through the cuticles of some aquatic insects. J Exp Biol 33: 107–118.
- View Article
- Google Scholar
76. Beament JWL (1961). The waterproofing mechanism of Arthropods. II. The permeability of the cuticle of some aquatic insects. J Exp Biol 38: 277–290.
- View Article
- Google Scholar
77. Armold MT, Blomquist GJ, Jackson LL (1969) Cuticular lipids of insects III. The surface lipids of the aquatic and terrestrial life forms of the bit stonefly, Pteronarcys californica Newport. Comp Biochem Physiol 31: 685–692.
- View Article
- Google Scholar
78. Jacob F, Hassen HP (1986) Distribution and variability of cuticular hydrocarbons whitin the Coleoptera. Biochem Sys Ecol 14: 207–210.
- View Article
- Google Scholar
79. Alaire Y, Joly H, Dennie D (1998) Cuticular hydrocarbon analysis of the aquatic beetle Agabus anthracinus Mannerheim (Coleoptera:Dytiscidae). Can Entomol 130: 615–629.
- View Article
- Google Scholar
80. Bradley TJ, Phillips JE (1977). The effect of external salinity on drinking rate and rectal secretion in the Larvae of the saline-water mosquito Aedes taeniorhynchus. J Exp Biol 66: 97–110. pmid:858994
- View Article
- PubMed/NCBI
- Google Scholar
81. Bradley TJ (2002) Excretion. In: Resh VH, Carde RT, editors. Encyclopedia of insects. Amsterdam: Academic Press. pp. 380–387.
82. Aikins MJ, Schooley DA, Begum K, Detheux M, Beeman RW, Park Y (2008) Vasopressin-like peptide and its receptor function in an indirect diuretic signaling pathway in the red flour beetle. Insect Biochem Mol Biol 38: 740–748. pmid:18549960
- View Article
- PubMed/NCBI
- Google Scholar
83. Stafflinger E, Hansen KK, Hauser F, Schneider M, Cazzamali G, Williamson M, et al. (2008). Cloning and identification of an oxytocin/vasopressin-like receptor and its ligand from insects. Proc Natl Acad Sci USA 105: 3262–3267. pmid:18316733
- View Article
- PubMed/NCBI
- Google Scholar
84. Gruber CW (2014) Physiology of invertebrate oxytocin and vasopressin neuropeptides. Exp Physiol 99: 55–61. pmid:23955310
- View Article
- PubMed/NCBI
- Google Scholar
85. Bradley TJ, Phillips JE (1977) The location and mechanism of hyperosmotic fluid secretion in the rectum of the saline-water mosquito larvae Aedes taeniorhynchus. J Exp Biol 66: 111–126. pmid:858991
- View Article
- PubMed/NCBI
- Google Scholar
86. Gutiérrez-Cánovas C, Millán A, Velasco J, Vaughan IP, Ormerod SJ (2013) Contrasting effects of natural and anthropogenic stressors on beta-diversity in river organisms. Global Ecol Biogeogr 22: 796–805.
- View Article
- Google Scholar
87. Scudder GE (1983) A review of factors governing the distribution of two closely related corixid in the saline lakes of British Columbia. Hydrobiologia 105, 143–154.
- View Article
- Google Scholar
88. Wilson DS, Keddy PA (1986) Species competitive ability and position along a natural stress/disturbance gradient. Ecology 67: 1236–1242.
- View Article
- Google Scholar
89. Bradley TJ (1994) The role of physiological capacity, morphology, and phylogeny in determining habitat use in mosquitoes. In: Wainwright PC, Reilly SM, editors. Ecological Morphology. Integrative Organismal Biology. Chicago: University of Chicago Press. pp. 303–318.
90. Edwards HA (1982) Aedes aegypti: Energetics of osmoregulation. J Exp Biol 101: 135–141.
- View Article
- Google Scholar
91. Beadle LC (1943) Osmotic regulation and the faunas of inland waters. Biol Rev 18: 172–183.
- View Article
- Google Scholar
92. Cloudsley-Thompson JL (1975) Adaptations of arthropoda to arid environments. Annu Rev Entomol 20: 261–283. pmid:1090239
- View Article
- PubMed/NCBI
- Google Scholar
93. Gibbs AG, Chippindale AK, Rose MR (1997) Physiological mechanisms of evolved desiccation resistance in Drosophila melanogaster. J Exp Biol 200: 1821–1832. pmid:9225453
- View Article
- PubMed/NCBI
- Google Scholar
94. Gómez-Mestre I, Tejedo M (2005) Adaptation or exaptation? An experimental test of hypotheses on the origin of salinity tolerance in Bufo calamita. J Evolution Biol 18: 847–855.
- View Article
- Google Scholar
95. Broza M, Borut A, Pener MP (1976) Osmoregulation in the desert tenebrionid beetle Trachyderma philistina Reiche during dehydration and subsequent rehydration. Israel J Med Sci 12: 868–871. pmid:977304
- View Article
- PubMed/NCBI
- Google Scholar
96. Riddle WA, Crawford CS, Zeitone AH (1976) Patterns of hemolymph osmoregulation in three desert arthropods. J Comp Physiol 112: 295–305.
- View Article
- Google Scholar
97. Cohen AC, March RB, Pinto JD (1986) Effects of water stress and rehydration on hemolymph volume and amino acid content in the blister beetle, Cysteodemus armatus. Comp Biochem Physiol 85: 743–746.
- View Article
- Google Scholar

