The effects of diatom polyunsaturated aldehydes on embryonic and larval zebrafish (Danio rerio)

doi:10.21203/rs.3.rs-4870231/v1

Download PDF

Research Article

The effects of diatom polyunsaturated aldehydes on embryonic and larval zebrafish (Danio rerio)

https://doi.org/10.21203/rs.3.rs-4870231/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Marine diatoms are pervasive in many planktonic and benthic environments and represent an important food source for a wide range of species. Some diatoms produce polyunsaturated aldehydes (PUAs) as defensive toxins. PUA exposure is known to reduce the fecundity of invertebrate grazers like copepods and echinoderm larvae, but little is known about the effects of PUAs on vertebrates. Many fish species are likely to come into close contact with diatoms. Many may deposit eggs on diatom-coated substrates, consume diatoms as larvae, and/or feed heavily on zooplankters that may be gut-loaded with diatoms. The purpose of this study was to test whether dissolved diatom PUAs affect the early life stages of a model fish, Danio rerio (zebrafish). To test this, zebrafish embryos and larvae were exposed to proportionally increasing mixtures of the dissolved diatom PUAs 2E,4E-decadienal, 2E,4E-octadienal and 2E,4E-heptadienal. Under PUA exposure, three metrics of fitness were assessed: embryo heart rate, larval size at hatch, and pre-feeding mortality rate. Zebrafish embryos exposed at 24 hours post fertilization (hpf) experienced decreased average heart rate after 2 days of PUA exposure. Embryos 24 hpf exposed to PUA mixtures for 6 days showed a reduction in size in comparison to embryos from controls. Embryos exposed to PUAs from 2 hpf until death showed lower survivorship compared to larvae in controls. The results of this study suggest that larval fish that are sympatric with PUA producing diatoms during their embryonic and larval stages may be susceptible to detrimental effects from PUA exposure.

Diatoms

polyunsaturated aldehydes

development

larval fishes

zebrafish

toxins

Diatoms are an important functional group in marine ecosystems because of their basal trophic position as primary producers and their prominent role in global biogeochemical cycles. They can be especially abundant in coastal waters due to the increased presence of nutrients from terrestrial environments and from seasonal upwelling (Malviya et al. 2016). This high biomass of diatoms in coastal environments means strong competition pressure for resources within diatom communities and from other primary producer functional groups (Zhou et al. 2017), and it exposes diatoms to a wide consortium of nearshore consumers.

To gain a competitive advantage for resources and reduce predation pressure, some diatoms produce and emit bioactive chemicals that have effects across trophic levels (Pohnert 2005; Franz$\:é$ et al. 2018). These chemicals can lead to reduced herbivore grazing and decreased growth of co-occurring phytoplankton populations (e.g. Ribalet et al. 2007; Miralto et al. 1999). Oxylipins, a compound within this array of diatom-produced chemicals, are a product of the lipoxidation of polyunsaturated fatty acids (PUFAs). Oxylipins show high bioactivity in plants, algae, and animals (Barbosa et al. 2016; Prost et al. 2005; Tourdot et al. 2014). Some diatoms, upon damage to the diatom cell wall, intracellularly produce oxylipins called polyunsaturated aldehydes (PUAs), biomolecules composed of a chain lipid with an attached oxygen atom (Caldwell 2009; Fontana et al. 2007). The lipid chain length determines the bioactivity of the molecule, with longer chained molecules being associated with increased bioactive effects on other organisms (Adolph et al. 2003; Ceballos and Ianora 2003). While these molecules can confer fitness benefits to diatoms, the full extent of each unique PUA’s effects and range of biological targets is still being explored (Galasso et al. 2020; Ruocco et al. 2020).

Bioactivity studies on diatom PUAs have historically focused on the invertebrate predators of diatoms, such as pelagic copepods and echinoderm larvae (Ianora et al. 2011; Koski et al. 2008; Lettieri et al. 2015; Romano et al. 2010; Ruocco et al. 2019; Varrella et al. 2014; Wolfram et al. 2014). A consistent observation in these studies is that PUAs negatively affect grazer fecundity (Caldwell 2009; Miralto et al. 1999). In a pioneering study on the effects of PUAs on copepod grazers, three specific diatom PUAs were identified as the compounds responsible for these deleterious effects: 2-trans-4-cis-7-cis-decatrienal, 2-trans-4-trans-7-cis-decatrienal, and 2-trans-4-trans-decadienal (Miralto et al. 1999). This study also highlighted a negative correlation between in situ diatom biomass and copepod reproductive success in the Adriatic Sea, suggesting that increased consumption of diatom PUAs negatively affected fecundity.

Since the identification of those three PUAs as a causative agent for reduced grazer fecundity, additional diatom PUAs have been isolated, identified, and explored for negative bioactive effects. Ianora and Miralto (2010) reviewed the effects of a range of diatoms on copepod fecundity and showed that, of the 21 species tested, all 21 produced negative effects on at least one of four different copepod reproductive criteria (egg production rate, hatching success, morphologically abnormal larvae, and development from naupliar stages through adulthood). In addition to the negative effects of diatom PUAs on copepod fecundity and offspring survival, antiproliferative effects on other invertebrates have also been explored. Larvae of the chordate invertebrate filter-feeder, Ciona intestinalis, developed malformed bodies in response to dissolved PUAs (Lettieri et al. 2015). Paracentrotus lividus (sea urchin) embryos displayed an increased number of abnormalities and deformations as embryos and larvae when exposed to PUAs (Romano et al. 2010). Furthermore, 2-trans-4-trans-decadienal caused larval development to stop at the gastrula stage and induced considerable numbers of dead or prematurely hatched urchin larvae.

PUAs also display toxic effects on vertebrate cells, including cancerous cells (Martinez Andrade et al. 2018). For example, PUAs exhibited anti-proliferative activity and activated programmed cell death (apoptosis) for cancer cells originating from human adenocarcinoma colon and lung cell lines (Sansone et al. 2014). 2-trans-4-trans-decadienal also caused the mitochondria of rat liver cells to experience dysfunction and oxidative stress (Sigolo et al. 2008). These observed negative effects of PUAs on vertebrate cells suggest that PUAs may also interact with the developing planktonic offspring of marine vertebrates.

Because diatoms are so pervasive in ocean ecosystems, many fishes are likely to be exposed to the defensive compounds that they produce, including PUAs. Benthic diatoms are extremely common on marine substrates where diatom colonies may cover extensive areas (Cotiyane-Pondo et al. 2021; Risjani et al. 2021; Witkowski et al. 2016). The eggs of fishes that lay demersal eggs or attach them to other surfaces are therefore likely to encounter diatoms. Although many fishes guard and clean their eggs, a large number abandon them after spawning (e.g., silversides, herring, surf smelt, sand lance, flying fishes) (Conover 1992; Dickey-Collas et al. 2001; Quinn et al. 2012; Robards et al. 1999; Kojima 1971), which places them at risk of diatom colonization that could expose the embryos to diatom toxins, including PUAs (Johnson et al. 2023).

