Biodiversity in marine invertebrate responses to acute warming revealed by a comparative multi-omics approach.

Understanding species' responses to environmental change underpins our abilities to make predictions on future biodiversity under any range of scenarios. In spite of the huge biodiversity in most ecosystems, a model species approach is often taken in environmental studies. To date, we still do not know how many species we need to study to input into models and inform on ecosystem-level responses to change. In this study, we tested current paradigms on factors setting thermal limits by investigating the acute warming response of six Antarctic marine invertebrates: a crustacean Paraceradocus miersi, a brachiopod Liothyrella uva, two bivalve molluscs, Laternula elliptica, Aequiyoldia eightsii, a gastropod mollusc Marseniopsis mollis and an echinoderm Cucumaria georgiana. Each species was warmed at the rate of 1 °C h-1 and taken to the same physiological end point (just prior to heat coma). Their molecular responses were evaluated using complementary metabolomics and transcriptomics approaches with the aim of discovering the underlying mechanisms of their resilience or sensitivity to warming. The responses were species-specific; only two showed accumulation of anaerobic end products and three exhibited the classical heat shock response with expression of HSP70 transcripts. These diverse cellular measures did not directly correlate with resilience to heat stress and suggested that each species may have a different critical point of failure. Thus, one unifying molecular mechanism underpinning response to warming could not be assigned, and no overarching paradigm was supported. This biodiversity in response makes future ecosystems predictions extremely challenging, as we clearly need to develop a macrophysiology-type approach to cellular evaluations of the environmental stress response, studying a range of well-rationalized members from different community levels and of different phylogenetic origins rather than extrapolating from one or two arbitrary model species.

Molecular snapshot of an intracellular freezing event in an Antarctic nematode.

The Antarctic nematode, Panagrolaimus sp. DAW1 (formerly called Panagrolaimus davidi), is the best documented example of an organism able to survive intracellular ice formation in all of its compartments. Not only is it able to survive such extreme physiological disruption, but it is able to produce progeny once thawed from such a state. In addition, under slower rates, or less extreme degrees, of cooling, its body remains unfrozen and the vapour pressure difference between the supercooled body fluids and the surrounding ice leads to a process termed cryoprotective dehydration. In contrast to a fairly large body of work in building up our molecular understanding of cryoprotective dehydration, no comparable work has been undertaken on intracellular freezing. This paper describes an experiment subjecting cultures of Panagrolaimus sp. DAW1 to a range of temperatures including a rapid descent to -10 °C, in a medium just prior to, and after, freezing. Through deep sequencing of RNA libraries we have gained a snapshot of which genes are highly abundant when P. sp. DAW1 is undergoing an intracellular freezing event. The onset of freezing correlated with a high production of genes involved in cuticle formation and subsequently, after 24 h in a frozen state, protease production. In addition to the mapping of RNA sequencing, we have focused on a select set of genes arising both from the expression profiles, as well as implicated from other cold tolerance studies, to undertake qPCR. Among the most abundantly represented transcripts in the RNA mapping is the zinc-metalloenzyme, neprilysin, which also shows a particularly strong upregulated signal through qPCR once the nematodes have frozen.

High-throughput sequencing reveals inbreeding depression in a natural population.

Proxy measures of genome-wide heterozygosity based on approximately 10 microsatellites have been used to uncover heterozygosity fitness correlations (HFCs) for a wealth of important fitness traits in natural populations. However, effect sizes are typically very small and the underlying mechanisms remain contentious, as a handful of markers usually provides little power to detect inbreeding. We therefore used restriction site associated DNA (RAD) sequencing to accurately estimate genome-wide heterozygosity, an approach transferrable to any organism. As a proof of concept, we first RAD sequenced oldfield mice (Peromyscus polionotus) from a known pedigree, finding strong concordance between the inbreeding coefficient and heterozygosity measured at 13,198 single-nucleotide polymorphisms (SNPs). When applied to a natural population of harbor seals (Phoca vitulina), a weak HFC for parasite infection based on 27 microsatellites strengthened considerably with 14,585 SNPs, the deviance explained by heterozygosity increasing almost fivefold to a remarkable 49%. These findings arguably provide the strongest evidence to date of an HFC being due to inbreeding depression in a natural population lacking a pedigree. They also suggest that under some circumstances heterozygosity may explain far more variation in fitness than previously envisaged.

