Biotic factors in induced defence revisited: cell aggregate formation in the toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> PCC 7806 is triggered by spent <Emphasis Type="Italic">Daphnia</Emphasis> medium and disrupted cells

Bioassays with the toxic cyanobacterium Microcystis aeruginosa PCC 7806, its non-toxic mutant ΔmcyB, and Daphnia magna as grazer were used to evaluate biotic factors in induced defence, in particular cyanobacterial and grazer-released info-chemicals. Three main questions were addressed in this study: Does Daphnia grazing lead to a loss of cyanobaterial biomass? Is the survival time of Daphnia shorter in a culture of the toxic cyanobacterium? Does direct grazing or the presence of spent Daphnia medium or a high number of disrupted toxic Microcystis cells in the assays lead to an increase in the cellular microcystin content in the remaining intact cells? The biovolume (growth) as well as size and abundance of Microcystis aggregates were determined by particle analysis, while the survival time of Daphnia individuals was recorded by daily observation and counting, with the relative concentration of cell-bound microcystin-LR, was measured by HPLC analysis. Compared to some recent studies in the field of induced defence, in this study, evidence was found for a direct grazing effect, i.e. the loss of biovolume in the toxic culture. In addition, Daphnia magna ingested more non-toxic than toxic cells, and survived longer with non-toxic cells. In terms of increased cell-bound toxin concentration as a means of defence reported in some studies, a higher cell-bound microcystin-LR content was not measured in this study in any of the treatments (P > 0.05). Under low light conditions with impaired growth of Microcystis, and the presence of a high number of particles with less than 1-μm diameter (possibly heterotrophic bacteria), Daphnia medium was associated with a strong reduction in cell-bound toxin concentration (P < 0.05). This study showed no increased cell aggregation under direct grazing (P > 0.05), but increased aggregation with spent Daphnia medium under high light conditions (P < 0.05). Further, the addition of cell-free extract from disrupted toxic Microcystis cells strongly increased the aggregation of the intact cells under low light (P < 0.05). These findings are discussed with the possible role of microcystin and other infochemicals in the expression of proteins and morphology changes in Microcystis.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

Roles of nitrogen and phosphorus in growth responses and toxin production (using LC-MS/MS) of tropical <Emphasis Type="Italic">Microcystis ichthyoblabe</Emphasis> and <Emphasis Type="Italic">M. flos-aquae</Emphasis>

In experiments investigating nutrient effects on tropical Microcystis, increasing nitrogen and phosphorus concentrations were found to have a significant positive effect on maximum cell yields of two strains of Microcystis ichthyoblabe (from Lower Peirce and Tengeh Reservoirs) and one strain of Microcystis flos-aquae isolated (Lower Peirce Reservoir) from Singapore. However, only increasing nitrogen concentration had a positive effect on growth rates of M. ichthyoblabe and M. flos-aquae from Lower Peirce Reservoir. MC-RR and MC-LR were produced by all three strains with MC-RR being the dominant variant. Phosphorus played an important role in MC production with increases in phosphorus from medium to high concentrations leading to decreases in MC-RR cell quotas for all three strains at the two highest nitrogen levels tested. The different growth and toxin production responses between M. ichthyoblabe strains could be due to location-specific differences.     

Freshwater anostracan, <Emphasis Type="Italic">Branchinella kugenumaensis</Emphasis>, as a potential controlling consumer species on toxic cyanobacteria <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

To evaluate the potential of Branchinella kugenumaensis for cyanobacterial bloom control relative to Daphnia, we conducted several feeding experiments on microcystin-free and microcystin-containing unicellular strains of Microcystis aeruginosa and colonial forms of Microcystis using B. kugenumaensis and Daphnia magna in a laboratory. Branchinella kugenumaensis showed higher filtration rates than those of D. magna in all treatments. In particular, the microcystin-containing unicellular strain supported the highest filtration rates of B. kugenumaensis among treatments. Daphnia magna reduced colonies less than 75 μm in length, whereas B. kugenumaensis could graze colonies less than 100 μm. The middle-sized group of B. kugenumaensis had a higher filtration rate than the small and large sized groups in a continuous feeding experiment for 4 days. In survival experiments, survivorships were not different between the two species, whereas ages at the beginning of the experiments affected their survival time. Our results showed that B. kugenumaensis grazed on toxic and colonial cyanobacteria at relatively high rates, indicating that locally abundant grazers like Branchinella may offer a better potential for bloom control than Daphnia.     

