Effects of Temperature on Early Stage Development of L. vulgaris

Based on the paper Ocean Warming Enhances Malformations, Premature Hatching, Metabolic Suppression and Oxidative Stress in the Early Life Stages of a Keystone Squid by Rosa et al., 2012

Warming and Squid Development

You may believe that global warming is a myth, or you may think the impending rise in sea levels due to melting icebergs will soon result in the submergence of the Sunshine State.  However, many scientists agree that there has been an increase in atmospheric temperatures (about 0.6oC) over the past century, and a definite rise in ocean surface temperatures.  Following this trend, this could mean a total increase of 3oC by the year 2100.

So what?

This warming trend implicates serious changes in coastal ecosystems worldwide.  Many marine species thrive under very specific temperature constraints, and even a slight variation can have very deleterious effects on development and behavior.  If the uncertain fate of thousands of species isn’t enough cause for alarm in and of itself, coastal marine ecosystems are among the most economically profitable.  While no one is certain what the future of this warming trend holds, it is to our benefit that we seek to understand what the consequences may be.

Why Loligo vulgaris as a model?

Loligo vulgaris, the European Squid, is an extensively commercially exploited organism.  Eggs are easily obtained and transported to labs to be manipulated under realistic physical conditions.  Most studies are easily applicable to other marine invertebrates as well as other fish.  Squids in particular are important in maintaining ecosystem stability, as they strongly influence ecosystem dynamics and function as biological pumps between ecosystems.  In addition, these squids have two distinct spawning seasons, one in winter and one in summer, each with different life strategies.  Winter spawners lay smaller eggs in comparison to summer spawners, suggesting a strategy of higher individual fitness for summer hatchlings.

Important concepts

Temperature is one of the most important regulating factors of embryonic development.  Factors such as hatching time, efficiency of yolk utilization, size and weight at first feeding, oxygen consumption rates, and enzymatic functions are all dependent on temperature.  In general, higher temperatures result in higher metabolism, which result in a higher demand for oxygen.  At the same time, as temperature increases, oxygen supply mechanisms become less and less effective at meeting metabolic demands.  Obviously, this presents an interesting dilemma for marine species under warming conditions.  The condition of oxygen supply deprivation to tissues is called hypoxia, and a common response to hypoxia in fish is premature hatching.  Additionally, under hypoxic conditions, organisms switch to anaerobic methods of respiration to reach their energy demands.  Because of the importance of temperature on development, most organisms thrive under specific thermal tolerance limits (an upward and lower bound on optimal temperature for survival).  Due to warming, most marine species are already living very close to the upward limit.

The Study

L. vulgaris eggs were collected from winter and summer spawning events and reared until the paralarval stage at current-day temperatures (13 and 17 degrees Celsius, respectively) and expected future temperatures (+2 degrees) to see how warming scenarios would affect the following: i) survival rates and development time, ii) embryo growth and yolk-sac depletion, iii) hatching, iv) metabolic rates and thermal sensitivity, v) anaerobic pathways, vi) thermal tolerance limits, vii) heat shock response, and viii) oxidative stress and peroxidation.

Figure 1. Effect of warming in the early ontogeny squid, Loligo vulgaris, namely: Development time (A), survival rates (B) and growth rates (% - C; mm day−1 - D) of embryos at the different temperature scenarios (red symbols highlight the future summer scenario).

As shown in Fig1A, development time plunged from 27±1 days under the current winter conditions to 14±2 under future warming scenarios.  Survival rates showed a drastic decrease from 96% and 94% at current-day temperatures to 71% at the summer warming temperature (Fig1B).  Growth rates increased with temperature then experienced a trend reversal at the summer warming temperature (Fig1C).

Figure 2 depicts some of the most commonly observed abnormalities.  The most common abnormalities among the embryos consisted of underdeveloped mantles, complete body deformities and eye dimorphism, elongated bodies and mantle deformities, and incidences of mantle detachment.  The likelihood of abnormalities was shown to significantly increase with temperature, with the highest incidence being seen at the future summer warming temperature (as shown in Fig3A).

Figure 2. Abnormalities found during the early development squid, Loligo vulgaris.

Not only did likelihood of deformity increase with temperature, but premature hatching as well (Fig3B), which is indicative of hypoxia in the eggs.  A striking consequence of premature hatching was an increase of hatchlings with their yolk sacs still attached (Fig3C).  While the yolk could still be consumed after hatching, the most efficient use of the yolk is during development in the embryo, where it functions to invest in the growth of tissue and provides energy for respiration.  The growth and energy loss is not easily compensated after hatching.

Figure 3. Abnormalities and premature hatching in the squid, Loligo vulgaris

Figure 4. Metabolic physiology of squid (Loligo vulgaris) late embryos and hatchlings: A) oxygen consumption rates (µmolO2h−1g−1), B) octopine concentration (µmol g−1), and C) individual energetic cost of the planktonic transition (cal day−1 ind−1), at the different temperature scenarios (red symbols highlight the future summer scenario).

Fig4&5)Oxygen Consumption Rates, Octopine Concentrations, Energetic costs, and Q10 Values

  • OCRs (Oxygen Consumption Rates) R(Tn) (see below formula) were calculated through periodical oxygen concentration measurements
  • Octopine- the end product of anaerobic respiration in many cephalopods.  Used to indicate reliance in anaerobic mechanisms
  • Energetic Cost- “metabolic burst” associated with transition from embryonic to paralarval stage
  • Q10- thermal sensitivity, which is the rate of change of the biological system from raising the temperature 10 degrees Celcius. Most systems function at Q10 values 2-3.  Calculated using the following formula: where R(T1) and R(T2) are the oxygen consumption rates at temperatures T1 and T2.

