Toxicity of triclosan, an antimicrobial agent, to a nontarget freshwater zooplankton species, Moina macrocopa.

The toxicity of triclosan (TCS) on the freshwater cladoceran Moina macrocopa was investigated by acute and chronic toxicity assessments followed by genotoxicity and oxidative stress response analyses. The 48-h LC50 of TCS for ≤24-h-old M. macrocopa was determined as 539 µg L-1 . Chronic exposure to TCS at concentrations ranging from 5 to 100 µg L-1 showed a stimulatory effect at low concentrations (≤10 µg L-1 ) and an inhibitory effect at high concentrations (≥50 µg L-1 ) on growth, reproduction, and population-growth-related parameters of M. macrocopa. The genotoxicity test results indicated that TCS concentrations ranging from 50 to 100 µg L-1 can alter individuals' DNA. Analysis of the antioxidant enzymes catalase (CAT) and glutathione s-transferase (GST) demonstrated increased levels of these enzymes at high TCS concentrations. Our results indicated that TCS concentrations found in the natural environment have minimal acute toxicity to M. macrocopa. However, TCS at even low concentrations can significantly affect its growth, reproduction, and population-growth-related characteristics. The observed responses suggest a hormetic dose-response pattern and imply a potential endocrine-disrupting effect of TCS. Our molecular and biochemical findings indicated that high concentrations of TCS have the potential to induce oxidative stress that may lead to DNA alterations in M. macrocopa.

Contamination of aquatic environment with anticancer reagents influences Daphnia magna - Ecotoxicogenomics approach.

Pharmaceuticals used in human medicine contaminate freshwater ecosystems. Chemotherapeutics applied in cancer treatment are found in freshwaters at low concentrations (in the range of ng L-1) which, however, can be toxic or mutagenic to aquatic organisms. The aim of this study was to determine the impact of the alkylating/crosslinking anticancer agents, cyclophosphamide (CP) and cisplatin (CDDP), at the concentration detected in water, on Daphnia magna life history, transcriptome, and proteome. This filter feeding cladoceran is an important member of the aquatic food webs controlling algal biomass and forming basic food for planktivorous fish. Here, observations of the D. magna growth rate, age at first reproduction, and the number of eggs produced were performed in the presence of CP or CDDP. The D. magna proteins and RNA were isolated and analysed by mass spectrometry and the mRNA-seq method, respectively. Five generations of contact with the pharmaceuticals in question significantly influenced the D. magna life history parameters with the growth rate and number of laid eggs decreased, whereas age at first reproduction was increased. A decrease in survivorship was observed when daphnids were exposed to CP. These changes are the result of modifications in the gene/transcript expression followed by differences in the proteome profile in comparison to the untreated control. The proteome changes were generally in accordance with the modified transcriptome. The ecotoxicogenomics approach makes it possible to get closer to a complete picture of the influence of CP and CDDP on Daphnia. We have gathered evidence that animals in the presence of anticancer pharmaceuticals attempt to cope with permanent stress by changing their proteome and transcriptome profile. Additionally, our analyses indicate that CDDP showed a stronger effect on tested organisms than CP.

Effect of heavy metals on the energy metabolism in the brackish water flea Diaphanosoma celebensis.

Heavy metals such as lead (Pb), cadmium (Cd), and arsenic (As) are of great concern in aquatic ecosystems because of their global distribution, persistence, and biomagnification via the food web. They can induce the expression of cellular protective systems (e.g., detoxification enzymes and antioxidant enzymes) to protect organisms from oxidative stress, which is a high-energy-consuming process. Thus, energy reserves (e.g., glycogen, lipids, and proteins) are utilized to maintain metabolic homeostasis. Although a few studies have suggested that heavy metal stress can modulate the metabolic cycle in crustaceans, information on changes in energy metabolism under metal pollution remains lacking in planktonic crustaceans. In the present study, the activity of digestive enzymes (amylase, trypsin, and lipase) and the contents of energy storage molecules (glycogen, lipid, and protein) were examined in the brackish water flea Diaphanosoma celebensis exposed to Cd, Pb, and As for 48 h. Transcriptional modulation of the three AMP-activated protein kinase (AMPK) and metabolic pathway-related genes was further investigated. Amylase activity was highly increased in all heavy metal-exposed groups, whereas trypsin activity was reduced in Cd- and As-exposed groups. While glycogen content was increased in all exposed groups in a concentration-dependent manner, lipid content was reduced at higher concentrations of heavy metals. The expression of AMPKs and metabolic pathway-related genes was distinct among heavy metals. In particular, Cd activated the transcription of AMPK-, glucose/lipid metabolism-, and protein synthesis-related genes. Our findings indicate that Cd can disrupt energy metabolism, and may be a potent metabolic toxicant in D. celebensis. This study provides insights into the molecular mode of action of heavy metal pollution on the energy metabolism in planktonic crustaceans.