Adequacy of planctomycetes as supplementary food source for <Emphasis Type="Italic">Daphnia magna</Emphasis>

The nutritional quality of daphnids diet can influence their growth, reproduction and survival. In aquatic ecosystems, bacteria can contribute significantly to Daphnia diet by supporting, for instances, their high needs for phosphorus. The laboratory feeding of the model organisms Daphnia spp. is algal based, but should be improved to allow their better performance. The aim of this study was to evaluate the potential of two planctomycetes, Gemmata obscuriglobus and Rhodopirellula rubra, from exponential and stationary growth phases as alternative or supplementary food source for Daphnia magna. The actinobacterium Arthrobacter sp. was used for comparison. The feeding with only bacteria showed the inefficacy of both planctomycetes and actinobacteria as the only food source. However, when used in supplement to Raphidocelis subcapitata, a decrease in the age of first reproduction, a significant increase in reproductive output, in somatic growth and in rate of population increase was found for the highest cell densities of bacteria tested. The typical pink coloration of these bacteria present in daphnids body and eggs confirmed bacterial absorption and metabolization of their pigment. Planctomycetes yielded better results than the actinobacteria Arthrobacter but G. obscuriglobus that possesses sterols did not induce a better performance comparatively to R. rubra. No relation could be established between the feeding treatments that allowed improvement of Daphnia performance and the different kind of Daphnia’ Fatty Acid Methyl Esters. The use of sonication to separate planctomycetal cells before feeding the daphnids proved to be efficient. We confirmed that R. subcapitata supplemented by bacteria allows a better growth performance of D. magna.     

Composition and distribution of sibling species of <Emphasis Type="Italic">Chironomus</Emphasis> Meigen, 1803 (Diptera,Chironomidae) in Curonian Lagoon of the Baltic Sea

The species composition of Chironomus in the Curonian Lagoon has been investigated. It was shown that three sibling species of the plumosus group, namely, Ch. plumosus, Ch. balatonicus, and Ch. muratensis, and a first-generation interspecific hybrid Ch. muratensis × Ch. plumosus lived here. The occurrence frequency in the lagoon was 87, 19, 6, and 3% for Ch. plumosus, Ch. balatonicus, Ch. muratensis × Ch. plumosus, and Ch. muratensis, respectively. The species Ch. plumosus was recorded in all the areas (southern, central, and northern), while Ch. balatonicus was found only in the northern area in the village of Juodkrante near the city of Klaipeda. Presumably, the distribution of these sibling species is due to the presence of a salinity gradient in the lagoon.     

Dynamic variation of toxic and non-toxic <Emphasis Type="Italic">Microcystis</Emphasis> proportion in the eutrophic Daechung Reservoir in Korea

This study was conducted to determine the environmental factors affecting the level of potentially toxic Microcystis. The long-term tendencies of temperature, precipitation, and water quality factors were analyzed to determine the environmental characteristics of the Daechung Reservoir in Korea, and water samples were directly collected to analyze the dynamics of toxic and non-toxic Microcystis at weekly intervals from May to October 2012. Microcystis was the dominant genus during the study period, and it was composed of potentially toxic and non-toxic Microcystis. The fraction of potentially toxic Microcystis ranged from 6.0% to 61.1%. The amount of toxic Microcystis was highly related to the intracellular microcystin concentration (r = 0.760, P < 0.01). Therefore, the fraction of potentially toxic Microcystis is an important concern in Microcystis blooming because the intracellular microcystin concentration may reflect microcystin levels in the water. The prevalence of potentially toxic Microcystis was highly related to water temperature in Daechung Reservoir (r = 0.585, P < 0.01). Thus, temperature increase during Microcystis blooming may lead to more frequent toxic Microcystis blooms in eutrophic water bodies.     