Deciphering the molecular adaptation of the king scallop (Pecten maximus) to heat stress using transcriptomics and proteomics.

BACKGROUND: The capacity of marine species to survive chronic heat stress underpins their ability to survive warming oceans as a result of climate change. In this study RNA-Seq and 2-DE proteomics were employed to decipher the molecular response of the sub-tidal bivalve Pecten maximus, to elevated temperatures. RESULTS: Individuals were maintained at three different temperatures (15, 21 and 25 °C) for 56 days, representing control conditions, maximum environmental temperature and extreme warming, with individuals sampled at seven time points. The scallops thrived at 21 °C, but suffered a reduction in condition at 25 °C. RNA-Seq analyses produced 26,064 assembled contigs, of which 531 were differentially expressed, with putative annotation assigned to 177 transcripts. The proteomic approach identified 24 differentially expressed proteins, with nine identified by mass spectrometry. Network analysis of these results indicated a pivotal role for GAPDH and AP-1 signalling pathways. Data also suggested a remodelling of the cell structure, as revealed by the differential expression of genes involved in the cytoskeleton and cell membrane and a reduction in DNA repair. They also indicated the diversion of energetic metabolism towards the mobilization of lipid energy reserves to fuel the increased metabolic rate at the higher temperature. CONCLUSIONS: This work provides preliminary insights into the response of P. maximus to chronic heat stress and provides a basis for future studies examining the tipping points and energetic trade-offs of scallop culture in warming oceans.

Variability among individuals is generated at the gene expression level.

Selection acts on individuals, specifically on their differences. To understand adaptation and responses to change therefore requires knowledge of how variation is generated and distributed across traits. Variation occurs on different biological scales, from genetic through physiological to morphological, yet it is unclear which of these carries the most variability. For example, if individual variation is mainly generated by differences in gene expression, variability should decrease progressively from coding genes to morphological traits, whereas if post-translational and epigenetic effects increase variation, the opposite should occur. To test these predictions, we compared levels of variation among individuals in various measures of gene expression, physiology (including activity), and morphology in two abundant and geographically widespread Antarctic molluscs, the clam Laternula elliptica and the limpet Nacella concinna. Direct comparisons among traits as diverse as heat shock protein QPCR assays, whole transcription profiles, respiration rates, burying rate, shell length, and ash-free dry mass were made possible through the novel application of an established metric, the Wentworth Scale. In principle, this approach could be extended to analyses of populations, communities, or even entire ecosystems. We found consistently greater variation in gene expression than morphology, with physiological measures falling in between. This suggests that variability is generated at the gene expression level. These findings have important implications for refining current biological models and predictions of how biodiversity may respond to climate change.

Adult acclimation to combined temperature and pH stressors significantly enhances reproductive outcomes compared to short-term exposures.

This study examined the effects of long-term culture under altered conditions on the Antarctic sea urchin, Sterechinus neumayeri. Sterechinus neumayeri was cultured under the combined environmental stressors of lowered pH (-0.3 and -0.5 pH units) and increased temperature (+2 °C) for 2 years. This time-scale covered two full reproductive cycles in this species and analyses included studies on both adult metabolism and larval development. Adults took at least 6-8 months to acclimate to the altered conditions, but beyond this, there was no detectable effect of temperature or pH. Animals were spawned after 6 and 17 months exposure to altered conditions, with markedly different outcomes. At 6 months, the percentage hatching and larval survival rates were greatest in the animals kept at 0 °C under current pH conditions, whilst those under lowered pH and +2 °C performed significantly less well. After 17 months, performance was not significantly different across treatments, including controls. However, under the altered conditions urchins produced larger eggs compared with control animals. These data show that under long-term culture adult S. neumayeri appear to acclimate their metabolic and reproductive physiology to the combined stressors of altered pH and increased temperature, with relatively little measureable effect. They also emphasize the importance of long-term studies in evaluating effects of altered pH, particularly in slow developing marine species with long gonad maturation times, as the effects of altered conditions cannot be accurately evaluated unless gonads have fully matured under the new conditions.

Interaction strengths in balanced carbon cycles and the absence of a relation between ecosystem complexity and stability.