[ref1] 1. Barton-Browne LB (1964) Water Regulation in Insects. Annu Rev Entomol 9: 63–82.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Evans DH (2008) Osmotic and ionic regulation: cells and animals. Boca Raton: CRC Press.

[ref3] 3. Kefford BJ, Papas PJ, Crowther M, Nugegoda D (2002) Are salts toxicants? Australas J Ecotoxicol 8: 63–68.
View Article
Google Scholar

[6] View Article

[7] Google Scholar

[ref4] 4. Bradley TJ (2008) Saline-water Insects: Ecology, Physiology and Evolution. In: Lancaster J, Briers R, editors. Aquatic Insects: Challenges to Populations. Wallingford: CAB International. pp. 20–35.

[ref5] 5. Bradley TJ (2009) Animal osmoregulation. New York: Oxford University Press.

[ref6] 6. Deaton LE (1981) Ion regulation in freshwater and brackish water bivalve molluscs. Physiol Zool 54: 109–121.
View Article
Google Scholar

[11] View Article

[12] Google Scholar

[ref7] 7. Evans DH (1981) Osmotic and ionic regulation by freshwater and marine fish. In: Ali MA, editor. Environmental Physiology of Fishes. New York: Plenum Press. pp 93–122.

[ref8] 8. Sang HM, Fotedar R (2004) Growth, survival, haemolymph osmolality and organosomatic indices of the western king prawn (Penaeus latisulcatus Kishinouye, 1896) reared at different salinities. Aquaculture 234: 601–614.
View Article
Google Scholar

[15] View Article

[16] Google Scholar

[ref9] 9. Serrano X, Serafy J, Grosell M (2011) Osmoregulatory capabilities of the gray snapper, Lutjanus griseus: salinity challenges and field observations. Mar Freshw Behav Physiol 44: 185–196.
View Article
Google Scholar

[18] View Article

[19] Google Scholar

[ref10] 10. Torres G, Giménez L, Anger K (2011) Growth, tolerance to low salinity, and osmoregulation in decapod crustacean larvae. Aquat Biol 12: 249–260.
View Article
Google Scholar

[21] View Article

[22] Google Scholar

[ref11] 11. Willmer P, Stone G, Johnston I (2005) Environmental Physiology of Animals. Malden: Blackwell Publishing.

[ref12] 12. Muñoz J, Gómez A, Green AJ, Figuerola J, Amat F, Rico C (2008) Phylogeography and local endemism of the native Mediterranean brine shrimp Artemia salina (Branchiopoda: Anostraca). Mol Ecol 17: 3160–3177. pmid:18510585
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref13] 13. Pinceel T, Brendonck L, Larmuseau MHD, Vanhove MPM, Timms BV, Vanschoenwinkel B (2013) Environmental change as a driver of diversification in temporary aquatic habitats: does the genetic structure of extant fairy shrimp populations reflect historic aridification? Freshw Biol 58: 1556–1572. pmid:23871383
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref14] 14. Arribas P, Andújar C, Abellán P, Velasco J, Millán A, Ribera I (2014) Tempo and mode of the multiple origins of salinity tolerance in a water beetle lineage. Mol Ecol 23: 360–373.
View Article
Google Scholar