Waterbird response indicates floodplain wetland restoration

Filamentous cyanobacteria disturb food collection in Daphnia by mechanical interference with the filtering apparatus by the long trichomes. The intensity of this interference depends on the water temperature and the Daphnia body size. However, Daphnia are capable of breaking down the filaments, therefore improving the palatability of the cyanobacteria. The main objective of this study was to test whether the shortening of cyanobacterial filaments and the ensuing clearance rate of Daphnia would increase at higher temperatures to a greater degree in small-bodied Daphnia species than in large-bodied one. Laboratory feeding experiments were conducted in order to measure variation in the length of Cylindrospermopsis raciborskii trichomes and to calculate clearance rate. The filament length and the cyanobacteria clearance rate by Daphnia were calculated following their exposure to grazing by large-bodied D. pulicaria and small-bodied D. longispina in 20, 24, and 28°C. Rising temperature did not affect the intensity of breakage of C. raciborskii trichomes by D. pulicaria and caused decrease in clearance rate of this species, whereas for D. longispina, the temperature increase enhanced both filament breakage and clearance rate. We suggest that these temperature-related changes may affect relative competitive performance of Daphnia species in the presence of cyanobacteria.     

Predator-induced plasticity does not alter the pathway from evolution to ecology among locally adapted populations of <Emphasis Type="Italic">Daphnia</Emphasis>

In the growing field of eco-evolutionary dynamics, evidence for an influence of rapid shifts in phenotype on ecological processes is accumulating, yet, the contributions of phenotypic plasticity versus genetic change to these observed ecological changes are unclear. In one of the best studied ecosystems in terms of eco-evolutionary dynamics, landlocked versus anadromous alewife (Alosa pseudoharengus) have caused strong evolutionary divergence in their key zooplankton prey (Daphnia ambigua). We previously showed that such evolutionary differences have cascading ecological effects on consumer-resource dynamics and primary production. Yet, these locally adapted populations of Daphnia also differ in trait plasticity, which may, in turn, modify the pathway from evolution to ecology. Here we compared Daphnia from lakes with landlocked versus anadromous alewife for differences in rates of population growth in the presence and absence of predator cues over the course of a 39-day experiment. We predicted that predator-induced shifts in life history traits would facilitate faster rates of population growth. Contrary to our expectations, predator cue exposure did not alter rates of population growth. We instead found that Daphnia from lakes with landlocked alewife ultimately attained higher population densities (and exhibited faster population growth) when compared with Daphnia from lakes with anadromous alewife. Based on our previous work, these population level responses were unexpected, as Daphnia from lakes with landlocked alewife exhibit slower rates of somatic growth and delayed maturation. We discuss our results in lieu of the known differences in plasticity and how the population growth patterns may be influenced by resource limitation.     

Horizontal distribution of the cell abundance and toxicity of <Emphasis Type="Italic">Microcystis</Emphasis> in a hypereutrophic Moroccan reservoir

The first results of the horizontal distribution of the cell abundance and toxicity of Microcystis in the hypereutrophic Moroccan reservoir Lalla Takerkoust are reported. An unexpected spatio-temporal heterogeneity has been shown between Microcystis abundance and microcystins concentrations. The principal determining factors were analyzed in order to identify the most likely sites for the proliferation and/or accumulation of Microcystis in this reservoir. The horizontal heterogeneity seems to be mainly influenced by the wind direction and inflows. The results can serve as reference data for monitoring cyanobacterial water blooms and associated cyanotoxins in the lake.     