The strength of interactions is crucial to the stability of ecological networks. However, the patterns of interaction strengths in mathematical models of ecosystems have not yet been based upon independent observations of balanced material fluxes. Here we analyse two Antarctic ecosystems for which the interaction strengths are obtained: (1) directly, from independently measured material fluxes, (2) for the complete ecosystem and (3) with a close match between species and 'trophic groups'. We analyse the role of recycling, predation and competition and find that ecosystem stability can be estimated by the strengths of the shortest positive and negative predator-prey feedbacks in the network. We show the generality of our explanation with another 21 observed food webs, comparing random-type parameterisations of interaction strengths with empirical ones. Our results show how functional relationships dominate over average-network topology. They make clear that the classic complexity-instability paradox is essentially an artificial interaction-strength result.

Transcriptome of the dead: characterisation of immune genes and marker development from necropsy samples in a free-ranging marine mammal.

BACKGROUND: Transcriptomes are powerful resources, providing a window on the expressed portion of the genome that can be generated rapidly and at low cost for virtually any organism. However, because many genes have tissue-specific expression patterns, developing a complete transcriptome usually requires a 'discovery pool' of individuals to be sacrificed in order to harvest mRNA from as many different types of tissue as possible. This hinders transcriptome development in large, charismatic and endangered species, many of which stand the most to gain from such approaches. To circumvent this problem in a model pinniped species, we 454 sequenced cDNA from testis, heart, spleen, intestine, kidney and lung tissues obtained from nine adult male Antarctic fur seals (Arctocephalus gazella) that died of natural causes at Bird Island, South Georgia. RESULTS: After applying stringent quality control criteria based on length and annotation, we obtained 12,397 contigs which, in combination with 454 data previously obtained from skin, gave a total of 23,096 unique contigs. Homology was found to 77.0% of dog (Canis lupus familiaris) transcripts, suggesting that the combined assembly represents a substantial proportion of this species' transcriptome. Moreover, only 0.5% of transcripts revealed sequence similarity to bacteria, implying minimal contamination, and the percentage of transcripts involved in cell death was low at 2.6%. Transcripts with immune-related annotations were almost five-fold enriched relative to skin and represented 13.2% of all spleen-specific contigs. By reference to the dog, we also identified transcripts revealing homology to five class I, ten class II and three class III genes of the Major Histocompatibility Complex and derived the putative genomic distribution of 17,121 contigs, 2,119 in silico mined microsatellites and 9,382 single nucleotide polymorphisms. CONCLUSIONS: Our findings suggest that transcriptome development based on samples collected post mortem may greatly facilitate genomic studies, not only of marine mammals but also more generally of species that are of conservation concern.

Physiological plasticity, long term resistance or acclimation to temperature, in the Antarctic bivalve, Laternula elliptica.

To further investigate the previously reported limited acclimation capacities of Antarctic marine stenotherms, the Antarctic mud clam, Laternula elliptica (King and Broderip, 1830-1831), was incubated at 3.0°C for 89days. The thermal windows of a suite of biochemical and physiological metrics that characterise tissue aerobic status, were then measured in response to acute temperature elevation (2-2.5°C increase per week). To test if acclimation had occurred at the higher temperature, results were compared with published data, from the preceding year, for L. elliptica which had been incubated at ambient temperature (0.0°C) and then subjected to the same acute temperature treatments. Incubation to 3.0°C led to a temperature induced increase of tissue aerobic status (reduced intracellular cCO(2) with increased O(2) consumption, PLA (phospho-L-arginine) and ATP). At the highest acute temperature (7.5°C) the increase in anaerobic pathways (summed acetate/succinate and propionate) was less after 3.0°C than 0.0°C incubation. No other metric shifted its reaction norm in response to acute temperature elevation and so whole animal acclimation had not occurred, even after 3months at 3.0°C. Combined with the constant mortality throughout the 3.0°C incubation period, these data suggest that the recorded physiological changes were either the early stages of acclimation or, more likely, time limited resistance mechanisms.

Skin healing and scale regeneration in fed and unfed sea bream, Sparus auratus.

