Effects of marine heatwaves and fish predator cues on the growth and development of pseudodiaptomus incisus (shen & lee, 1963) under laboratory conditions (tt)

VIETNAM NATIONAL UNIVERSITY, HANOIVNU UNIVERSITY OF SCIENCES FACULTY OF BIOLOGY Vu Ngoc Anh Major: Biology Major code: 8420101 ABSTRACT EFFECTS OF MARINE HEATWAVES AND FISH PREDATOR CUES

VIETNAM NATIONAL UNIVERSITY, HANOI

VNU UNIVERSITY OF SCIENCES

FACULTY OF BIOLOGY

Vu Ngoc Anh

Major: Biology Major code: 8420101

ABSTRACT

EFFECTS OF MARINE HEATWAVES AND

FISH PREDATOR CUES ON THE GROWTH AND DEVELOPMENT OF

Pseudodiaptomus incisus (Shen & Lee, 1963) UNDER LABORATORY CONDITIONS

Submitted in partial fulfillment of the requirements for the degree of

Master of Science in Biology

Hanoi – 08/2024

Climate change, especially marine heat waves (MHW), is one of the greatestthreats to global biodiversity because extreme warm sea surface temperature duringMHW are often beyond the optimal thermal range and last longer than onegeneration of tropical coastal species Moreover, the pace of changingenvironmental conditions is much faster than the ability of organisms to developadaptive responses However, we know little about the role of MHWtransgenerational acclimation in shaping coastal species, particularly an ecologicallyrelevant context with biotic interactions such as predation stress

Recent advancements in eco-evolutionary studies have revealed the criticalimportant role of transgenerational plasticity (TGP), where the environmentexperienced by the parental generation may improve offspring performance in thesame environment TGP generally occurs through epigenetic changes, habitatselection, or niche construction TGP is especially crucial for organisms to copewith new, predictable but fast changing and short-term environmental changesacross generations, which is relevant to the duration of an MHW that often lastslonger than one generation for nearly all tropical zooplankton species

In the shallow tropical coastal ecosystems such as mangroves, seagrasses,and coral reefs, the predation stress is typically high as these ecosystems are thespawning and nursery ground of marine species Non-consumptive predation stressfrom voracious fish larvae and juveniles can significantly influence morphology,behavior, physiology, growth, and reproduction of the prey Parental exposure topredators may also induce an increase in the reproduction of offspring generationand this effect may last two generations after exposure to predators However, theTGP of prey species to predation stress may reduce in the degree of plasticity with

an increasing number of exposed generations exposed to predators

Investigations of the transgenerational effect of MHWs in an ecologicallyrelevant context, such as the presence of fish predator cues (FPC) on key

environments Understanding whether copepods are resilient or vulnerable toMHWs in the context of predation stress is important, given that they are a keypathway for the transfer of energy and resources from photosynthesizing organisms

to higher trophic levels, and ultimately the productivity of the coastal ecosystems.However, the combined effect of heatwaves and non-consumptive predation risk onprey species across generations is still a major knowledge gap in current ecologicalresearch Our previous study shows that FPC induced a higher individual

performance of the calanoid copepod Pseudodiaptomus incisus under control

temperature, but it magnified the deleterious impacts of MHW on grazing andreproductive success In this study, we address the knowledge gap identified above

by assessing the immediate effect during the exposure together with the effects ofparental exposure, TGP to MHW, FPC, and their interactions in a full orthogonalmanner with 4 treatments in F1 and 16 treatments in F2 generation

LITERATURE REVIEW

Marine heatwaves (MHWs) are discrete periods of abnormally hightemperatures (≥5 days) above the 90th percentile of 30-year sea surfacetemperatures measured at the locality [23, 31, 41] In the last decade, MHWs haveemerged as a major driver reshaping marine biodiversity, causing the tropicalisation

of temperate coastal ecosystems, mass coral bleaching, and mass mortality ofcoastal invertebrates worldwide [17, 18, 24, 40] The effects of MHWs on marineecosystems and biota are becoming more severe under ongoing climate change [40]

In the Southeast Asian region, coastal marine organisms are increasingly beingexposed to episodes of MHWs [15, 47] The average duration of MHWs in theSoutheast Asian seas is 18–22 days per event [47], which is equivalent to ~2–3generations of tropical coastal invertebrates A few recent studies have found strongnegative effects of MHWs on marine organisms such as copepods and fish in theSoutheast Asian region (e.g., [11,56]), but these were mostly limited to one-exposedgeneration It remains to be explored how transgenerational plasticity to MHWsmight ameliorate the effects of warming on offspring generation (see, e.g., in coralfish, [13])