Is bigger better? A possibility for adaptation of <Emphasis Type="Italic">Daphnia</Emphasis> to filamentous cyanobacteria in the face of global warming

Body size is a key determinant of fitness in Daphnia. Bigger size means higher feeding efficiency and reproduction. However, filamentous cyanobacteria have a more detrimental effect on larger daphnids. Predicted global warming is expected to reduce size of Daphnia and facilitate frequent occurrence of cyanobacteria. Therefore, we asked two questions: (i) does elevated temperature cause reduced size of daphnids, and (ii) is temperature-dependent size reduction adaptive in an environment dominated by filamentous cyanobacteria? To address these issues, we obtained 8 clones of the Daphnia longispina complex from artificially heated lakes and 8 clones from control lakes, and exposed them to a favorable food Scenedesmus obliquus and a mixture of S. obliquus and the filamentous cyanobacterium Cylindrospermopsis raciborskii in life table experiments at 16, 20, and 24°C. Individuals from heated lakes attained larger body size than those from control lakes. Moreover, exposure to the filamentous cyanobacterium led to reduced fecundity of Daphnia from the non-heated lakes and did not affect reproduction of Daphnia from the heated lakes. We conclude that Daphnia display some evolutionary adaptations to cope with filamentous cyanobacteria, linked to long-term exposition to elevated temperature. Our results violate broadly accepted assumptions that ectotherms are smaller in warmer environments.     

Demands of eicosapentaenoic acid (EPA) in <Emphasis Type="Italic">Daphnia</Emphasis>: are they dependent on body size?

Fatty acids contribute to the nutritional quality of the phytoplankton and, thus, play an important role in Daphnia nutrition. One of the polyunsaturated fatty acids (PUFAs)––eicosapentaenoic acid (EPA)––has been shown to predict carbon transfer between primary producers and consumers in lakes, suggesting that EPA limitation of Daphnia in nature is widespread. Although the demand for EPA must be covered by the diet, the demand of EPA in Daphnia that differ in body size has not been addressed yet. Here, we hypothesize that the demand for EPA in Daphnia is size-dependent and that bigger species have a higher EPA demand. To elucidate this, a growth experiment was conducted in which at 20 °C three Daphnia taxa (small-sized D. longispina complex, medium-sized D. pulicaria, and large-bodied D. magna) were fed Synechococcus elongatus supplemented with cholesterol and increasing concentrations of EPA. In addition, fatty acid analyses of Daphnia were performed. Our results show that the saturation threshold for EPA-dependent growth increased with increasing body size. This increase in thresholds with body size may provide another mechanism contributing to the prevalence of small-bodied cladocera in warm habitats and to the midsummer decline of large cladocera in eutrophic water bodies.     

Genetic polymorphism,haplotype distribution,and phylogeny of <Emphasis Type="Italic">Daphnia</Emphasis> (Cladocera: Anomopoda) species from the water bodies of russia as inferred from the 16S mtDNA gene sequencing

The data on the genetic polymorphism of the most widespread Daphnia species occupying different water bodies of Russia are presented. The phylogenetic relationships between the examined species were established, and the haplotype networks were constructed. A fragment of the 16S mitochondrial DNA gene was used as a genetic marker. The results of molecular phylogenetic analysis generally coincided with modern concepts in the systematics of the genus Daphnia. The representatives of the divergent mitochondrial lineages within the D. longispina, D. pulex, and D. magna complex remain poorly investigated morphologically. For D. dentifera, a new habitat on the territory of Russia, namely, the water bodies of the Lake Baikal basin, was identified. A conclusion was made that the 16S mtDNA gene could be successfully used in phylogeographic analysis of the genus Daphnia.     

Morphological analyses allow to separate <Emphasis Type="Italic">Branchipus</Emphasis> species (Branchiopoda,Anostraca) from different geographic regions