BACKGROUND: Fish scales are an important reservoir of calcium and phosphorus and together with the skin function as an integrated barrier against environmental changes and external aggressors. Histological studies have revealed that the skin and scales regenerate rapidly in fish when they are lost or damaged. In the present manuscript the histological and molecular changes underlying skin and scale regeneration in fed and fasted sea bream (Sparus auratus) were studied using a microarray 3 and 7 days after scale removal to provide a comprehensive molecular understanding of the early stages of these processes. RESULTS: Histological analysis of skin/scales revealed 3 days after scale removal re-epithelisation and formation of the scale pocket had occurred and 53 and 109 genes showed significant up or down-regulation, respectively. Genes significantly up-regulated were involved in cell cycle regulation, cell proliferation and adhesion, immune response and antioxidant activities. 7 days after scale removal a thin regenerated scale was visible and only minor changes in gene expression occurred. In animals that were fasted to deplete mineral availability the expression profiles centred on maintaining energy homeostasis. The utilisation of fasting as a treatment emphasised the competing whole animal physiological requirements with regard to barrier repair, infection control and energy homeostasis. CONCLUSIONS: The identification of numerous genes involved in the mitotic checkpoint and cell proliferation indicate that the experimental procedure may be useful for understanding cell proliferation and control in vertebrates within the context of the whole animal physiology. In response to skin damage genes of immune surveillance were up-regulated along with others involved in tissue regeneration required to rapidly re-establish barrier function. Additionally, candidate fish genes were identified that may be involved in cytoskeletal re-modelling, mineralization and stem cells, which are of potential use in aquaculture and fish husbandry, as they may impact on the ability of the fish to produce structural proteins, such as muscle, efficiently.

Correction: Proteomics of intracellular freezing survival.

[This corrects the article DOI: 10.1371/journal.pone.0233048.].

Proteomics of intracellular freezing survival.

Panagrolaimus sp. DAW1, a nematode cultured from the Antarctic, has the extraordinary physiological ability to survive total intracellular freezing throughout all of its compartments. While a few other organisms, all nematodes, have subsequently also been found to survive freezing in this manner, P. sp. DAW1 has so far shown the highest survival rates. In addition, P. sp. DAW1 is also, depending on the rate or extent of freezing, able to undergo cryoprotective dehydration. In this study, the proteome of P. sp DAW1 is explored, highlighting a number of differentially expressed proteins and pathways that occur when the nematodes undergo intracellular freezing. Among the strongest signals after being frozen is an upregulation of proteases and the downregulation of cytoskeletal and antioxidant activity, the latter possibly accumulated before freezing much in the way the sugar trehalose has been shown to be stored during acclimation.

Discovering genes associated with dormancy in the monogonont rotifer Brachionus plicatilis.

BACKGROUND: Microscopic monogonont rotifers, including the euryhaline species Brachionus plicatilis, are typically found in water bodies where environmental factors restrict population growth to short periods lasting days or months. The survival of the population is ensured via the production of resting eggs that show a remarkable tolerance to unfavorable conditions and remain viable for decades. The aim of this study was to generate Expressed Sequence Tags (ESTs) for molecular characterisation of processes associated with the formation of resting eggs, their survival during dormancy and hatching. RESULTS: Four normalized and four subtractive libraries were constructed to provide a resource for rotifer transcriptomics associated with resting-egg formation, storage and hatching. A total of 47,926 sequences were assembled into 18,000 putative transcripts and analyzed using both Blast and GO annotation. About 28-55% (depending on the library) of the clones produced significant matches against the Swissprot and Trembl databases. Genes known to be associated with desiccation tolerance during dormancy in other organisms were identified in the EST libraries. These included genes associated with antioxidant activity, low molecular weight heat shock proteins and Late Embryonic Abundant (LEA) proteins. Real-time PCR confirmed that LEA transcripts, small heat-shock proteins and some antioxidant genes were upregulated in resting eggs, therefore suggesting that desiccation tolerance is a characteristic feature of resting eggs even though they do not necessarily fully desiccate during dormancy. The role of trehalose in resting-egg formation and survival remains unclear since there was no significant difference between resting-egg producing females and amictic females in the expression of the tps-1 gene. In view of the absence of vitellogenin transcripts, matches to lipoprotein lipase proteins suggest that, similar to the situation in dipterans, these proteins may serve as the yolk proteins in rotifers. CONCLUSION: The 47,926 ESTs expand significantly the current sequence resource of B. plicatilis. It describes, for the first time, genes putatively associated with resting eggs and will serve as a database for future global expression experiments, particularly for the further identification of dormancy related genes.

Estradiol and genistein effects on the sea bass (Dicentrarchus labrax) scales: Transcriptome dataset.