Genome Assembly of a Relict Arabian Species of Daphnia O. F. Müller (Crustacea: Cladocera) Adapted to the Desert Life.

The water flea Daphnia O.F. Müller 1776 (Crustacea: Cladocera) is an important model of recent evolutionary biology. Here, we report a complete genome of Daphnia (Ctenodaphnia) arabica (Crustacea: Cladocera), recently described species endemic to deserts of the United Arab Emirates. In this study, genome analysis of D. arabica was carried out to investigate its genomic differences, complexity as well as its historical origins within the subgenus Daphnia (Ctenodaphnia). Hybrid genome assembly of D. arabica resulted in ~116 Mb of the assembled genome, with an N50 of ~1.13 Mb (BUSCO score of 99.2%). From the assembled genome, in total protein coding, 5374 tRNA and 643 rRNA genes were annotated. We found that the D. arabica complete genome differed from those of other Daphnia species deposited in the NCBI database but was close to that of D. cf. similoides. However, its divergence time estimate sets D. arabica in the Mesozoic, and our demographic analysis showed a great reduction in its genetic diversity compared to other Daphnia species. Interestingly, the population expansion in its diversity occurred during the megadrought climate around 100 Ka ago, reflecting the adaptive feature of the species to arid and drought-affected environments. Moreover, the PFAM comparative analysis highlights the presence of the important domain SOSS complex subunit C in D. arabica, which is missing in all other studied species of Daphnia. This complex consists of a few subunits (A, B, C) working together to maintain the genome stability (i.e., promoting the reparation of DNA under stress). We propose that this domain could play a role in maintaining the fitness and survival of this species in the desert environment. The present study will pave the way for future research to identify the genes that were gained or lost in this species and identify which of these were key factors to its adaptation to the harsh desert environment.

Metabolomic analysis of combined exposure to microplastics and methylmercury in the brackish water flea Diaphanosoma celebensis.

Owing to their widespread distribution and high bioaccumulation, microplastics (MPs) and mercury (Hg) are considered major threats to the ocean. MP interacts with Hg because of its high adsorption properties. However, their toxicological interactions with marine organisms, especially combined effects at the molecular level, are poorly understood. This study investigated the single and combined effects of MP and Hg on the metabolic profile of the brackish water flea Diaphanosoma celebensis. A total of 238 metabolites were significantly affected by MP, Hg, or MP + Hg. Metabolite perturbation patterns showed that toxicity of Hg and MP + Hg was similar and that of MP was not significant. Among the 223 metabolites affected by Hg, profiles of 32 unannotated metabolites were significantly different from those of MP + Hg, and combined effects of MP + Hg decreased the effect of Hg on 25 of these metabolites. Only 11 annotated metabolites were significantly affected by Hg or MP + Hg and were related to carbohydrate, lipid, vitamin, and ecdysteroid metabolism. Ten metabolites were decreased by Hg and MP + Hg and were not significantly different between the exposure groups. Enrichment analysis showed that galactose, starch, and sucrose metabolism were the most affected pathways. These findings suggest that MP has negligible toxic effect, and Hg can induce energy depletion, membrane damage, and disruption of growth, development, and reproduction. Although the impact of MP was negligible, the combined effects of MP + Hg could be metabolite specific. This study provides better understanding of the combined effects of MP and Hg on marine organisms.

Maternal Effect of Polyethylene Microplastic Fragments Containing Benzophenone-3 in Different Ages and Broods of Daphnia Magna.