[33] View Article

[34] Google Scholar

[ref15] 15. Bayly I (1972) Salinity tolerance and osmotic behavior of animals in athalassic saline and marine hypersaline waters. Annu Rev Ecol Syst 3: 233–268.
View Article
Google Scholar

[36] View Article

[37] Google Scholar

[ref16] 16. Millán A, Velasco J, Gutiérrez-Cánovas C, Arribas P, Picazo F, Sánchez-Fernández D, et al. (2011) Mediterranean saline streams in southeast Spain: What do we know? J Arid Environ 75: 1352–1359.
View Article
Google Scholar

[39] View Article

[40] Google Scholar

[ref17] 17. Velasco J, Millán A, Hernández J, Gutiérrez-Cánovas C, Abellán P, Sánchez-Fernández D, et al. (2006) Response of biotic communities to salinity changes in a Mediterranean hypersaline stream. Saline Syst 12: 1–15.
View Article
Google Scholar

[42] View Article

[43] Google Scholar

[ref18] 18. Bradley TJ, Briscoe AD, Brady SG, Contreras HL, Danforth BN, Dudley R, et al. (2009) Episodes in insect evolution. Integr Comp Biol 49: 590–606. pmid:21665843
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref19] 19. Albers MA, Bradley TJ (2011) On the evolution of saline tolerance in the larvae of mosquitoes in the genus Ochlerotatus. Physiol Biochem Zool 84: 258–267. pmid:21527816
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

[51] Google Scholar

[ref20] 20. Bradley TJ (1987) Physiology of osmoregulation in mosquitoes. Annu Rev Entomol 32: 439–462. pmid:2880552
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

[55] Google Scholar

[ref21] 21. Treherne JE (1954) Osmoregulation in the larvae of Helodes (Coleoptera: Helodidae). Trans R Ent Soc Lond 105: 117–130.
View Article
Google Scholar

[57] View Article

[58] Google Scholar

[ref22] 22. Nemenz H (1969) Physiologische und ökologische untersuchungen an einem hydrophiliden aus hyperhalinen gewässern (Berosus spinosus Stephen) (Coleoptera). Vie Milieu 20: 171–230.
View Article
Google Scholar

[60] View Article

[61] Google Scholar

[ref23] 23. Tones PI, Hammer UT (1975) Osmoregulation in Trichocorixa verticalis interiores Sailer (Hemiptera.Corixidae)-an inhabitant of Saskatchewan saline lakes, Canada. Can J Zool 53: 1207–1212.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref24] 24. Tones PI (1977) Osmoregulation in adults and larvae of Hygrotus salinarius Wallis (Coleoptera, Dytiscidae). Comp Biochem Physiol A Comp Physiol 60: 247–250.
View Article
Google Scholar

[66] View Article

[67] Google Scholar

[ref25] 25. Nicholls SP (1983) Ionic and osmotic regulation of the haemolymph of the dragonfly, Libellula quadrimaculata (Odonata: Libellulidae). J Insect Physiol 29: 541–546.
View Article
Google Scholar

[69] View Article

[70] Google Scholar

[ref26] 26. Frisbie MP, Dunson WA (1988) Seasonal aspects of sodium, potassium, and water balance in the predaceous diving beetle Dytiscus verticalis. Can J Zool 66: 1553–1561.
View Article
Google Scholar

[72] View Article

[73] Google Scholar

[ref27] 27. Frisbie MP, Dunson WA (1988) Sodium and water balance in larvae of the predaceous diving beetle, Dytiscus verticalis: An air-breather resistant to acid-induced sodium loss. Comp Biochem Physiol A 89: 409–414.
View Article
Google Scholar

[75] View Article

[76] Google Scholar

[ref28] 28. Wigglesworth VB (1938) The regulation of osmotic pressure and chloride concentration in the haemolymph of mosquito larvae. J Exp Biol 15: 235–247.
View Article
Google Scholar