Fish scales are mineralized structures that play important roles in protection and mineral homeostasis. This tissue expresses multiple estrogen receptor subtypes and can be targeted by estrogens or estrogenic endocrine-disrupting compounds, but their effects are poorly explored. The transcriptome data here presented support the findings reported in the research article "Genistein and estradiol have common and specific impacts on the sea bass (Dicentrarchus labrax) skin-scale barrier" [1]. Juvenile sea bass were exposed to estradiol and the phytoestrogen genistein for 1 and 5 days, by intraperitoneal injections, and the effects on scale transcript expression were analysed by RNA-seq using an Illumina Hi-seq 1500. The raw reads of the 30 libraries produced have been deposited in the NCBI-SRA database with the project accession number SRP102504. Mapping of RNA-seq reads against the sea bass reference genome using the Cufflinks/TopHat package identified 371 genes that had significant (FDR<0.05) differential expression with the estradiol or genistein treatments in relation to the control scales at each exposure time, 254 of which presented more than a 2-fold change in expression. The identity of the differentially expressed genes was obtained using both automatic and manual annotations against multiple public sequence databases and they were grouped according to their patterns of expression using hierarchical clustering and heat-maps. The biological processes and KEGG pathways most significantly affected by the estradiol and/or genistein treatments were identified using Cytoscape/ClueGO enrichment analyses.

Genistein and estradiol have common and specific impacts on the sea bass (Dicentrarchus labrax) skin-scale barrier.

Teleost fish scales play important roles in animal protection and homeostasis. They can be targeted by endogenous estrogens and by environmental estrogenic endocrine disruptors. The phytoestrogen genistein is ubiquitous in the environment and in aquaculture feeds and is a disruptor of estrogenic processes in vertebrates. To test genistein disrupting actions in teleost fish we used a minimally invasive approach by analysing scales plucked from the skin of sea bass (Dicentrarchus labrax). Genistein transactivated all three fish nuclear estrogen receptors and was most potent with the Esr2, had the highest efficacy with Esr1, but reached, in all cases, transactivation levels lower than those of estradiol. RNA-seq revealed 254 responsive genes in the sea bass scales transcriptome with an FDR < 0.05 and more than 2-fold change in expression, 1 or 5 days after acute exposure to estradiol or to genistein. 65 genes were specifically responsive to estradiol and 106 by genistein while 83 genes were responsive to both compounds. Estradiol specifically regulated genes of protein/matrix turnover and genistein affected sterol biosynthesis and regeneration, while innate immune responses were affected by both compounds. This comprehensive study revealed the impact on the fish scale transcriptome of estradiol and genistein, providing a solid background to further develop fish scales as a practical screening tool for endocrine disrupting chemicals in teleosts.

Molecular mechanisms underpinning transgenerational plasticity in the green sea urchin Psammechinus miliaris.

The pre-conditioning of adult marine invertebrates to altered conditions, such as low pH, can significantly impact offspring outcomes, a process which is often referred to as transgenerational plasticity (TGP). This study describes for the first time, the gene expression profiles associated with TGP in the green sea urchin Psammechinus miliaris and evaluates the transcriptional contribution to larval resilience. RNA-Seq was used to determine how the expression profiles of larvae spawned into low pH from pre-acclimated adults differed to those of larvae produced from adults cultured under ambient pH. The main findings demonstrated that adult conditioning to low pH critically pre-loads the embryonic transcriptional pool with antioxidants to prepare the larvae for the "new" conditions. In addition, the classic cellular stress response, measured via the production of heat shock proteins (the heat shock response (HSR)), was separately evaluated. None of the early stage larvae either spawned in low pH (produced from both ambient and pre-acclimated adults) or subjected to a separate heat shock experiment were able to activate the full HSR as measured in adults, but the capacity to mount an HSR increased as development proceeded. This compromised ability clearly contributes to the vulnerability of early stage larvae to acute environmental challenge.

Signatures of the Evolution of Parthenogenesis and Cryptobiosis in the Genomes of Panagrolaimid Nematodes.