Daphnia perform diel vertical migration (DVM), a predator-avoidance strategy to migrate towards deeper and colder layers in the water column in the morning and movement to the algae-rich surface layers in the evening. However, individuals performing DVM incur several trade-offs since they might suffer from resource limitation and a slower instantaneous birth rate in deeper depths. DVM patterns may be modified by abiotic factors such as temperature, food concentration, or pH and vary among different Daphnia species and genotypes. Furthermore, Daphnia host a variety of microparasites that might pose an additional factor influencing DVM behaviour. For infected individuals, migration into cooler temperature layers might slow down parasite growth. Moreover, parasites can increase opacity of their hosts. Non-migrating individuals might then be selectively purged from the upper layers by visually hunting predators. With these premises we asked, whether epidemics of the ichthyosporean parasite Caullerya mesnili affect or are affected by the DVM behaviour of Daphnia in Lake Greifensee, Switzerland by analysing the vertical distribution of Daphnia during day and night on two dates. Furthermore, we were interested whether a potential interaction depends on host genotype. We therefore studied the genotypic composition of the integrated population in regular sampling intervals over the course of one year and on a fine-grained vertical resolution during the Caullerya epidemic in late summer. Since Caullerya-infected Daphnia migrated equally well as uninfected ones, the findings of this study suggest that Caullerya epidemics neither affected nor were affected by the DVM behaviour of Daphnia. We observed clonal succession in the lake but could not link this succession to the Caullerya epidemic; all except one of the common multilocus genotypes were under-infected. In addition, outbreak and course of this Caullerya epidemic seemed to rely mainly on environmental cues. Because this first study only provides a snapshot of time, we hope that further studies will be done to verify our results.     

<Emphasis Type="Italic">Lecanicillium muscarium</Emphasis> and <Emphasis Type="Italic">Adalia bipunctata</Emphasis> combination for the control of black bean aphid<Emphasis Type="Italic">, Aphis fabae</Emphasis>

The predator Adalia bipunctata (Coleoptera: Coccinellidae) and the entomopathogenic fungus Lecanicillium muscarium, have been considered as potential biological control against aphids. While it can be difficult to achieve a high control level of Aphis fabae Scopoli (Hemiptera: Aphididae) using only a single beneficial agent, the research presented here aimed to determine the interaction between L. muscarium and A. bipunctata potential for control against A. fabae. Lecanicillium muscarium was found to cause about 30% mortality in A. bipunctata and with a reduction in feeding by about 15%. However, co-application of A. bipunctata and L. muscarium can cause an addititive effect in reducing aphid populations, resulting in about 90% reduction in aphid populations compared with control treatment. Thus, these two biocontrol agents have the potential to be complementary. This research study demonstrates that it is possible to combine A. bipunctata with L. muscarium to provide a sustainable method for management of A. fabae on broad bean cropping system and that field studies are required.     

Threshold Concentrations of Cations in the Water Necessary for Maintaining the Ionic Balance Between Organism of <Emphasis Type="Italic">Chironomus balatonicus</Emphasis> Devai et al. Larvae and Environment

Threshold concentrations of sodium, potassium, calcium, and magnesium in water determining the borders of the Chironomus balatonicus range in waterbodies are 0.6–0.62, 0.11–0.12, 0.071–0.073, and 0.022–0.028 mmol/L, respectively. In the freshwater section of the Curonian Lagoon of the Baltic Sea and other waterbodies with weakly mineralized water where the content of ions is lower than the threshold values, larval Ch. balatonicus cannot dwell because of the impossibility of maintaining the ionic balance between the organism and environment. The threshold concentrations and rates of loss of ions from an organism in various species of aquatic organisms are comparatively analyzed.     

Potential for biocontrol of the exotic starfish<Emphasis Type="Italic">, Asterias amurensis</Emphasis>, using a native starfish

Asterias amurensis is a starfish native to the northern Pacific that was introduced into southern Australia in the 1980s. It is widely viewed as one of Australia’s most serious invasive marine pests, but there are few methods available to control new or established populations. The role of Coscinasterias muricata in controlling the distribution of Asterias in Port Phillip Bay, and its potential to eliminate new infestations of Asterias were investigated. Laboratory feeding trials, where alternative mussel prey were available, showed Coscinasterias consumed Asterias at the rate of ~45/year. Field surveys in Port Phillip Bay showed that most Coscinasterias occurred at depths shallower than 15 m, while most Asterias occurred at depths greater than 15 m, and the ratio of Asterias/Coscinasterias only exceeded 45 at depths greater than 15 m. Consequently, if laboratory and field feeding rates are similar, Coscinasterias would be expected to exert significant control over Asterias populations at depths shallower than 15 m, and augmenting Coscinasterias populations at sites of new Asterias infestations may help eliminate newly-established populations.     