Diatom exposure may be even more likely after hatching when fish begin to actively feed. The majority of marine fishes have planktonic larvae (Cooper et al. 2020) and diatoms are ubiquitous in the upper reaches of the ocean (Malviya et al. 2016). Many fish species are known to consume diatoms during their larval stages, especially at smaller body sizes when they may be less efficient at capturing zooplankton (Friedenberg et al. 2012; Nirazuka et al. 2021; Giraldo et al. 2011; Salas-Berrios et al. 2013; Rossi et al. 2006; Napiórkowska-Krzebietke 2017; Denis et al. 2016). The defensive toxins produced by planktonic diatoms may therefore pose a risk for marine fishes during their earliest free-living stages of development.

Whether or not the PUAs produced by diatoms have toxic effects on early fish development is unknown. Determining whether PUAs affect the embryogenesis, development, and/or survival of fishes is therefore an important step toward understanding their role in aquatic ecosystems. To this end, we tested for the effects of PUAs on the development of a model species frequently used in toxicological studies, the zebrafish (Danio rerio; Hamilton 1822). Embryos and larvae were exposed to a range of dissolved PUA concentrations to ascertain whether: 1) PUA exposure affects heart physiology in zebrafish embryos; 2) PUA exposure impairs zebrafish growth in early development; and 3) PUA exposure increases larval zebrafish mortality.

Animal care

AB wildtype zebrafish were maintained in a standard zebrafish system (model ZS554, Aquaneering, San Diego, CA, USA) at Western Washington University’s (WWU) Department of Biology under approved animal care protocol 20 − 007. Fish were kept under standard conditions (14/10 light/dark cycle; 28 ± 1°C; 6.75–7.5 pH; daily 10% water changes) and fed twice daily with commercial tropical fish flakes and live brine shrimp (Artemia sp.) enriched with algae. Fertilized eggs were obtained by placing 4–6 male/female pairs in 1.7 L sloped breeding tanks overnight (Techniplast, West Chester, PA, USA). Embryos were maintained at 28°C in 100 mm diameter petri dishes with E3 embryo water (doi:10.1101/pdb.rec066449; Cold Spring Harbor Protocols 2011).

PUA mixture and solvent choice

Zebrafish embryos and larvae were treated with mixtures of three PUAs, 2-trans-4-trans-decadienal (C₁₀H₁₆O, Cas No. 25152-84-5), 2-trans-4-trans-octadienal (C₈H₁₂O, Cas No. 30361-28-5; 5577-44-6), and 2-trans-4-trans-heptadienal (C₇H₁₀O, Cas No. 4313-03-5), hereafter referred to as decadienal, octadienal, and heptadienal, following Ruocco et al. (2019). The effects of binary and ternary combinations of these PUAs on the development of the sea urchin Paracentrotus lividus were found to be synergistic, with negative effects increasing with exposure to increased concentrations of combined rather than to individual PUAs (Ruocco et al. 2019). The decision to examine the effects of these PUAs in combination, rather than individually, was reinforced by the fact that the three commonly co-occur when diatoms produce chemical defenses in response to grazing (Bartual et al. 2020; Vidoudez et al. 2011). PUA mixtures were dissolved in methanol, which increases the miscibility of lipid chains (Ribalet et al. 2007; Varella et al. 2014), and homogenized in sterile E3 embryo water. Although methanol is toxic to vertebrates, at low concentrations it is less toxic (no toxicity up to 1.0% concentration) than dimethyl sulfoxide (toxic at 0.3% concentration), which is the amphipathic solvent most used in toxicological studies of zebrafish development (Christou et al. 2020).

Heart rate

To test for PUA effects on zebrafish physiology, embryonic heart rates were measured after exposure to a range of PUA concentrations (Table 1). At 24 hours post fertilization (hpf) zebrafish embryos were transferred to new petri dishes containing one of three concentrations of PUA mixtures dissolved in 1.0% methanol (Table 1) and exposed for 2 days at 28^°C (25 embryos per plate; 3 replicate petri dishes per treatment). The PUA treatments are referred to as PUA1, PUA2, and PUA3 in order of increasing concentration. Solutions were replaced daily to account for possible PUA evaporation, remove zebrafish waste products (e.g., ammonia), and repress bacterial and/or fungal growth. We used two control treatments, a 1.0% methanol solvent control (referred to as S), and an embryo water only (referred to as EW).

Before the end of 2 days, there was 100% mortality in PUA3 treatment, and 100% mortality in one replicate (1 petri dish) of PUA2 treatment. After 2 days of PUA exposure, ten specimens were randomly selected in each of the remaining petri dishes that contained live embryos. Their heart rates (beats per minute; bpm) were then calculated from 10 s video recordings made using a FLIR Blackfly USB3 Vision camera running the Micro-Manager 2.0.0 program and mounted on an Olympus SZ-CTV dissecting microscope. Embryos were then euthanized by immersion in a 0^°C ice/embryo water mixture before disposal in compliance with WWU animal care protocol 20–700.

The effect of PUA concentration on heart rate was analyzed with a linear mixed model (LMM) in R version 4.3.0 using R package lme4 (v1.1-26; Bates et al., 2015) and MuMIn (v0.12.0; Barton 2009). A restricted maximum likelihood approach was used to make unbiased estimates of variance and covariance. To optimize a function with multiple arguments we used nlminb optimizer. The model included within-treatment variance between replicates as a random effect. Dish variability was weighted using the varIdent function, which improved model fit as measured by Akaike information criterion (AIC) values. Parameters were standardized by fitting the model on a standardized version of the dataset. Confidence Intervals (95%) and p-values were computed using a Wald t-distribution approximation.

Morphology

We used the same experimental design described above to test for PUA effects on the morphological development of zebrafish, except that embryos and larvae were treated with PUAs for 6 days (Table 1). This time frame allowed for 100% hatching. After 6-days of PUA treatment larvae (post-hatch fish are larvae) were euthanized as described above, transferred to a 75% glycerol solution, and photographed in a lateral position using a FLIR Blackfly USB3 Vision camera interfaced with Olympus SZ-CTV dissecting microscope. The ImageJ program was used to collect three measurements from specimen images: 1) standard length (SL; the tip of the snout to the end of the spine); 2) body depth measured through the center of the yolk sac (BD1); and body depth at the anus (BD2; Fig. 1).

All body measurements were analyzed using the same approach described above for the heart rate data. We also employed a permutational multivariate analysis of variance (PERMANOVA) to assess differences in approximate larval size between PUA treatments using the R packages vegan (v1.15-1; Oksanen et al. 2008) and MVN (Korkmaz et al. 2014). PERMANOVA was used instead of MANOVA due to the data’s departure from assumptions of multivariate normality and homogeneity (Anderson 2017). Because all morphological data were collected in one experiment, identical LMMs were used to describe all body measurements. The LMM addressed the nested design and variance needs of the data, with models containing petri dishes as a random effect, and weighted dishes leading to improved model fit based on compared AIC values (lme4 and MuMIn packages).