Most animal species reproduce sexually and fully parthenogenetic lineages are usually short lived in evolution. Still, parthenogenesis may be advantageous as it avoids the cost of sex and permits colonization by single individuals. Panagrolaimid nematodes have colonized environments ranging from arid deserts to Arctic and Antarctic biomes. Many are obligatory meiotic parthenogens, and most have cryptobiotic abilities, being able to survive repeated cycles of complete desiccation and freezing. To identify systems that may contribute to these striking abilities, we sequenced and compared the genomes and transcriptomes of parthenogenetic and outcrossing panagrolaimid species, including cryptobionts and non-cryptobionts. The parthenogens are triploids, most likely originating through hybridization. Adaptation to cryptobiosis shaped the genomes of panagrolaimid nematodes and is associated with the expansion of gene families and signatures of selection on genes involved in cryptobiosis. All panagrolaimids have acquired genes through horizontal gene transfer, some of which are likely to contribute to cryptobiosis.

Cold hardening processes in the Antarctic springtail, Cryptopygus antarcticus: clues from a microarray.

The physiology of the Antarctic microarthropod, Cryptopygus antarcticus, has been well studied, particularly with regard to its ability to withstand low winter temperatures. However, the molecular mechanisms underlying this phenomenon are still poorly understood. 1180 sequences (Expressed Sequence Tags or ESTs) were generated and analysed, from populations of C. antarcticus. This represents the first publicly available sequence data for this species. A sub-set (672 clones) were used to generate a small microarray to examine the differences in gene expression between summer acclimated cold tolerant and non-cold tolerant springtails. Although 60% of the clones showed no sequence similarity to annotated genes in the datasets, of those where putative function could be inferred via database homology, there was a clear pattern of up-regulation of structural proteins being associated with the cold tolerant group. These structural proteins mainly comprised cuticle proteins and provide support for the recent theory that summer SCP variation within Collembola species could be a consequence of moulting, with moulting population having lowered SCPs.

Investigating trehalose synthesis genes after cold acclimation in the Antarctic nematode Panagrolaimus sp. DAW1.

Panagrolaimus sp. DAW1 is a freeze-tolerant Antarctic nematode which survives extensive intracellular ice formation. The molecular mechanisms of this extreme adaptation are still poorly understood. We recently showed that desiccation-enhanced RNA interference (RNAi) soaking can be used in conjunction with quantitative polymerase chain reaction (qPCR) to screen for phenotypes associated with reduced expression of candidate genes in Panagrolaimus sp. DAW1. Here, we present the use of this approach to investigate the role of trehalose synthesis genes in this remarkable organism. Previous studies have shown that acclimating Panagrolaimus sp. DAW1 at 5°C before freezing or desiccation substantially enhances survival. In this study, the expression of tps-2 and other genes associated with trehalose metabolism, as well as lea-1, hsp-70 and gpx-1, in cold-acclimated and non-acclimated nematodes was analyzed using qPCR. Pd-tps-2 and Pd-lea-1 were significantly upregulated after cold acclimation, indicating an inducible expression in the cold adaptation of Panagrolaimus sp. DAW1. The role of trehalose synthesis genes in Panagrolaimus sp. DAW1 was further investigated by RNAi. Compared to the controls, Pd-tps-2a(RNAi)-treated and cold-acclimated nematodes showed a significant decrease in mRNA, but no change in trehalose content or freezing survival. The involvement of two other trehalose synthesis genes (tps-2b and gob-1) was also investigated. These findings provide the first functional genomic investigation of trehalose synthesis genes in the non-model organism Panagrolaimus sp. DAW1. The presence of several trehalose synthesis genes with different RNAi sensitivities suggests the existence of multiple backup systems in Panagrolaimus sp. DAW1, underlining the importance of this sugar in preparation for freezing.

Beyond connectedness: why pairwise metrics cannot capture community stability.

The connectedness of species in a trophic web has long been a key structural characteristic for both theoreticians and empiricists in their understanding of community stability. In the past decades, there has been a shift from focussing on determining the number of interactions to taking into account their relative strengths. The question is: How do the strengths of the interactions determine the stability of a community? Recently, a metric has been proposed which compares the stability of observed communities in terms of the strength of three- and two-link feedback loops (cycles of interaction strengths). However, it has also been suggested that we do not need to go beyond the pairwise structure of interactions to capture stability. Here, we directly compare the performance of the feedback and pairwise metrics. Using observed food-web structures, we show that the pairwise metric does not work as a comparator of stability and is many orders of magnitude away from the actual stability values. We argue that metrics based on pairwise-strength information cannot capture the complex organization of strong and weak links in a community, which is essential for system stability.