The maternal effect of microplastics (MPs) toxicity is likely influenced by age and brood of test species. This study investigated the maternal effect of polyethylene MP fragments (18.23 ± 8.02 µm) with benzophenone-3 (BP-3; 2.89 ± 0.20% w/w) on chronic toxicity to Daphnia magna over two generations. Neonate (< 24 h old) and adult (5 d old) daphnids in the F0 generation were exposed until 21 d old, then first and third brood neonates in the F1 generation were recovered in clean M4 medium for 21 d. Higher chronic toxicity and maternal effect of MP/BP-3 fragments were observed in the adult group compared with the neonate group, reducing growth and reproduction in both F0 and F1 generations. First brood neonates in the F1 generation showed a higher maternal effect of MP/BP-3 fragments than third brood ones, resulting in enhanced growth and reproduction relative to the control. This study provided insights into the ecological risk of MPs containing plastic additives in the natural environment.

Daphnia as a Sentinel Species for Environmental Health Protection: A Perspective on Biomonitoring and Bioremediation of Chemical Pollution.

Despite available technology and the knowledge that chemical pollution damages human and ecosystem health, chemical pollution remains rampant, ineffectively monitored, rarely prevented, and only occasionally mitigated. We present a framework that helps address current major challenges in the monitoring and assessment of chemical pollution by broadening the use of the sentinel species Daphnia as a diagnostic agent of water pollution. And where prevention has failed, we propose the application of Daphnia as a bioremediation agent to help reduce hazards from chemical mixtures in the environment. By applying "omics" technologies to Daphnia exposed to real-world ambient chemical mixtures, we show improvements at detecting bioactive components of chemical mixtures, determining the potential effects of untested chemicals within mixtures, and identifying targets of toxicity. We also show that using Daphnia strains that naturally adapted to chemical pollution as removal agents of ambient chemical mixtures can sustainably improve environmental health protection. Expanding the use of Daphnia beyond its current applications in regulatory toxicology has the potential to improve both the assessment and the remediation of environmental pollution.

Methylmercury bioaccumulation in water flea Daphnia carinata by AIEgen.

Mercury ion (Hg2+) is a toxic heavy metal ion and Hg2+ is convertible to methylmercury (MeHg) by many aquatic microorganisms, leading to bioaccumulation and biomagnification in aquatic organisms, which can interfere with brain development and function in humans. This study employs a newly developed AIEgen (Aggregation-induced emission fluorogen) to quantify and visualise the process of MeHg bioaccumulation in vivo on the species of water flea Daphnia carinata. Two approaches to MeHg absorption were taken, either by direct incubation in a MeHg solution or by indirect consumption of algae contaminated with MeHg. We analysed the relationship between the ratio of photoluminescence (PL) intensities (I585/I480) and MeHg concentration (CMeHg) and generated a master curve for determining MeHg concentration based on the measurement of PL intensities. Fluorescent image analysis showed the occurrence of MeHg in D. carinata to be mainly in the compound eyes, optic nerve and carapace. This study indicates that MeHg absorption can be quantified and visualised in the body of zooplankton, and the MeHg transfer to zooplankton is more likely through direct exposure than via indirect food intake. The accumulation of MeHg in the eye and the nervous system could be the cause of the high mortality of D. carinata exposed to MeHg in water.

Acute toxicity of praziquantel to fish Danio rerio and planktonic crustacean Daphnia magna.

This study evaluated the toxicity of the pyrazino isoquinoline anthelmintic praziquantel (PZQ) to the Danio rerio zebrafish and Daphnia magna water flea. The estimated 24 h and 96 h LC50 of PZQ to the zebrafish was 39.9 mg/l and 30.4 mg/l, respectively. The highest 24 h and 96 h non-lethal concentration (LC0) was 21.7 mg/l and 21.2 mg/l, respectively. The mobility inhibition test of the juvenile Daphnia magna revealed a 48 h EC50 of 42.7 mg/l.

Mechanisms of thermal treatment on two dominant copepod species in O3/BAC processing of drinking water.