[78] View Article

[79] Google Scholar

[ref29] 29. Patrick ML, Bradley TJ (2000) The physiology of salinity tolerance in larvae of two species of Culex mosquitoes: the role of compatible solutes. J Exp Biol 203: 821–830. pmid:10648224
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref30] 30. Garrett MA, Bradley TJ (1984) The pattern of osmotic regulation in larvae of the mosquito Culiseta inornata. J Exp Biol 113: 133–141. pmid:6151963
View Article
PubMed/NCBI
Google Scholar

[85] View Article

[86] PubMed/NCBI

[87] Google Scholar

[ref31] 31. Garrett MA, Bradley TJ (1987) Extracellular accumulation of proline, serine and trehalose in the haemolymph of osmoconforming brackish-water mosquitoes. J Exp Biol 129: 231–238. pmid:3108441
View Article
PubMed/NCBI
Google Scholar

[89] View Article

[90] PubMed/NCBI

[91] Google Scholar

[ref32] 32. Havird JC, Santos SR, Henry RP (2014) Osmoregulation in the Hawaiian anchialine shrimp Halocaridina rubra (Crustacea: Atyidae): expression of ion transporters, mitochondria-rich cell proliferation, and hemolymph osmolality during salinity transfers. J Exp Biol https://doi.org/10.1242/jeb.103051

[ref33] 33. Nayar JK, Sauerman DM (1974) Osmoregulation in larvae of the salt-marsh mosquito, Aedes taeniorhynchus. Entomol Exp Appl 17: 367–380.
View Article
Google Scholar

[94] View Article

[95] Google Scholar

[ref34] 34. Bradley TJ, Phillips JE (1975) The secretion of hyperosmotic fluid by the rectum of a saline-water mosquito larva, Aedes taeniorhynchus. J Exp Biol 63: 331–342. pmid:1202126
View Article
PubMed/NCBI
Google Scholar

[97] View Article

[98] PubMed/NCBI

[99] Google Scholar

[ref35] 35. Neumann D (1976) Adaptations of Chironomids to Intertidal Environments. Annu Rev Entomol 21: 387–414.
View Article
Google Scholar

[101] View Article

[102] Google Scholar

[ref36] 36. Kokkinn MJ (1986) Osmoregulation, salinity tolerance and the site of ion excretion in the halobiont chironomid, Tanytarsus barbitarsis Freeman. Aust J Mar Fresh Res 37: 243–250.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref37] 37. Herbst DB, Conte FP, Brookes VJ (1988) Osmoregulation in an alkaline salt lake insect, Ephydra (Hydropyrus) hians Say (Diptera: Ephydridae) in relation to water chemistry. J Insect Physiol 34: 903–909.
View Article
Google Scholar

[107] View Article

[108] Google Scholar

[ref38] 38. Herbst DB (1999) Biogeography and physiological adaptations of the brine fly genus Ephydra (Diptera: Ephydridae) in saline waters of the Great Basin. Gt Basin Nat 59: 127–135.
View Article
Google Scholar

[110] View Article

[111] Google Scholar

[ref39] 39. Herbst DB (2001) Gradients of salinity stress, environmental stability and water chemistry as a templet for defining habitat types and physiological strategies in inland salt waters. Hydrobiologia 466: 209–219.
View Article
Google Scholar

[113] View Article

[114] Google Scholar

[ref40] 40. Jäch MA, Balke M (2008) Global diversity of water beeltes (Coleoptera) in freshwater. Hydrobiologia 595: 419–442.
View Article
Google Scholar

[116] View Article

[117] Google Scholar

[ref41] 41. Shull VL, Vogler AP, Baker MD, Maddison DR, Hammond PM (2001) Sequence alignment of 18S ribosomal RNA and the basal relationships of adephagan beetles: evidence for monophyly of aquatic families and the placement of Trachypachidae. Syst Biol 50: 945–969. pmid:12116642
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref42] 42. Hunt T, Bergsten J, Levkanicova Z, Papadopoulou A, John OS, Wild R, et al. (2007) A comprehensive phylogeny of beetles reveals the evolutionary origins of a superradiation. Science 318: 1913–1916. pmid:18096805
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref43] 43. Bernhard D, Schmidt C, Korte A, Fritzsch G, Beutel RG (2006). From terrestrial to aquatic habitats and back again—molecular insights into the evolution and phylogeny of Hydrophiloidea (Coleoptera) using multigene analyses. Zool. Scripta 35, 597–606.
View Article
Google Scholar