The results also show how OCRs increase significantly with temperature, from 13.0 µmol O2/h/g at the winter temperature to 24.1 µmol O2/h/g at the future summer warming scenario for the embryos, and more drastically from 28.1 µmol O2/h/g to 59.1 µmol O2/h/g for the paralarvae (Fig4A).   This is consistent with the concept that higher temperature leads to higher metabolism, which was previously mentioned.  In addition, it was seen that octopine concentrations were higher in the embryos in comparison to hatchlings, showing that they relied more on anaerobic mechanisms (Fig4B).  This shows that the eggs experienced more difficulty meeting metabolic demands, possibly due to less effective methods of ambient oxygen extraction mechanisms of the eggs.  Figure 4C shows that a higher energetic cost is associated with higher temperature, consistent with the fact that both life stages have increased OCRs as temperatures increase.  Furthermore, the thermal sensitivity was measured for hatchlings and embryos, showing values ranging from 2-3 for hatchlings, and values around 1.5 above 15 degrees for embryos, indicating metabolic suppression (Fig5).  Expected OCRs were calculated using the expected Q10, and are shown as dashed lines in Fig4A.

Figure 5. Thermal sensitivity (Q10) of late embryonic stages and hatchlings of Loligo vulgaris at the different temperature scenarios (red symbols highlight the future summer scenario).

Figure 6. Thermal tolerance limits (A - Lt50, °C; B - Lt100, °C) and heat shock response (HSP70/HSC70, ng mg−1) (C) of late embryonic stages and hatchlings of the squid Loligo vulgaris at the different temperature scenarios (red symbols highlight the future summer scenario).

Fig6)Thermal Tolerance Limits and Heat Shock Response

  • LT50- median lethal temperature
  • LT100- absolute lethal temperature
  • HSP70/HSC70- heat shock protein, which can eliminate or change reactive oxygen species; formed as a result of increased metabolic demands

Both LT50 and LT100 were affected by temperature, with higher thresholds in embryos than in hatchlings (Fig6A-B).  The heat shock response increased with temperature in both stages, and all but winter embryos showed acclimation to temperature from embryo to hatchling through elevated heat shock response (Fig6B).

Oxidative Stress Response by Antioxidant Enzymes GST, CAT, and SOD

  • Peroxidation- oxygen degradation of lipids by ROS
  • ROS (reactive oxygen species)- oxygen radicals that damage cells by “stealing” electrons
  • GST  (Glutathione S-Transferase)- breaks down xenobiotics and detoxifies peroxidized lipids
  • CAT (catalase )- catalyzes the decomposition of hydrogen peroxide to water
  • SOD (superoxide dismutase)- catalyzes dismutation of superoxide to oxygen and hydrogen peroxide
  • MDA (Malondialdehyde)-  end product of oxidative degradation of lipids; marker of peroxidation

Figure 7. Antioxidant response of Loligo vulgaris’ late embryos and hatchlings at the different temperature scenarios: A) GST (nmol min−1mg−1), B) CAT (pmol mg−1), C) SOD (U g−1) and d) malondialdehyde (MDA; pmol mg−1).

GST activity was shown to be much higher in hatchlings, showing a positive trend, with a reversal at the summer warming temperature (Fig7A).  In contrast, the increase in hatchling CAT concentrations was non-significant, while the embryos showed a slight negative trend in CAT concentrations (Fig7B).  SOD concentrations did not vary for embryos, but showed a positive trend in hatchlings (Fig7C).  Lastly, Figure 7D shows that MDA concentrations rose significantly with temperature, with a more profound increase in hatchlings.  These findings indicate higher levels of metabolism, resulting in higher levels of ROS, which , fortunately, was followed by a subsequent rise in antioxidant activity, with marked increases in hatchlings.


Based on the results, higher temperature resulted in higher metabolic demand driven by augmented growth rate and earlier hatching time.  Under these conditions, it was difficult for the embryos to meet their increased oxygen demands, aerobically or anaerobically, and the hypoxic stress often lead to premature hatching, abnormalities, and a higher mortality rate.  In addition, after hatching, paralarvae must feed, and their method of locomotion (a jet-propulsion mechanism) is demanding in regards to oxygen consumption, especially in reference to the low level of metabolic reserves.   Because of this, hatchlings must be able to find food within a shorter period of time.  Not only that, but increased temperature led to more heat stress on enzymatic processes, which was met by a compensatory response by hatchling (but not embryo) HSPs.  While the outcome may look bleak for marine biotas and the survival of coastal ecosystems and economies, L. vulgaris did show compensatory mechanisms and the ability to acclimate to temperatures.  All in all, species are plastic, and there is hope that future development will be marked by mechanisms to support higher energy demands resultant of higher temperatures.


This study was one of the very few that studied early life stage response to warming, in contrast to studies done on adult stages.  This approach may be more beneficial in the long run, since embryonic development and paralarval stages may be more vulnerable in the face of long-term environmental changes.  However, so far, it seems that this article hasn’t received much attention from the scientific community as a whole.  However, similar studies by the same authors (Pimentel et al., 2012) indicate comparable results in other squid species, as well as other fish.  In contrast, fish do not experience as negative effects from increased temperature, possibly because of the extremely high metabolic demands of the planktonic life strategy exhibited by squid species.  In addition, an interesting anomaly was that catalase activity was the only variable that did not vary significantly with temperature.  It might be worthwhile to determine the cause, and if this finding could be the springboard for future studies on the enzyme’s response to temperature increases in biological systems.


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