Phyllodiaptomus tunguidus and Heliodiaptomus falxus are dominant copepods species in drinking water processing plants in southern China. With a potential penetration risk, the breeding and leakage of copepods are drawing more and more attention in recent years. The current study provided a thermal treatment method to control copepods and their eggs. Results showed that: (1) the immediate death rates of P. tunguidus and H. falxus after heated to 34-40 °C for 5 min are positively correlated to the treatment temperatures (P < 0.01), and all individuals of the both species were eliminated after heated at 40 °C for 5 min; (2) overall hatching rates of P. tunguidus eggs were negatively correlated with treatment temperatures (P < 0.01) between 39-45 °C, with zero percent hatched after treatment at 45 °C for 5 min; (3) hatching rates of H. falxus were negatively correlated with treatment temperatures (P < 0.01) between 37-41 °C, with no nauplii hatched when treated at 41 °C for 5 min; (4) paraffin section histological examination indicated that thermal treatment caused severe damage to internal organs and egg structure. Finally, based on the experimental data, the application of the thermal treatment method was discussed in ozonation combined with biological activated carbon (O3/BAC) processing of drink water treatment.

Identification of Mitochondrial DNA (NUMTs) in the Nuclear Genome of Daphnia magna.

This is the first study in which the Daphnia magna (D. magna) nuclear genome (nDNA) obtained from the GenBank database was analyzed for pseudogene sequences of mitochondrial origin. To date, there is no information about pseudogenes localized in D. magna genome. This study aimed to identify NUMTs, their length, homology, and location for potential use in evolutionary studies and to check whether their occurrence causes co-amplification during mitochondrial genome (mtDNA) analyses. Bioinformatic analysis showed 1909 fragments of the mtDNA of D. magna, of which 1630 were located in ten linkage groups (LG) of the nDNA. The best-matched NUMTs covering >90% of the gene sequence have been identified for two mt-tRNA genes, and they may be functional nuclear RNA molecules. Isolating the total DNA in mtDNA studies, co-amplification of nDNA fragments is unlikely in the case of amplification of the whole tRNA genes as well as fragments of other genes. It was observed that TRNA-MET fragments had the highest level of sequence homology, thus they could be evolutionarily the youngest. The lowest homology was found in the D-loop-derived pseudogene. It may probably be the oldest NUMT incorporated into the nDNA; however, further analysis is necessary.

The concentration of dissolved organic matter impacts the metabolic response in Daphnia magna exposed to 17α-ethynylestradiol and perfluorooctane sulfonate.

The pharmaceutical 17α-ethynylestradiol (EE2) and the industrial chemical perfluorooctane sulfonate (PFOS) are organic contaminants frequently detected in freshwater environments. It is hypothesized that hydrophobic organic contaminants can sorb to dissolved organic matter (DOM) and this may reduce the toxicity of these contaminants by reducing the contaminants' bioavailability. To investigate this hypothesis, 1H nuclear magnetic resonance (NMR)-based metabolomics was used to determine how the metabolome of Daphnia magna changes when a range of DOM concentrations are added during EE2 and PFOS exposure experiments. D. magna were exposed for 48 h to sub-lethal concentrations of 1 mg/L EE2 or 30 mg/L PFOS in the presence of 0, 1, 2, 3 and 4 mg dissolved organic carbon (DOC)/L. EE2 exposure resulted in increased amino acids and decreased glucose in D. magna. All DOM concentrations were able to lessen these metabolite disturbances from EE2 exposure, likely due to reductions in the bioavailability of EE2 through interactions with DOM. Exposure to PFOS resulted in decreased amino acids, and the presence of 1 mg DOC/L did not alter this metabolic response. However, PFOS exposure with the higher DOM concentrations resulted in a different pattern of metabolite changes which may be due to combined impacts of PFOS and DOM on the metabolome or due to an increase in PFOS bioavailability and uptake in D. magna. These results suggest that the concentration of DOM influences the sensitive biochemical changes in organisms that occur during acute sub-lethal exposure to organic contaminants.

Acute toxicity of bisphenol A and its structural analogues and transcriptional modulation of the ecdysone-mediated pathway in the brackish water flea Diaphanosoma celebensis.