Survival

To test for PUA effects on survival we treated zebrafish embryos with PUAs at 2 hours post fertilization (hpf). We used an experimental design similar to those described above, with the following exceptions: 1) PUA treatments began at 2 hpf; 2) the number of replicates per treatment was increased to four; 3) the number of embryos per replicate was increased to 30; and 4) PUA concentrations were decreased in the two treatments that used the highest concentrations (Table 1) to account for the greater sensitivity of early-stage embryos. The PUA treatments will be referred to as PUA1 (also used in the heart rate and morphology experiments), PUA2, and PUA3 in order of increasing concentration. Mortality was recorded daily until it reached 100%.

These data were examined using a time-to-event approach by performing a non-parametric Kaplan-Meier survival analysis with the survminer package in R (Jager et al. 2008: Kassambra et al. 2018). Survival probabilities were generated for each PUA concentration by calculating the probability of an event (mortality) at each time interval (experimental day) and iteratively recalibrating probabilities based on past occurrences using the following equation (Di Paolo et al. 2015; Jager et al. 2008; LaMorte 2016):

$$\:\:{S}_{t+1}\:=\:{S}_{t}*\left(\frac{{N}_{t}+1-{D}_{t}+1}{{N}_{t}}+1\right)$$

where:

$\:{S}_{t+1}$ = the cumulative probability of surviving beyond time interval t,

$\:{N}_{t}$ = the number of subjects at risk at time interval t,

$\:{D}_{t}$ = the number of deaths during time interval t

A log-rank test was used to identify significant differences between curves, followed by value-adjusted pairwise comparisons to clarify significant differences between curves.

Heart rate

Zebrafish embryo heart rates significantly decreased with increasing PUA concentration (Fig. 1: Table 2). The mean heart rates for each treatment were: 121.2 bpm (EW); 107.2 bpm (S); 106.2 bpm (PUA1); and 77.2 bpm (PUA2). The 36% reduction in heart rate in PUA2 relative to the control was the only significant difference between treatments (p = 0.006; Table 2). The explanatory power of the model without random effects was 0.562 (marginal R²; Table 2), and increased to 0.949 when random effects were included (conditional R²; Table 2).

Morphology

Standard length

Larval zebrafish SL significantly decreased under PUA exposure (Fig. 1; Table 2). The mean SL from each treatment were: 3.39 mm (EW); 3.36 mm (S); 3.36 mm (PUA1); 2.83 mm (PUA2); and 2.75 mm (PUA3). Larvae from both the PUA2 and PUA3 treatments were significantly shorter than those from the control treatment (p = 0.006 and 0.003, respectively; Table 2; Fig. 1). The explanatory power of the model without random effects was 0.585 (marginal R²), and increased to 0.879 when random effects were included (conditional R²; Table 2).

Body depth at yolk sac

Larval zebrafish BD1 significantly decreased with increasing PUA concentration (Fig. 1; Table 2). The mean BD1 measurements from each treatment were: 0.45 mm (EW); 0.43 mm (S); 0.43 mm (PUA1); 0.35 mm (PUA2); and 0.32 mm (PUA3). Larvae from both the PUA2 and PUA3 treatments had significantly shorter BD1 measurements than those from the control treatment (p = 0.006 and 0.001, respectively; Table 2; Fig. 1). The explanatory power of the model without random effects was 0.477 (marginal R²), and increased to 0.701 when random effects were included (conditional R²; Table 2).

Body depth at anus

Larval zebrafish BD2 was unaffected by PUA exposure (Fig. 1; Table 2). Mean larval BD2 measurements from each treatment were: 0.14 mm (EW); 0.08 mm (S); 0.09 mm (PUA1); 0.10 mm (PUA2); and 0.10 mm (PUA3). There were no significant differences between treatments (Table 2; Fig. 1). The explanatory power of the model without random effects was 0.110 (marginal R²), and increased to 0.506 when random effects were included (conditional R²; Table 2).

Multivariate analysis of larval body shape

PUA concentrations caused significant differences in larval sizes and were responsible for 65% of the overall size variation between treatment concentrations (Table 3). Pairwise comparisons identified seven significant differences. There was a significant difference between the embryo water and solvent control treatments (adjusted p = 0.010), but no significant differences between either of these treatments and PUA1 (adjusted p = 0.110 and adjusted p = 1.000, respectively; Table 3). The body sizes in the PUA2 and PUA3 treatments were significantly different from those in all other treatments (see Table 3 for p-values), but the body sizes of specimens in these two treatments were not significantly different (adjusted p = 0.830). These differences can be seen in Fig. 2, where all body metrics cluster closely for EW, S, and PUA1 treatments.

Survival

PUA exposure significantly increased the mortality rate for pre-feeding zebrafish larvae (Fig. 3). Larval zebrafish achieved 100% mortality in each treatment group at the following times: 15 dpf (EW); 15 dpf (S); 12 dpf (PUA1); 12 dpf (PUA2); and 7 dpf (PUA3). Over 75% of the larvae in PUA3 died after 1 day of exposure (Table 4, Fig. 3). Survival in the EW and S controls were not significantly different (p = 0.5976), yet survival in each of these treatments was significantly different from that in all other treatments (Table 4). Survival in PUA1 and PUA2 treatments were not significantly different from each other (p = 0.1742), but survival in each of these treatments was significantly different from that in all other treatments (Table 4).

We discovered that PUAs reduced zebrafish embryo heart rates, overall larval size, and pre-feeding larval survival. Findings presented here suggest that diatom-derived PUAs are likely to impair the development of other early life history stages of fishes that are sympatric with PUA-producing diatoms. In the following sections we discuss the implications of these findings on zebrafish, and assuming that observations are transferable to other fishes, we discuss the implications for the fitness of marine fishes exposed to PUAs during development.

Zebrafish Embryo Physiology

Zebrafish embryos exposed to higher PUA concentrations had lower heart rates than those in control treatments. Embryonic fish hearts are particularly vulnerable to environmental toxins, such as PUAs, due to their highly permeable epithelia and the absence of organs responsible for blood filtration (e.g., liver and kidneys) that have yet to develop (Incardona and Scholz 2017). Vertebrates exposed to oxylipins, such as PUAs, are known to develop atherosclerosis, experience problems with platelet aggregation and vascular constriction, and be prone to cardiac injury (Caligiuri et al. 2017; Nayeem 2018). Embryonic zebrafish hearts should be especially susceptible to the effects of PUA toxicity due to the ease with which these toxins can reach heart tissues and the lack of compensatory mechanisms that will arise in later development. Therefore, it is reasonable to assume that PUAs will have similar effects on other embryonic fishes and their cardiac health when exposed to PUAs in nature.