[127] View Article

[128] Google Scholar

[ref44] 44. Short AEZ, Fikáček M (2006) Molecular phylogeny, evolution and classification of the Hydrophilidae (Coleoptera). Syst Entomol 38: 723–752.
View Article
Google Scholar

[130] View Article

[131] Google Scholar

[ref45] 45. Coutchie PA, Crowe JH (1979) Transport of water vapor by tenebrionid beetles. II. Regulation of the osmolality and composition of the haemolymph. Physiol Zool 52: 88–100.
View Article
Google Scholar

[133] View Article

[134] Google Scholar

[ref46] 46. Nicolson SW (1980) Water balance and osmoregulation in Onymacris plana, a tenebrionid beetle from the Namib Desert. J Insect Physiol 26: 315–320.
View Article
Google Scholar

[136] View Article

[137] Google Scholar

[ref47] 47. Riddle W (1986) Hemolymph osmoregulation in three species of beetles. Comp Biochem Physiol A 83: 619–626.
View Article
Google Scholar

[139] View Article

[140] Google Scholar

[ref48] 48. Naidu S (2001) Water balance and osmoregulation in Stenocara gracilipes, a wax-blooming tenebrionid beetle from the Namib Desert. J Insect Physiol 47: 1429–1440. pmid:12770149
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref49] 49. Naidu S (2006) Haemolymph amino acid, sugar and glycerol levels in the Namib Desert tenebrionid Physadesmia globosa (Coleoptera: Tenebrionidae) during dehydration. Eur J Entomol 103: 305–310.
View Article
Google Scholar

[146] View Article

[147] Google Scholar

[ref50] 50. Hofmann GE, Todgham AE (2010) Living in the now: physiological mechanisms to tolerate a rapidly changing environment. Annu Rev Physiol 72: 127–145. pmid:20148670
View Article
PubMed/NCBI
Google Scholar

[149] View Article

[150] PubMed/NCBI

[151] Google Scholar

[ref51] 51. Somero GN (2011) Comparative physiology: a “crystal ball” for predicting consequences of global change. Am J Physiol Regul Integr Comp Physiol 301: R1–R14. pmid:21430078
View Article
PubMed/NCBI
Google Scholar

[153] View Article

[154] PubMed/NCBI

[155] Google Scholar

[ref52] 52. Kefford BJ, Papas PJ, Metzeling L, Nugegoda D (2004) Do laboratory salinity tolerances of freshwater animals correspond with their field salinity? Environ Pollut 129: 355–362. pmid:15016457
View Article
PubMed/NCBI
Google Scholar

[157] View Article

[158] PubMed/NCBI

[159] Google Scholar

[ref53] 53. Cañedo-Argüelles M, Kefford BJ, Piscart C, Prat N, Schäfer RB, Schulz CJ (2013) Salinisation of rivers: an urgent ecological issue. Environ Pollut 173: 157–167. pmid:23202646
View Article
PubMed/NCBI
Google Scholar

[161] View Article

[162] PubMed/NCBI

[163] Google Scholar

[ref54] 54. Sala OE, Chapin FS, Armesto JJ, Berlow R, Bloomfield J, Dirzo R, et al. (2000) Global biodiversity scenarios for the year 2100. Science 287: 1770–1774. pmid:10710299
View Article
PubMed/NCBI
Google Scholar

[165] View Article

[166] PubMed/NCBI

[167] Google Scholar

[ref55] 55. Montes C, Martino P (1987) Las lagunas salinas españolas. In: Bases científicas para la protección de los humedales españoles. Madrid: Real Academia de Ciencias Exactas, Físicas y Naturales de Madrid. pp. 95–145. pmid:3270297

[ref56] 56. Arribas P, Gutiérrez-Cánovas C, Abellán P, Sánchez-Fernández D, Picazo F, Velasco J, et al. (2009) Tipificación de los ríos salinos ibéricos. Ecosistemas 18: 1–13.
View Article
Google Scholar

[170] View Article

[171] Google Scholar

[ref57] 57. Millán A, Abellán P, Ribera I, Sánchez-Fernández D, Velasco J (2006) The Hydradephaga of the Segura basin (SE Spain): twenty five years studying water beetles. Monograph on Hydroadephaga. Memorie Soc Entom Ital 85: 137–158.
View Article
Google Scholar

[173] View Article

[174] Google Scholar

[ref58] 58. Quinn GP, Keough MJ (2002) Experimental design and data analysis for biologists. Cambridge: Cambridge University Press.