Bisphenol A (BPA) is a representative endocrine disrupting chemical (EDC) that has estrogenic effects in aquatic animals. In recent years, due to the continuing usage of BPA, its analogues have been developed as alternative substances to replace its use. The molting process is a pivotal point in the development and reproduction of crustaceans. However, studies of the effects of EDCs on molting in crustaceans at the molecular level are scarce. In the present study, we examined the acute toxicity of BPA and its analogues bisphenol F (BPF) and S (BPS) to the brackish water flea Diaphanosoma celebensis. We further identified four ecdysteroid pathway - related genes (cyp314a1, EcRA, EcRB, and USP) in D. celebensis, and investigated the transcriptional modulation of these genes during molting and after exposure to BPA and its analogues for 48 h. Sequencing and phylogenetic analyses revealed that these four genes are highly conserved among arthropods and may be involved in development and reproduction in the adult stage. The mRNA expression patterns of cyp314a1, EcRA and USP were matched with the molting cycle, suggesting that these genes play a role in the molting process in the adult stage in cladocerans. Following relative real-time polymerase chain reaction (RT-PCR) analyses, BPA and its analogues were found to modulate the expression of each of these four genes differently, indicating that these compounds can disrupt the normal endocrine system function of D. celebensis. This study improves our understanding of the molecular mode of action of BPA and its analogues in D. celebensis.

Evaluation of 4-nonylphenol and bisphenol A toxicity using multiple molecular biomarkers in the water flea Daphnia magna.

Alkylphenols are well-known endocrine disruptors and may cause developmental and reproductive disorders in aquatic organisms. Daphnia magna is commonly used in ecotoxicological studies as a promising model species to investigate the effects of endocrine distruptors. In the present study, transcriptional modulation of eleven potential molecular indicators related to detoxification, antioxidant, development, and cellular stress was analyzed in D. magna exposed to different concentrations of bisphenol A (BPA) and 4-nonylphenol (4-NP) for 24 h and 48 h, using real-time qPCR. A hierarchical clustering analysis was applied to investigate relations among molecular markers depending on the compound, exposure duration, and concentration. Our findings suggested that GSH-related systems and stress proteins may be involved in cellular defense against BPA and 4-NP-mediated toxicity with different modes of action. Furthermore, these compounds may interrupt molting and reproduction in daphnids. In particular, D. magna GSH-related genes seem to be strongly affected by 4-NP exposure, indicating their potential as molecular biomarkers.

Time-Dependent Responses of Oxidative Stress, Growth, and Reproduction of Daphnia magna Under Thermal Stress.

The present study evaluated the effects of temperature (20 and 25°C) on the oxidative stress responses and life-history traits of Daphnia magna depending on exposure time. Daphnid exposed to an elevated temperature for 21 days had notably higher activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase while the enzyme activities did not differ significantly between the two temperature groups for daphnid exposed for 5 days. However, the results of body length were opposite where only the 5 days exposure daphnid had significantly longer bodies at 25°C compared to those at 20°C (p < 0.05). Despite the earlier reproduction for daphnids at 25°C, the cumulative number of offspring per female for 21 days was not significantly different from those at 20°C (p < 0.05). These findings suggest that D. magna undergo strategic changes in oxidative stress response, growth, and reproduction throughout the exposure period of 21 days.

Identification and characterization of homeobox (Hox) genes and conservation of the single Hox cluster (324.6 kb) in the water flea Daphnia magna.

We report the complete sequence analysis of the entire complement of eight typical homeobox (Hox) genes (Lab, Pb, Dfd, Scr, Antp, Ubx, Abd-A, and Abd-B) and two other genes (Hox3 and Ftz) in a 324.6-kb region in the water flea Daphnia magna. In the cluster of D. magna Hox genes, we found one long interspersed nuclear element (LINE)/R2-NeSL between Ubx and Abd-A that was not present in Daphnia pulex Hox genes. In basal expression of Hox genes at different developmental stages, biothorax complex genes (Ubx, Abd-A, and Abd-B) and some antennapedia complex genes (Lab, Scr, Antp) were moderately expressed, but the Hox3 gene was barely expressed. Three homeobox genes (Antp, Ubx, Abd-A) were highly expressed at 6-7 days after release from the brood chamber and/or in the adult stage. The structural array and transcribed orientation of Dm-Hox genes were identical to those of the sister species D. pulex (∼340 kb), indicating that the Hox gene structure in daphnids is highly conserved. However, Dm- and Dp-Hox3, -deformed (Dfd), and -fushi tarazu (Ftz) genes varied from orthologous genes in pancrustacean species.

De novo transcriptome assembly of brackish water flea Diaphanosoma celebensis based on short-term cadmium and benzo[a]pyrene exposure experiments.