The consequences of early cardiac injury in fishes can be catastrophic. For example, the lake trout, Salvelinus namaycush, (Walbaum in Artedi 1972) population of the Great Lakes collapsed because of heart-related issues caused by the exposure of trout embryos to the industrial pollutant dioxin (Cook et al. 2003). Chemical exposure to dioxin in lake trout caused symptoms similar to those that were seen in zebrafish exposed to PUAs in this study. For lake trout, stress from dioxin presents first as pre-hatch heart abnormalities before progressing into problems with circulatory dysregulation, anemia, and hypoxia, eventually resulting in premature death from attendant lesions in the brain, retina, liver, and other organs (Spitsbergen et al. 1991). This circulatory dysregulation was also shown to cause yolk sac edema and impair swim bladder development (Guiney et al. 1997; Lanham et al. 2014; Spitsbergen et al. 1991). While not quantified in this study, we observed that zebrafish treated with PUAs frequently exhibited cardiac edema and underdeveloped swim bladders, which suggests that these fish may have experienced circulatory dysregulation symptoms beyond decreased heart rate. If embryonic and larval stages of marine fishes are exposed to PUA concentrations used in this study, they would plausibly experience similar, life-threatening disruptions to circulatory development.

Morphometrics

PUA exposure affected the morphological development of zebrafish by reducing body length, body depth, and overall size. If the mechanisms which retarded zebrafish growth after PUA exposure are similar in marine fishes, then PUA exposure may affect fish fitness through multiple pathways. For example, larval fish survival is coupled with larval body size, with smaller larvae experiencing higher rates of predation (Cowan et al. 1996; Paradis 1996). For any planktonic organism, the risk of predation is significant, and this is also true for ichthyoplankton, where predation is one of the most important causes of mortality (Miller et al. 1988; Bailey and Houde 1989; Paradis 1996; Peterson and Wroblewski 1984). As such, high selective pressure exists on planktonic organisms to grow bigger rapidly. If PUA exposure causes larval fishes to grow slower, then this increases their exposure to predation which can eventually affect recruitment success (Houde 2008).

Smaller fishes also tend to have slower swimming speeds and lower feeding efficiencies in comparison to larger conspecifics (Houde 2008; Gleason and Bangston 1996; Houde and Schekter 1980; Hare and Cowen 1997). In bay anchovies (Anchoa mitchilli; Valenciennes 1848) feeding efficiency increased with size (Gleason and Bengston 1996; Houde and Schekter 1980). In bluefish (Pomatomus saltatrix; Linnaeus 1766) larger size was correlated with higher survival rates from yolk-sac-stage embryos to first-feeding larvae (Hare and Cowen 1997). Raventos and MacPherson (2005) found that larval size was an accurate predictor of post-settlement survival for two species of reef fishes, the five-spotted wrasse (Symphodus roissali; Risso 1810) and the ocellated wrasse (Symphodus ocellatus;Linnaeus 1758).

Fish larvae experience slower swimming speeds and reduced feeding efficiencies relative to later life stages, likely a result of the relationship between larval size and fluid dynamics (Van Leeuwen et al. 2015; Voesenek et al. 2018). After hatching most larval fishes continue to develop using maternally-provided yolk and lipid reserves (Kamler 2008). They quickly develop functional mouth parts, allowing them to feed and acquire energy exogenously (Yúfera and Darias 2007). Larval stages of fishes encompass a size range that places them in an intermediate hydrodynamic regime between laminar (viscous) and turbulent (inertial) flow (Voesenek et al. 2018). The relative dominance of viscous and inertial forces is estimated by calculating the Reynolds number (Van Leeuwen et al. 2015). Larval sizes < 5mm, representative of zebrafish measured here, exist in a low Reynolds number environment where viscous forces dominate (Van Leeuwen et al. 2015). Because movement through more viscous fluids requires a higher cost of transport (Schmidt-Nielson 1972), and because higher viscosities lower maximum swimming speeds and reduce a larvae’s ability to avoid predators, prolonging the time that young fishes spend in a low Reynolds number environment can greatly reduce survival (Voesenek et al. 2018). Further, it is a taxation on endogenous energy reserves. If these reserves are exhausted before transitioning to exogenous feeding, starvation will likely result (Yin & Blaxter 1987).

Existing in a high-viscosity environment also requires small larval fishes to employ higher forces (relative to body size) when suction feeding, which is usually the only feeding strategy available (China and Holzman 2014; Cooper et al. 2020). The ratio of energy expended to energy acquired during feeding will therefore be higher for smaller fishes. Additionally, the movement required by small fish larvae in viscous environments requires the beating of their tails at a high frequency, which in turn is dependent on the use of fast-twitch muscles. These muscles are fueled by mitochondria-dense fibers with a high ATP demand and energy reserves (Huriaux et al. 1996; Van Leeuwen et al. 2015). Prolonged residence in a high-viscosity environment increases the likelihood of starvation for larval fishes, which is already the largest source of mortality for most larval fishes (Hjort 1914; Houde 2002).

Pre-feeding survival

In our experiments where pre-feeding larvae were exposed to PUAs and reared until starvation mortality, PUA exposure at all concentrations accelerated rates of larval mortality in comparison to control treatments. Most specimens exposed to high PUA concentrations did not survive until they would otherwise have begun to feed exogenously, suggesting that PUAs impart stress to developing zebrafish larvae. Energetic taxation to larvae from stress during pre-feeding stages in fishes can negatively affect the population demographics of fishes. The transition from lecithotrophic to planktotrophic feeding is a critical phase in marine fishes and has been described as a governing factor determining year class strength (Hjort 1914). For this transition to occur with high fidelity, all anatomical features necessary for food capture, assimilation, and digestion need to develop from maternally provided yolk and other energy reserves, and be in place and functional prior to the point where irreversible starvation prevents feeding even when presented with adequate food (Osse 1990; Osse et al. 1997). If exposure to dissolved PUAs imparts stress necessitating utilization of endogenous energy to compensate, likely, the sensory, predatory, and digestive machinery needed for exogenous feeding and larval growth will not be present and ready when endogenous reserves are exhausted. This can lead to high larval mortality and, possibly, compromised adult recruitment.

This study provides some of the first evidence that diatom-derived PUAs can negatively affect the development of fishes. Given the sympatry of diatoms with embryonic and larval stages of marine fishes in most temperate marine ecosystems, and the clear effects of PUAs on a larval fish, the likelihood that PUAs will negatively affect the fitness of marine fishes seems high. If true, this pathway represents an unexplored mechanism that might explain some of the interannual variation in fish population dynamics. Our findings also suggest pathways for diatom-derived PUAs to affect marine food webs and marine secondary production beyond the diatom-copepod link.