Species and clone-dependent effects of tilapia fish (Cichlidae) on the morphology and life-history of temperate and tropical <Emphasis Type="Italic">Daphnia</Emphasis>

In aquatic systems, tilapia introductions may result in marked changes in the structure of prey communities. In this study, we experimentally examined the effects of tilapia-mediated water at the individual and population levels of prey by exposing three Daphnia species to predation cues. We hypothesized that tilapia-mediated water determines reduced age and size at primipara, greater and faster reproduction, enhanced intrinsic rates of population increase (r), and longer tail spines in Daphnia; but that the magnitude of these changes would be species and clone-dependent. When three tropical D. laevis and one temperate D. similis clones were exposed to predation cues, adaptive changes were observed in some of the aforementioned parameters for each clone. The three D. laevis clones exhibited changes in all life-history and morphological measures. Temperate Daphnia spinulata displayed no changes but decreased r values in the presence of predators. The observed changes in the species and clones tested here suggest that, overall, both temperate and tropical Daphnia can detect and adaptively react to the risk of tilapia predation. However, only a fraction of the possible defenses may be displayed by individual clones. In contrast, D. spinulata seems more vulnerable to tilapia predation, given its long body length and absence of adaptive changes. Our study indicates that Daphnia can respond to tilapia-mediated water, and that interspecific and clonal variation exists between temperate and tropical species.     

Low temperature and <Emphasis Type="Italic">Daphnia</Emphasis>-associated infochemicals promote colony formation of <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> and its harvesting

Objective

To explore the combined effects of temperature and Daphnia-associated infochemicals on colony formation of Scenedesmus obliquus to faciliate harvesting the algal biomass.

Results

A three-parameter modified Gaussian model fitted the changes of the number of cells per particle in S. obliquus induced by Daphnia culture filtrate well under any temperature. Decreases in temperature enhanced the induced–colony formation of Scenedesmus. The maximum colony size at 15–25 °C was significantly larger than those at 30–35 °C. An additional 1 or 2 days at low temperature was needed to reach the maximum colony size, which indicates the best harvest time for algal biomass.

Conclusion

Induced-colony formation of Scenedesmus by Daphnia culture filtrate at 15–25 °C is recommended to settle algal cells. This condition facilitates harvesting the biomass.

     

Trophic plasticity among spring vs. cave populations of <Emphasis Type="Italic">Gammarus minus:</Emphasis> examining functional niches using stable isotopes and C/N ratios

In some environments, species may exhibit trophic plasticity, which allows them to extend beyond their assigned functional group. For Gammarus minus, a freshwater amphipod classified as a shredder or detritivore, cave populations have been observed consuming heterotrophs as well as shredding leaves, and therefore may be exhibiting trophic plasticity. To test this possibility, we examined the C and N stable isotope and C/N ratios for cave and spring populations of G. minus. A 15-day feeding experiment using leaves and G. minus from a spring population established that the diet-tissue discrimination factor was 3.2 ‰ for δ¹⁵N. Cave G. minus were 8 ‰ higher in δ¹⁵N relative to cave leaves, indicating they did not derive nitrogen from leaves, whereas field collected spring populations were 2–3 ‰ higher than spring leaves, indicating that they did. Cave G. minus were 2.6 ‰ higher in δ¹⁵N than the cave isopod, Caecidotea holsingeri. Relative to spring populations, Organ Cave G. minus were ¹⁵N enriched by 6 ‰, suggesting they occupied a different trophic level, or incorporated an isotopically distinct N source. While stable isotopes cannot tell what the cave G. minus are eating, the isotopes certainly show that G. minus are not eating leaves and are trophically distinct form the surface populations. Differences in C/N ratios were observed, but reflect the size of the G. minus examined and not feeding group or habitat. The isotope data strongly support the hypothesis that cave populations of G. minus have become generalist or omnivorous by including animal protein in their diet.