To develop a brackish water flea as a promising model for marine monitoring, Diaphanosoma celebensis were exposed to two pollutants, cadmium (Cd) and benzo[a]pyrene (BaP), which have different chemical characteristics and distinct modes of metabolic action on aquatic animals. Twenty-four hours after exposure to Cd (2 mg/L) or BaP (25 µg/L), whole body transcriptomes were analyzed. In total, 99.6 Mbp were assembled from nine libraries, resulting in 98,458 transcripts with an N50 of 1883 bp and an average contig length of 968 bp. Functional gene annotations were performed using Gene Ontology, Eukaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes pathway analyses. Cd significantly modulated endocrine and digestive enzyme system. Following BaP treatment, DNA repair and circadian rhythm related metabolisms were significantly modulated. Both the chemicals induced stress response and detoxification metabolism. This brackish water flea genomic information will be useful to monitor estuaries and coastal regions, as water fleas have been confirmed as promising sentinel models in freshwater ecosystems.

Ethoxyquin: a feed additive that poses a risk for aquatic life.

Ethoxyquin (EQ) is an antioxidant that has, to date, been commonly used in feed production. Reports on the detrimental effects of this substance on vertebrates are growing, but effects in aquatic systems have rarely been described. Therefore, the present study was conducted using serial concentrations of EQ ranging from 0.03 to 16.5 mg l-1 to determine effects on 3 types of aquatic organisms. In zebrafish, 5 mg l-1 EQ caused mortality (25%) and a further 62.5% of the embryos showed yolk sac edema as well as deformed bodies or missing eyes. Furthermore, all the investigated EQ concentrations decreased the heart rate of the embryos. The lowest observed effect level was 0.31 mg l-1. In addition to zebrafish, the study also used water fleas Daphnia magna and green algae (Scenedesmus obliquus and Chlorella vulgaris). These treatments revealed that daphnids are also sensitive to EQ, exhibiting detrimental effects with a half-maximal effective concentration (EC50) of 2.65 mg l-1 after 48 h of exposure. The algae appeared to be at least 2 times less sensitive to EQ than fish embryos or daphnids. The results were used to calculate the risk for aquatic life resulting in a maximum tolerable level of 1 µg l-1 for fish embryos and daphnids, with a safety factor of 300. According to current knowledge, this does not exceed environmental concentrations of this substance. However, this study raises further concern about the (until recently) legal maximum tolerable EQ levels in fish feeding and the rather slow pace at which authorization to use EQ as a feed additive for diverse animals in Europe is being suspended.

Safety of Bacillus thuringiensis Cry1C protein for Daphnia magna based on different functional traits.

Cry1C is a Bacillus thuringiensis (Bt) insecticidal protein and it can be produced by transgenic rice lines developed in China. Cladocera species are common aquatic arthropods that may be exposed to insecticidal proteins produced in Bt-transgenic plants through ingestion of pollen or crop residues in water. As the cladoceran Daphnia magna plays an important role in the aquatic food chain, it is important to assess the possible effects of Bt crops to this species. To evaluate the safety of the Cry1C protein for D. magna, individuals were exposed to different concentrations of purified Cry1C protein in M4 medium for 21 days. Potassium dichromate (K2Cr2O7), a known toxicant to D. magna, was added to M4 medium as a positive control treatment, and pure M4 medium was used as a negative control. Our results show that developmental, reproductive, and biochemical parameters of D. magna were not significantly different between Cry1C and negative control treatments but were significantly inhibited by the positive control. We thus conclude that D. magna is insensitive to Cry1C.

Temperature-dependent benefits of bacterial exposure in embryonic development of Daphnia magna resting eggs.

The environments in which animals develop and evolve are profoundly shaped by bacteria, which affect animals both indirectly through their role in biogeochemical processes and directly through antagonistic or beneficial interactions. The outcomes of these activities can differ according to environmental context. In a series of laboratory experiments with diapausing eggs of the water flea Daphnia magna, we manipulated two environmental parameters, temperature and presence of bacteria, and examined their effect on development. At elevated temperatures (≥ 26 °C), resting eggs developing without live bacteria had reduced hatching success and correspondingly higher rates of severe morphological abnormalities compared with eggs with bacteria in their environment. The beneficial effect of bacteria was strongly reduced at 20 °C. Neither temperature nor the presence of bacteria affected directly developing parthenogenetic eggs. The mechanistic basis of this effect of bacteria on development is unclear, but these results highlight the complex interplay of biotic and abiotic factors influencing animal development after diapause.