While not quantified in this study, zebrafish embryos seemingly hatched later if they had been exposed to high PUA concentrations and many needed assistance rupturing the chorion in order to be liberated from the egg. These observations suggest that the hatching success of embryos born in areas of high diatom density could be compromised. As such, hatching success might be a useful metric for quantifying the effects of PUAs on the development of fish species. Findings here also suggest that future experiments could include other meaningful markers of PUA stress on fish fitness like the number of edematous larvae, swim bladder development, and observations of later life history stages. Given the clear potential presented here for negative interactions between PUAs and developing fish, further exploration of PUA exposure will likely add to our current understanding of the nearshore ecology of marine fishes.

Author Contribution

Authors RR, WJC, and MBO contributed to the study conception and design. Material preparation, data collection and analysis were performed by RR and SMJ. RR prepared all figures. The first draft of the manuscript was written by RR and MBO and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Acknowledgement

The authors thank Dr. karin Lemkau for guidance on PUA chemisty. We thank P. Thut and WWU's College of Science and Engineering and its Biology Department for laborastory space.

Adolph S, Poulet SA, Pohnert G (2003) Synthesis and biological activity of α,β,γ,δ-unsaturated aldehydes from diatoms. Tetrahedron 59:3003–3008. https://doi.org/10.1016/S0040-4020(03)00382-X
Anderson MJ (2017) Permutational Multivariate Analysis of Variance (PERMANOVA). Wiley StatsRef: Statistics Reference Online. 1–15. https://doi.org/10.1002/9781118445112.stat07841
Bailey KM, Houde ED (1989) Predation on eggs and larvae of marine fishes and the recruitment problem. In Blaxter JHS and & Southward AJ (eds.) Advances in Marine Biology 25:1–83. Academic Press. https://doi.org/10.1016/S0065-2881(08)60187-X
Barbosa M, Valentão P, Andrade PB (2016) Biologically active oxylipins from enzymatic and nonenzymatic routes in macroalgae. Mar Drugs 14:23 https://doi.org/10.3390/md14010023
Bartoń K (2009) Mu-MIn: Multi-model inference. R Package Version 0.12.2/r18. http://R-Forge.R-project.org/projects/mumin/
Bartual A, Hernanz-Torrijos M, Sala I, Ortega MJ, González-García C, Bolado-Penagos M, López-Urrutia, Romero-Martínez L, Lubián LM, Bruno M, Echevarría F, García CM (2020) Types and distribution of bioactive polyunsaturated aldehydes in a gradient from mesotrophic to oligotrophic waters in the Alborán Sea (Western Mediterranean). Mar Drugs 18:159. https://doi.org/10.3390/md18030159
Bates D, Mächler M, Bolker B, Walker S (2015). Fitting Linear Mixed-Effects Models Using lme4. J Stat Softw 67:1–48. https://doi.org/10.18637/jss.v067.i01
Caldwell GS (2009) The influence of bioactive oxylipins from marine diatoms on invertebrate reproduction and development. Mar Drugs 7:367–400. https://doi.org/10.3390/md7030367
Caligiuri SP, Parikh M, Stamenkovic A, Pierce GN, Aukema HM (2017) Dietary modulation of oxylipins in cardiovascular disease and aging. Am J Physiol Heart Circ Physiol 313:H903–H918. https://doi.org/10.1152/ajpheart.00201.2017
Ceballos S, Ianora A (2003) Different diatoms induce contrasting effects on the reproductive success of the copepod Temora stylifera. J Exp Mar Biol Ecol 294:189–202.
China V, Holzman R (2014) Hydrodynamic starvation in first-feeding larval fishes. PNAS 111:8083–8088. https://doi.org/10.1073/pnas.1323205111
Christou M, Kavaliauskis A, Ropstad E, Fraser TWK (2020) DMSO effects larval zebrafish (Danio rerio) behavior, with additive and interaction effects when combined with positive controls. Sci Total Environ 709:134490. https://doi.org/10.1016/j.scitotenv.2019.134490
Conover DO (1992) Seasonality and the scheduling of life-history at different latitudes. J Fish Biol 41:161–178. https://doi.org/10.1111/j.1095-8649.1992.tb03876.x
Cook PM, Robbins JA, Endicott DD, Lodge KB, Guiney PD, Walker MK, Zabel EW, Peterson RE (2003) Effects of aryl hydrocarbon receptor-mediated early life stage toxicity on lake trout populations in Lake Ontario during the 20th century. Environ Sci Technol 37:3864–3877. https://doi.org/10.1021/es034045m
Cooper WJ, Vanhall R, Sweet E, Milewski H, Deleon Z, Verderber A, Deleon A, Galindo D, Lazono O (2020) Functional morphogenesis from embryos to adults: Late development shapes trophic niche in coral reef damselfishes. Evol Dev 22:221–40. https://doi.org/10.1111/ede.12321
Cotiyane-Pondo P, Bornman TG, Dabek P (2021) Insight into nearshore diatom assemblages from an island ecosystem: Composition, spatial variation, and benthic contribution to the pelagic community (Bird Island, South Africa). Reg Stud Mar Sci:44. https://doi.org/10.1016/j.rsma.2021.101762
Cowan JH, Houde ED, Rose KA (1996) Size-dependent vulnerability of marine fish larvae to predation: An individual-based numerical experiment. ICES J Mar Sci 53:23–37. https://doi.org/10.1006/jmsc.1996.0003
Denis J, Vallet C, Courcot L, Lefebvre V, Caboche J, Antajan E, Marchal P, Loots C (2016) Feeding strategy of Downs herring larvae (Clupea harengus). J Sea Res 115:33–46. https://doi.org/10.1016/j.seares.2016.07.003
Di Paolo C, Groh KJ, Zennegg M, Vermeirssen EL, Murk AJ, Eggen RI, Hollert H, Werner I, Schirmer K (2015) Early life exposure to PCB126 results in delayed mortality and growth impairment in the zebrafish larvae. Aquat Toxicol 169:168–178. https://doi.org/10.1016/j.aquatox.2015.10.014
Dickey-Collas M, Nash RDM, Brown J (2001) The location of spawning of Irish Sea herring (Clupea harengus). J Mar Bio Assoc U K 81:713–714. http://dx.doi.org/10.1017/S0025315401004489
Fontana A, d’Ippolito G, Cutignano A, Miralto A, Ianora A, Romano G, Cimino G (2007) Chemistry of oxylipin pathways in marine diatoms. Pure Appl Chem 79:481–490. https://doi.org/10.1351/pac200779040481
Franzè G, Pierson JJ, Stoecker DK, Lavrentyev PJ (2018) Diatom-produced allelochemicals trigger trophic cascades in the planktonic food web. Limnol Oceanogr 63:1093–1108. https://doi.org/10.1002/lno.10756
Friedenberg LE, Bollens SM, Rollwagen-Bollens G (2012) Feeding dynamics of larval Pacific herring (Clupea pallasi) on natural prey assemblages: the importance of protists. Fish Oceanogr 21:95–108. http://dx.doi.org/10.1111/j.1365-2419.2011.00611.x