[ref59] 59. Charmantier G, Charmantier-Daures M, Aiken DE (1984) Variation des capacités osmoreégulatrices des larves et postlarves de Homarus americanus Milnes-Edwards, 1837 (Crustacea, Decapoda). C R Acad Sci Paris 299: 863–866.
View Article
Google Scholar

[177] View Article

[178] Google Scholar

[ref60] 60. Lignot JH, Spanings-Pierrot C, Charmantier G (2000) Osmoregulatory capacity as a tool in monitoring the physiological condition and the effect of stress in crustaceans. Aquaculture 191: 209–245.
View Article
Google Scholar

[180] View Article

[181] Google Scholar

[ref61] 61. Calosi P, Ugolini A, Morritt D (2005) Physiological responses to hyposmotic stress in the supralittoral amphipod Talitrus saltator (Crustacea: Amphipoda). Comp Biochem Physiol A Mol Integr Physiol 142: 267–275. pmid:16183309
View Article
PubMed/NCBI
Google Scholar

[183] View Article

[184] PubMed/NCBI

[185] Google Scholar

[ref62] 62. Chown SL, Nicolson S (2004) Insect Physiological Ecology: Mechanisms and patterns. Oxford: Oxford University Press.

[ref63] 63. Frisbie MP, Dunson WA (1988) The effect of food consumption on sodium and water balance in the predaceous diving beetle, Dytiscus verticalis. J Comp Physiol B 158: 91–98.
View Article
Google Scholar

[188] View Article

[189] Google Scholar

[ref64] 64. Pannabecker T (1995) Physiology of the Malpighian tubule. Annu Rev Entomol 40: 493–510.
View Article
Google Scholar

[191] View Article

[192] Google Scholar

[ref65] 65. O’Donnell MJ (1997) Mechanisms of excretion and ion transport in invertebrates. In: Dantzler WH, editor. Handbook of Physiology. Vol. 2. Oxford: Oxford University Press. pp. 1207–1289.

[ref66] 66. Wichard W, Komnick H (1974) Fine structure and function of the rectal chloride epithelia of damselfly larvae. J Insect Physiol 20: 1611–1621. pmid:4852050
View Article
PubMed/NCBI
Google Scholar

[195] View Article

[196] PubMed/NCBI

[197] Google Scholar

[ref67] 67. Donini A, O’Donnell MJ (2005) Analysis of Na⁺, Cl^-, K⁺, H⁺ and NH4⁺ concentration gradients adjacent to the surface of anal papillae of the mosquito Aedes aegypti: application of self-referencing ion-selective microelectrodes. J Exp Biol 208: 603–610. pmid:15695753
View Article
PubMed/NCBI
Google Scholar

[199] View Article

[200] PubMed/NCBI

[201] Google Scholar

[ref68] 68. Patrick ML, Aimanova K, Sanders HR, Gill SS (2006) P-type Na⁺/K⁺-ATPase and V-type H⁺-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti. J Exp Biol 209: 4638–4651. pmid:17114398
View Article
PubMed/NCBI
Google Scholar

[203] View Article

[204] PubMed/NCBI

[205] Google Scholar

[ref69] 69. Larsen EH, Deaton LE, Onken H, O´Donnell M, Grosell M, Dantzler WH, et al. (2014) Osmoregulation and Excretion. Compr Physio 4:405–573.
View Article
Google Scholar

[207] View Article

[208] Google Scholar

[ref70] 70. Céspedes V, Pallarés S, Arribas P, Millán A, Velasco J (2013) Water beetle tolerance to salinity and anionic composition and its relationship to habitat occupancy. J Insect Physiol 59: 1076–1084. pmid:23973816
View Article
PubMed/NCBI
Google Scholar