Galasso C, Celentano S, Costantini M, D’Aniello S, Ianora A, Sansone C, Romano G (2020) Diatom-derived polyunsaturated aldehydes activate similar cell death genes in two different systems: Sea urchin embryos and human cells. Int J of Mol Sci 21:5201 https://doi.org/10.3390/ijms21155201
Giraldo C, Cherel Y, Vallet C, Mayzaud P, Tavernier E, Moteki M, Hosie G, Koubbi P (2011) Ontogenic changes in the feeding ecology of the early life stages of the Antarctic silverfish (Pleuragramma antarcticum) documented by stable isotopes and diet analysis in the Dumont d'Urville Sea (East Antarctica). Pol Sci 5:252–263. https://doi.org/10.1016/j.polar.2011.04.004
Gleason TR, Bengtson DA (1996) Growth, survival and size-selective predation mortality of larval and juvenile inland silversides, Menidia beryllina (Pisces; Atherinidae). J Exp Mar Biol Ecol 199:165–177. https://doi.org/10.1016/0022-0981(95)00194-8
Guiney PD, Smolowitz RM, Peterson RE, Stegeman JJ (1997) Correlation of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in vascular endothelium with toxicity in early life stages of lake trout. Toxicol Appl Pharmacol 14:256–273. https://doi.org/10.1006/taap.1996.8051
Hare JA, Cowen RK (1997) Size, growth, development, and survival of the planktonic larvae of Pomatomus Saltatrix (pisces: Pomatomidae). Ecology 78:2415–2431. https://doi.org/10.1890/0012-9658(1997)078[2415:SGDASO]2.0.CO;2
Hjort J (1914) Fluctuations in the great fisheries of northern Europe viewed in the light of biological research. ICES
Houde ED (2002) Mortality. In Fulman LA and Werner RG (eds) Fishery Science: the Unique Contributions of Early Life Stages. pp. 64–87 Blackwell Science
Houde ED (2008) Emerging from Hjort’s Shadow. J Northwest Atl Fish Sci 41:53–70. https://doi.org/10.2960/J.v41.m634
Houde ED, Schekter RC (1980) Feeding by marine fish larvae: Developmental and functional responses. Environ Biol Fishes 5:315–334. https://doi.org/10.1007/BF00005186
Huriaux F, Mélot F, Vandewalle P, Collin S, Focant B (1996) Parvalbumin isotypes in white muscle from three teleost fish: Characterization and their expression during development. Comp Biochem Physiol B: Biochem Mol Biol 113:475–484. https://doi.org/10.1016/0305-0491(95)02066-7
Ianora A, Bentley MG, Caldwell GS, Casotti R, Cembella AD, Engström-Öst J, Halsband C, Sonnenschein E, Legrand C, Llewellyn CA, Paldavičienë A, Pilkaityte R, Pohnert G, Razinkovas A, Romano G, Tillmann U, Vaiciute D (2011) The relevance of marine chemical ecology to plankton and ecosystem function: An emerging field. Mar Drugs 9:1625–1648. https://doi.org/10.3390/md9091625
Ianora A, Miralto A (2010) Toxigenic effects of diatoms on grazers, phytoplankton and other microbes: A review. Ecotoxicol 19:493–511. https://doi.org/10.1007/s10646-009-0434-y
Incardona J, Scholz N (2017) Environmental Pollution and the Fish Heart. Fish Physiol 36:373–433. https://doi.org/10.1016/bs.fp.2017.09.006
Jager K, van Dijk PC, Zoccali C, Dekker FW (2008) The analysis of survival data: The Kaplan–Meier method. Kidney Int 74:560–565. https://doi.org/10.1038/ki.2008.217
Johnson J, Olson MB, Parker I, Hoffmeister I, Lemkau K (2024) Widespread Production of Polyunsaturated Aldehydes by Benthic Diatoms of the North Pacific Ocean’s Salish Sea. J Chem Ecol 50:290–298. https://doi.org/10.1007/s10886-024-01496-9
Kamler E (2008) Resource allocation in yolk-feeding fish. Rev Fish Biol Fish 18:143–200. http://dx.doi.org/10.1007/s11160-007-9070-x
Kassambara A, Kosinski M, Biecek P, Fabian S (2018) survminer: Survival Analysis and Visualization. https://rpkgs.datanovia.com/survminer/index.html
Kojima S (1971) Studies on the migration and spawning habit of the flying-fish-IV. Distribution of eggs in the spawning ground. Nippon Suisan Gakkaishi 37:284–288. https://doi.org/10.2331/suisan.37.284
Korkmaz S, Goksuluk D, Zararsiz G (2014) MVN: An R Package for Assessing Multivariate Normality.” R J 6:151–162. https://journal.r-project.org/archive/2014-2/korkmaz-goksuluk-zararsiz.pdf
Koski M, Wichard T, Jónasdóttir SH (2008) “Good” and “bad” diatoms: Development, growth and juvenile mortality of the copepod Temora longicornis on diatom diets. Mar Biol 154:719–734. https://doi.org/10.jager1007/s00227-008-0965-4
LaMorte WW (2016) Survival Analysis.
Lanham KA, Plavicki J, Peterson RE, Heideman W (2014) Cardiac myocyte-specific AHR activation phenocopies TCDD-induced toxicity in zebrafish. Toxicol Sci 141:141–54.
Lettieri A, Esposito R, Ianora A, Spagnuolo A (2015) Ciona intestinalis as a marine model system to study some key developmental genes targeted by the diatom-derived aldehyde decadienal. Mar Drugs 13:1451–1465. https://doi.org/10.3390/md13031451
Malviya S, Scalco E, Audic S, Vincent F, Veluchamy A, Poulain J, Wincker P, Iudicone D, de Vargas C, Bittner L, Zingone A, Bowler C (2016) Insights into global diatom distribution and diversity in the world’s ocean. Proc Natl Acad Sci 113:E1516–E1525. https://doi.org/10.1073/pnas.1509523113
Martínez Andrade KA, Lauritano C, Romano G, Ianora A (2018) Marine microalgae with anti-cancer properties. Mar Drugs 16:165. https://doi.org/10.3390/md16050165
Miller TJ, Crowder LB, Rice JA, Marschall EA (1988) Larval size and recruitment mechanisms in fishes: toward a conceptual framework. Can J Fish Aquat Sci 45:1657–1670. https://doi.org/10.1139/f88-197
Miralto A, Barone G, Romano G, Poulet SA, Ianora A, Russo GL, Buttino I, Mazzarella G, Laabir M, Cabrini M, Giacobbe MG (1999) The insidious effect of diatoms on copepod reproduction. Nature 402:173–176. https://doi.org/10.1038/46023
Napiórkowska-Krzebietke A (2017) Phytoplankton as a basic nutritional source in diets of fish. J Elem 22:831–841. http://dx.doi.org/10.5601/jelem.2016.21.4.1375
Nayeem MA (2018) Role of oxylipins in cardiovascular diseases. Acta Pharmacol Sin 39:1142–1154.
Nirazuka S, Makabe R, Swadling KM, Moteki M (2021) Phyto-detritus feeding by early-stage larvae of Electrona antarctica (Myctophidae) off Wilkes Land in the Southern Ocean, austral summer 2017. Pol Biol 44:1415–1425. https://doi.org/10.1007/s00300-021-02880-x
Oksanen J, Kindt R, Legendre P, O'Hara B, Simpson GL, Solymos P, Stevens MHH, Wagner H (2008) vegan: Community Ecology Package (R package version 1.15-1)MVN R package
Osse JWM (1990) Form changes in fish larvae in relation to changing demands of function. Neth J Zool 40: 362–385. https://doi.org/10.1163/156854289X00354
Osse JWM, vandenBoogaart JGM, vanSnik GMJ, vanderSluys L (1997) Priorities during early growth of fish larvae. Aquac 155: 249–258. https://doi.org/10.1016/S0044-8486(97)00126-9
Paradis AR, Pepin P, Brown JA (1996) Vulnerability of fish eggs and larvae to predation: Review of the influence of the relative size of prey and predator. Can J Fish Aquat Sci 53:1226–1235. https://doi.org/10.1139/cjfas-53-6-1226
Peterson I, Wroblewski J (1984) Mortality rate of fishes in the pelagic ecosystem. Can J Fish Aquat Sci 41:1117–1120. https://doi.org/10.1139/f84-131
Pohnert G (2005) Diatom/copepod interactions in plankton: The indirect chemical defense of unicellular algae. ChemBioChem 6:946–959. https://doi.org/10.1002/cbic.200400348
Prost I, Dhondt S, Rothe G, Vicente J, Rodriguez MJ, Kift N, Carbonne F, Griffiths G, Esquerré-Tugayé MT, Rosahl S, Castresana C, Hamberg M, Fournier J (2005) Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens. Plant Physiol 139:1902–1913. https://doi.org/10.1104/pp.105.066274
Quinn T, Krueger K, Pierce K, Pentilla D, Perry K, Hicks T, Lowry D (2012) Patterns of Surf Smelt, Hypomesus pretiosus, Intertidal Spawning Habitat Use in Puget Sound, Washington State. Estuaries Coast 35:1214–1228. http://dx.doi.org/10.1007/s12237-012-9511-1
Raventos N, Macpherson E (2005) Effect of pelagic larval growth and size-at-hatching on post-settlement survivorship in two temperate labrid fish of the genus Symphodus. Mar Ecol Prog Ser 285:205–211. https://doi.org/10.3354/meps285205
Ribalet F, Berges JA, Ianora A, Casotti R (2007) Growth inhibition of cultured marine phytoplankton by toxic algal-derived polyunsaturated aldehydes. Aquat Toxicol 85:219–227. https://doi.org/10.1016/j.aquatox.2007.09.006
Risjani Y, Witkowski A, Kryk A, Yuniata, Górecka E, Krywda M, Safitri I, Sapar A, Dabek P, Arsad S, Gusev E, Rudiyansyah, Peszek L, Wróbel RJ (2021) Indonesian coral reef habitats reveal exceptionally high species richness and biodiversity of diatom assemblages. Estuar Coast Shelf Sci 261:107551. http://dx.doi.org/10.1016/j.ecss.2021.107551
Robards MD, Piatt JF, Rose GA (1999) Maturation, fecundity, and intertidal spawning of Pacific sand lance in the northern Gulf of Alaska. J Fish Biol 54:1050–1068. https://doi.org/10.1111/j.1095-8649.1999.tb00857.x
Romano G, Miralto A, Ianora A (2010) Teratogenic effects of diatom metabolites on sea urchin Paracentrotus lividus embryos. Mar Drugs 8:950–967. https://doi.org/10.3390/md8040950
Rossi S, Sabatés A, Latasa M, Reyes E (2006) Lipid biomarkers and trophic linkages between phytoplankton, zooplankton and anchovy (Engraulis encrasicolus) larvae in the NW Mediterranean. J Plankton Res 28:551–562. https://doi.org/10.1093/plankt/fbi140
Ruocco N, Annunziata C, Ianora A, Libralato G, Manfra L, Costantini S, Costantini M (2019) Toxicity of diatom-derived polyunsaturated aldehyde mixtures on sea urchin Paracentrotus lividus development. Sci Rep 9:517. https://doi.org/10.1038/s41598-018-37546-y
Ruocco N, Albarano L, Esposito R, Zupo V, Costantini M, Ianora A (2020) Multiple roles of diatom-derived oxylipins within marine environments and their potential biotechnological applications. Mar Drugs 18:342. https://doi.org/10.3390/md18070342
Salas-Berrios F, Valdés-Aguilera J, Landaeta MF, Bustos CA, Pérez-Vargas A, Balbontín F (2013) Feeding habits and diet overlap of marine fish larvae from the peri-Antarctic Magellan region. Pol Biol 36:1402–1414. http://dx.doi.org/10.1007/s00300-013-1359-8
Sansone C, Braca A, Ercolesi E, Romano G, Palumbo A, Casotti R, Francone M, Ianora A (2014) Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells. PLoS One, 9:e101220. https://doi.org/10.1371/journal.pone.0101220
Schmidt-Nielsen K (1972) Locomotion: Energy cost of swimming, flying, and running. Science 177:222–228. https://doi.org/10.1126/science.177.4045.222
Spitsbergen JM, Walker MK, Olson JR, Peterson RE (1991) Pathologic alterations in early life stages of lake trout, Salvelinus namaycush, exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin as. Aquat Toxicol 19:41–71. https://doi.org/10.1016/0166-445X(91)90027-7
Tourdot B, Ahmed I, Holinstat M (2014) The emerging role of oxylipins in thrombosis and diabetes. Front Pharmacol 4:176. https://www.frontiersin.org/articles/10.3389/fphar.2013.00176
Van Leeuwen JL, Voesenek CJ, Müller UK (2015) How body torque and Strouhal number change with swimming speed and developmental stage in larval zebrafish. J R Soc Interface 12:20150479. https://doi.org/10.1098/rsif.2015.0479
Varrella S, Romano G, Ianora A, Bentley M, Ruocco N, Costantini M (2014) Molecular response to toxic diatom-derived aldehydes in the sea urchin Paracentrotus lividus. Mar Drugs 12:2089–2113. https://doi.org/10.3390/md12042089
Vidoudez C, Casotti R, Bastianini M, Pohnert G (2011) Quantification of dissolved and particulate polyunsaturated aldehydes in the Adriatic Sea. Mar Drugs 9:500–513. https://doi.org/10.3390/md9040500
Voesenek CJ, Muijres FT, van Leeuwen JL (2018) Biomechanics of swimming in developing larval fish. J Exp Biol 221:jeb149583. https://doi.org/10.1242/jeb.149583
Witkowski A, Li CL, Zgłobicka I, Yu SX, Ashworth M, Dąbek P, Qin S, Tang C, Krzywda M, Ruppel M, Theriot EC, Jansen RK, Car A, Płociński T, Wang YC, Sabir JSM, Daniszewska-Kowalczyk G, Kierzek A, Hajrah NH (2016) Multigene Assessment of Biodiversity of Diatom (Bacillariophyceae) Assemblages from the Littoral Zone of the Bohai and Yellow Seas in Yantai Region of Northeast China with some Remarks on Ubiquitous Taxa. J Coast Res 74:166–195. https://doi.org/10.2112/SI74-016.1
Wolfram S, Nejstgaard JC, Pohnert G (2014) Accumulation of polyunsaturated aldehydes in the gonads of the copepod Acartia tonsa revealed by tailored fluorescent probes. PLoS One 9:e112522. https://doi.org/10.1371/journal.pone.0112522
Yin MC, Blaxter JHS (1987) Feeding ability and survival during starvation of marine fish larvae reared in the laboratory. J Exp Mar Biol Ecol 105:73–83. https://doi.org/10.1016/S0022-0981(87)80030-8
Yúfera M, Darias MJ (2007) The onset of exogenous feeding in marine fish larvae. Aquacult 268:53–63. https://doi.org/10.1016/j.aquaculture.2007.04.050
Zhou Y, Zhang Y, Li F, Tan L, Wang J (2017) Nutrients structure changes impact the competition and succession between diatom and dinoflagellate in the East China Sea. Sci Total Environ 574:499–508. https://doi.org/10.1016/j.scitotenv.2016.09.092