[210] View Article

[211] PubMed/NCBI

[212] Google Scholar

[ref71] 71. Carbonell JA, Millán A, Velasco J (2012) Concordance between realised and fundamental niches in three Iberian Sigara species (Hemiptera: Corixidae) along a gradient of salinity and anionic composition. Freshw Biol 57: 2580–2590.
View Article
Google Scholar

[214] View Article

[215] Google Scholar

[ref72] 72. Herbst DB, Bradley TJ (1988) Osmoregulation in dolichopodid larvae (Hydrophorus plumbeus) from a saline lake. J Insect Physiol 34: 369–372.
View Article
Google Scholar

[217] View Article

[218] Google Scholar

[ref73] 73. Gainey LF (1984) Osmoregulation in the larvae of Odontomyia cincta (Diptera: Stratiomyidae). Physiol Zool 57: 663–672.
View Article
Google Scholar

[220] View Article

[221] Google Scholar

[ref74] 74. Scudder GE (1976) Water-boatmen of saline waters (Hemiptera: Corixidae). In: Cheng L, editor. Marine Insects. New York: Elsevier. pp. 265–290

[ref75] 75. Holdgate MW (1956) Transpiration through the cuticles of some aquatic insects. J Exp Biol 33: 107–118.
View Article
Google Scholar

[224] View Article

[225] Google Scholar

[ref76] 76. Beament JWL (1961). The waterproofing mechanism of Arthropods. II. The permeability of the cuticle of some aquatic insects. J Exp Biol 38: 277–290.
View Article
Google Scholar

[227] View Article

[228] Google Scholar

[ref77] 77. Armold MT, Blomquist GJ, Jackson LL (1969) Cuticular lipids of insects III. The surface lipids of the aquatic and terrestrial life forms of the bit stonefly, Pteronarcys californica Newport. Comp Biochem Physiol 31: 685–692.
View Article
Google Scholar

[230] View Article

[231] Google Scholar

[ref78] 78. Jacob F, Hassen HP (1986) Distribution and variability of cuticular hydrocarbons whitin the Coleoptera. Biochem Sys Ecol 14: 207–210.
View Article
Google Scholar

[233] View Article

[234] Google Scholar

[ref79] 79. Alaire Y, Joly H, Dennie D (1998) Cuticular hydrocarbon analysis of the aquatic beetle Agabus anthracinus Mannerheim (Coleoptera:Dytiscidae). Can Entomol 130: 615–629.
View Article
Google Scholar

[236] View Article

[237] Google Scholar

[ref80] 80. Bradley TJ, Phillips JE (1977). The effect of external salinity on drinking rate and rectal secretion in the Larvae of the saline-water mosquito Aedes taeniorhynchus. J Exp Biol 66: 97–110. pmid:858994
View Article
PubMed/NCBI
Google Scholar

[239] View Article

[240] PubMed/NCBI

[241] Google Scholar

[ref81] 81. Bradley TJ (2002) Excretion. In: Resh VH, Carde RT, editors. Encyclopedia of insects. Amsterdam: Academic Press. pp. 380–387.

[ref82] 82. Aikins MJ, Schooley DA, Begum K, Detheux M, Beeman RW, Park Y (2008) Vasopressin-like peptide and its receptor function in an indirect diuretic signaling pathway in the red flour beetle. Insect Biochem Mol Biol 38: 740–748. pmid:18549960
View Article
PubMed/NCBI
Google Scholar

[244] View Article

[245] PubMed/NCBI

[246] Google Scholar

[ref83] 83. Stafflinger E, Hansen KK, Hauser F, Schneider M, Cazzamali G, Williamson M, et al. (2008). Cloning and identification of an oxytocin/vasopressin-like receptor and its ligand from insects. Proc Natl Acad Sci USA 105: 3262–3267. pmid:18316733
View Article
PubMed/NCBI
Google Scholar

[248] View Article

[249] PubMed/NCBI

[250] Google Scholar

[ref84] 84. Gruber CW (2014) Physiology of invertebrate oxytocin and vasopressin neuropeptides. Exp Physiol 99: 55–61. pmid:23955310
View Article
PubMed/NCBI
Google Scholar