Table 1. Experimental conditions. PUA concentrations in µg/ml; *mortality of all embryos in one replicate prior to measurement; **mortality of all embryos prior to measurement.

	Age at exposure	Exposure duration	PUA	PUA1	PUA2	PUA3
Heart rate (n=25 per replicate)	24 hpf	3 dpf	Octadienal	1.12	5.59	11.18
			Heptadienal	0.66	3.30	6.61
			Decadienal	0.38	1.90	3.81
			Total	2.16	10.80*	21.59**

Morphology	24 hpf	6 dpf	Octadienal	1.12	5.59	11.18
(n=25 per replicate)			Heptadienal	0.66	3.30	6.61
			Decadienal	0.38	1.90	3.81
			Total	2.16	10.80	21.59

Survival (n=30 per replicate)	2 hpf	Until 100% mortality	Octadienal	1.12	2.24	3.36
			Heptadienal	0.66	1.32	1.98
			Decadienal	0.38	0.76	1.14
			Total	2.16	3.24	6.48

Table 2. Results of linear mixed models for heart rate, standard length (SL), body depth one (BD1), and body depth two (BD2). Numbers in parentheses are model reductions in comparison to embryo water control (EW). S: solvent control. CI: confidence intervals.

	Heart rate			SL
Predictors	Estimates	CI	P	Estimates	CI	P
EW control (intercept)	121.20	106.83 – 135.57	<0.001	3.39	3.16 – 3.62	<0.001
S control	107.2 (-15.00)	-39.22 – 9.22	0.187	3.36 (-0.03)	-0.40 – 0.33	0.841
PUA1	106.2 (-14.20)	-38.42 – 10.02	0.208	3.36 (-0.03)	-0.40 – 0.33	0.851
PUA2	77.2 (-44.10)	-71.18 – -17.02	0.006	2.83 (-0.57)	-0.93 – -0.20	0.006
PUA3				2.75 (-0.64)	-1.00 – -0.27	0.003

Observations	110			326
Marginal R²/Conditional R²	0.56/0.95			0.59/0.88

	BD1			BD2
Predictors	Estimates	CI	P	Estimates	CI	P
EW control (intercept)	0.45	0.41 – 0.49	<0.001	0.14	0.12 – 0.15	<0.001
S control	0.43 (-0.02)	-0.08 – 0.04	0.463	0.08 (-0.06)	-0.04 – 0.02	0.402
PUA1	0.43 (-0.02)	-0.08 – 0.04	0.471	0.09 (-0.05)	-0.03 – 0.02	0.671
PUA2	0.35 (-0.10)	-0.16 – -0.04	0.006	0.10 (-0.04)	-0.02 – 0.03	0.466
PUA3	0.32 (-0.13)	-0.19 – -0.06	0.001	0.10 (-0.04)	-0.02 – 0.03	0.526

Observations	326			326
Marginal R²/Conditional R²	0.48/0.70			0.11/0.51

Table 3. PERMANOVA pairwise comparisons of larval morphology. Sig = p < 0.05.

Pair	Df	SS	F Model	R2	p-value	p-adjusted	Sig
PUA3 vs PUA2	1.000	0.009	2.674	0.022	0.083	0.830
PUA3 vs PUA1	1.000	0.349	186.363	0.593	0.001	0.010	*
PUA3 vs S	1.000	0.343	184.448	0.596	0.001	0.010	*
PUA3 vs EW	1.000	0.393	221.308	0.634	0.001	0.010	*
PUA2 vs PUA1	1.000	0.243	134.487	0.512	0.001	0.010	*
PUA2 vs S	1.000	0.237	132.489	0.515	0.001	0.010	*
PUA2 vs EW	1.000	0.279	163.046	0.560	0.001	0.010	*
PUA1 vs S	1.000	0.000	0.288	0.002	0.755	1.000
PUA1 vs EW	1.000	0.002	5.014	0.035	0.011	0.110
S vs EW	1.000	0.003	9.461	0.065	0.001	0.010	*

Table 4. P-values from pairwise comparisons of 5 different treatment group survival curves from Kaplan-Meier analysis.

PUA	PUA1	PUA2	PUA3	EW
Mid	0.1742	-	-	-
High	<0.0001	<0.0001	-	-
EW	0.0028	<0.0001	<0.0001	-
S	0.0076	<0.0001	<0.0001	0.5976

No competing interests reported.

Download PDF

Reviewers agreed at journal
06 Sep, 2024
Reviewers agreed at journal
04 Sep, 2024
Reviewers invited by journal
03 Sep, 2024
Editor assigned by journal
07 Aug, 2024
Submission checks completed at journal
07 Aug, 2024
First submitted to journal
06 Aug, 2024

You are reading this latest preprint version

The effects of diatom polyunsaturated aldehydes on embryonic and larval zebrafish (Danio rerio)

Status:

Version 1

Abstract

Figures

INTRODUCTION

METHODS

Animal care

PUA mixture and solvent choice

Heart rate

Morphology

Survival

RESULTS

Heart rate

Morphology

Standard length

Body depth at yolk sac

Body depth at anus

Multivariate analysis of larval body shape

Survival

DISCUSSION

Conclusions and future directions

Declarations

Author Contribution

Acknowledgement

References

Tables

Additional Declarations

Status:

Version 1