[252] View Article

[253] PubMed/NCBI

[254] Google Scholar

[ref85] 85. Bradley TJ, Phillips JE (1977) The location and mechanism of hyperosmotic fluid secretion in the rectum of the saline-water mosquito larvae Aedes taeniorhynchus. J Exp Biol 66: 111–126. pmid:858991
View Article
PubMed/NCBI
Google Scholar

[256] View Article

[257] PubMed/NCBI

[258] Google Scholar

[ref86] 86. Gutiérrez-Cánovas C, Millán A, Velasco J, Vaughan IP, Ormerod SJ (2013) Contrasting effects of natural and anthropogenic stressors on beta-diversity in river organisms. Global Ecol Biogeogr 22: 796–805.
View Article
Google Scholar

[260] View Article

[261] Google Scholar

[ref87] 87. Scudder GE (1983) A review of factors governing the distribution of two closely related corixid in the saline lakes of British Columbia. Hydrobiologia 105, 143–154.
View Article
Google Scholar

[263] View Article

[264] Google Scholar

[ref88] 88. Wilson DS, Keddy PA (1986) Species competitive ability and position along a natural stress/disturbance gradient. Ecology 67: 1236–1242.
View Article
Google Scholar

[266] View Article

[267] Google Scholar

[ref89] 89. Bradley TJ (1994) The role of physiological capacity, morphology, and phylogeny in determining habitat use in mosquitoes. In: Wainwright PC, Reilly SM, editors. Ecological Morphology. Integrative Organismal Biology. Chicago: University of Chicago Press. pp. 303–318.

[ref90] 90. Edwards HA (1982) Aedes aegypti: Energetics of osmoregulation. J Exp Biol 101: 135–141.
View Article
Google Scholar

[270] View Article

[271] Google Scholar

[ref91] 91. Beadle LC (1943) Osmotic regulation and the faunas of inland waters. Biol Rev 18: 172–183.
View Article
Google Scholar

[273] View Article

[274] Google Scholar

[ref92] 92. Cloudsley-Thompson JL (1975) Adaptations of arthropoda to arid environments. Annu Rev Entomol 20: 261–283. pmid:1090239
View Article
PubMed/NCBI
Google Scholar

[276] View Article

[277] PubMed/NCBI

[278] Google Scholar

[ref93] 93. Gibbs AG, Chippindale AK, Rose MR (1997) Physiological mechanisms of evolved desiccation resistance in Drosophila melanogaster. J Exp Biol 200: 1821–1832. pmid:9225453
View Article
PubMed/NCBI
Google Scholar

[280] View Article

[281] PubMed/NCBI

[282] Google Scholar

[ref94] 94. Gómez-Mestre I, Tejedo M (2005) Adaptation or exaptation? An experimental test of hypotheses on the origin of salinity tolerance in Bufo calamita. J Evolution Biol 18: 847–855.
View Article
Google Scholar

[284] View Article

[285] Google Scholar

[ref95] 95. Broza M, Borut A, Pener MP (1976) Osmoregulation in the desert tenebrionid beetle Trachyderma philistina Reiche during dehydration and subsequent rehydration. Israel J Med Sci 12: 868–871. pmid:977304
View Article
PubMed/NCBI
Google Scholar

[287] View Article

[288] PubMed/NCBI

[289] Google Scholar

[ref96] 96. Riddle WA, Crawford CS, Zeitone AH (1976) Patterns of hemolymph osmoregulation in three desert arthropods. J Comp Physiol 112: 295–305.
View Article
Google Scholar

[291] View Article

[292] Google Scholar

[ref97] 97. Cohen AC, March RB, Pinto JD (1986) Effects of water stress and rehydration on hemolymph volume and amino acid content in the blister beetle, Cysteodemus armatus. Comp Biochem Physiol 85: 743–746.
View Article
Google Scholar

[294] View Article

[295] Google Scholar

Figures

Abstract

Introduction

Material and Methods

Studied species

Animal collection, maintenance and experimental design

Measurements of haemolymph osmolality

Data analysis

Results

Pattern of osmotic regulation

Osmotic capacity

Discussion

Supporting Information

S1 Table. Equivalent osmolalities of the experimental conductivities.

Acknowledgments

Author Contributions

References