Advancements in eco-evolutionary studies on adaptations of organisms totoxic algae, warming, ocean acidification, and contaminants have further exploredthe critical role of transgenerational plasticity (TGP) where the environmentexperienced by the parental generation may improve offspring performance in thesame environment [13, 16, 19, 26, 29, 42] For example, the tropical coraldamselfish A polyacanthus reduced the aerobic scope by 15–30% under acuteexposure to an elevated temperature of +1.5°C and + 3°C, but the offspring'saerobic scope fully recovered when reared at the same temperature [13] TGP isespecially crucial for organisms to cope with new, predictable but fast-changing andshort-term environmental changes across generations [14] The duration of anMHW often lasts longer than one generation for nearly all tropical zooplankton

temperature [36] or long-term warming [5, 9] TGP generally occurs throughepigenetic changes, habitat selection, or niche construction (reviewed in [12]) Inparticular, epigenetic changes such as the methylation of genes encoding oxygenconsumption, mitochondrial activity, and energy homeostasis play crucial functions

in restoring the performance of stress-exposed organisms across generations [35].Besides TGP, the parental effect is another non-genetic transgenerational effect, asthe transfer of nutrients from mothers to eggs may also affect offspring performance(reviewed in [22]) Parental effects of warming may negatively affect offspringperformance, which has been shown in mosquito larvae with increased mortalityand delayed development [44] Alternatively, genetic selection may occur throughstressor-induced mortality [26] or selection in favor of higher hatching success [5],which removes the most sensitive genotypes or increases the tolerant genotypes inthe population, and may contribute to increased fitness of the offspring generation

In the shallow tropical coastal ecosystems such as seagrasses, mangroves,and coral reefs, the predation stress is typically high as these ecosystems are thespawning and nursery ground of marine species Non-consumptive predation stressfrom voracious fish larvae and juveniles can significantly influence preymorphology, behavior, physiology, growth, and reproduction (e.g., [3, 45])

Calanoid copepods commonly show an increase in growth (e.g., Temora longicornis, [3]) and reproduction (e.g., P incisus, [45]) as general life-history trait responses to

fish kairomones Parental exposure to predators may also induce an increase in the

reproduction of offspring generation (e.g., Daphnia magna) and this effect may last

two generations after exposure to predators [46] However, the TGP of prey species

to predation stress may reduce in the degree of plasticity with an increasing number

of exposed generations exposed to predators For example, the pea aphid

(Acyrthosiphon pisum) responds to the presence of the predator ladybirds (Harmonia axyridis) by producing a higher frequency of the winged offspring, but

this TGP response decreases across 22 exposed generations [38]

Investigations of the transgenerational effect of MHWs in an ecologicallyrelevant context, such as the presence of fish predator cues (FPC) on key

zooplankton species, are relevant and timely with the increasing frequency, severity,and duration of MHWs and the intense predation stress of tropical coastalenvironments Understanding whether copepods are resilient or vulnerable toMHWs in the context of predation stress is important, given that they are a keypathway for the transfer of energy and resources from photosynthesizing organisms

to higher trophic levels, and ultimately the productivity of the coastal ecosystems[4] However, the combined effect of heatwaves and non-consumptive predationrisk on prey species across generations is still a major knowledge gap in currentecological research Our previous study shows that FPC induced a higher individual

performance of the calanoid copepod Pseudodiaptomus incisus under control

temperature, but it magnified the deleterious impacts of MHW on grazing andreproductive success [45]

MATERIALS AND METHODS Objectives of the study

The combined effects of MHW and non-consumptive predation risk on preyspecies across generations remain a major knowledge gap in current ecologicalresearch Therefore, in this study, we unravel the aforementioned question byassessing the direct effect during the exposure [8], together with the effects ofparental exposure, TGP (parental and offspring exposure) to MHW, FPC, and their

interactions on the growth and development of the copepods Pseudodiaptomus incisus through survival rates of both sexes, clutch sizes, percentage of female with

hatched eggs, the number of hatched nauplii per clutch, cumulative nauplii andfaecal pellets in five days The experiment was conducted in a full orthogonalmanner with 4 treatments in F1 and 16 treatments in F2 generation (Figure 1)

Figure 1.The schematic overview of the transgenerational experiment for the

direct and transgenerational MHW and FPC effects on Pseudodiaptomus incisus

(*CT: control temperature)

We tested the susceptibility of P incisus to MHW and/or FPC in two

generations by following eight hypotheses:

H1: MHW reduces the performance of P incisus due to energetic constraints

under extreme warming [45]

H2: FPC increases the performance of P incisus as a general antipredator

response [3, 45]

H3: FPC-induced increase in performance of P incisus is not sustained under

MHW due to the energetic constraints [45]

H4: Parental exposure to MHW reduces offspring performance in the controltemperature due to poor maternal provisioning [44]

H5: Parental exposure to FPC increases offspring performance in the absence ofFPC [46]

H6: TGP to MHW ameliorates the MHW effect on P incisus offspring [13] H7: TGP to FPC decreases the P incisus offspring performance toward

Adult copepod Pseudodiaptomus incisus were collected from a coastal pond

in Cam Ranh Bay, in July 2020 as F0 generation, using a zooplankton net (meshsize = 200 μm) The coordinates of the pond are (11.82397°N, 109.1233°E).During the collection, the pond water salinity and temperature were 27 - 28°C and

35 PSU, respectively

Fish predator cue stock

Barramundi (Lates calcarifer) larvae (15 individuals) were reared in 1–L

bottle for three days The total length of barramundi is 14 ± 1 mm A previous studyhas shown that barramundi larvae and juveniles with a total length of 4 – 20 mm

virtually prey upon copepods [6] Fish larvae were fed with P incisus After three

days, fish larvae were removed, the remaining water containing fish predator cueswas collected, divided into aliquots and frozen at -20 °C FPC was thawed beforeused in the experiment The effect of FPC still remains after being frozen [28]

Experimental methods

Copepod acclimation

F0 adult copepods were acclimated in water baths to the temperature of 30°C

or 34°C for three days 30°C was chosen as the control temperature since it is themean sea surface temperature in the southern Vietnam coastal sea (see Appendix S1,[10]) The MHW condition was manipulated at a temperature of 4°C since it isabout ~2°C higher than the 90% temperature variations measured in the Cam RanhBay (Doan X.N., Pham, Q.H and Dinh K.V., unpublished data) Adult males andfemales were sorted and then divided into 5-L bottles, approximately 1200individuals per bottle The temperature was gently increased by 1°C every 12 hoursuntil reaching the experimental temperatures During the acclimation, the salinity,light: dark cycle, and dissolved oxygen concentration were kept at 30 PSU,12L:12D, and >5 mg L-1 by aerations, respectively (see also [45]) F0 P incicus were fed two times a day with Isochrysis galbana at 30,000-33,000 cell L-1 (~800 –

850 μg carbon per litter, [10])

Experimental design and set up

To start the experiment, acclimatized F0 female P incisus carrying egg sacs

(prosomal length = 797.43 ± 2.17 µm, clutch size = 16 ± 2 eggs) were assigned to

1.2-L plastic bottles (15 females each bottle) and fed with I galbana for incubation After 30 h, when the hatching success of Speudodiaptomus species was > 90% [18].

F1 nauplii (180 - 240 individuals per bottle) were collected for the main experiment.There are 4 treatments in F1 generation: 2 temperatures (30°C or 34°C) × 2 FPC(absence or presence) × 10 replicates (Fig 1, Fig 2) FPC solution or filteredseawater (1 ml) was added to each experimental bottle The rearing medium andFPC and algae were renewed daily to minimize the change in the FPC concentrationand the indirect effect of MHW on the algal quality [45].

To generate F2 generation, once F1 developed into adults, 20 F1 femalescarrying egg sacs in each of 10 bottles of each treatment (200 individuals per F1treatment) were collected and transferred into 20 bottles (10 F1 females per bottle)and incubated for 30 h F2 offspring in every F1 treatment were divided into fourgroups, corresponding to four experimental conditions 30°C – no FPC, 30°C – FPC,34°C - no FPC and 34°C - FPC, resulting in 16 treatments (Fig 1, Fig 2)

Figure 2 The schematic of experimental design testing the direct and

transgenerational MHW and FPC effects on Pseudodiaptomus incisus.

Data analyses

In both generations, we analysed clutch size (from fixed females carrying anegg sac), hatching success, the survival of males and females, cumulative naupliiand faecal pellets over five days In addition to the aforementioned parameters, to

fully assess the growth of P incisus, we are currently measuring size at the maturity

of both male and female copepods

RESULTS AND DISCUSSION Results

Effects of MHW, FPC, and their interactions on F1 generation of P incisus

Exposure to MHW reduced survival of both males and females by 17-18%(MHW, Table 1, Fig 3) The lethal effect of MHW was independent of FPCexposure, indicated by an insignificant MHW × FPC interaction (Table 1, Fig 3)

Figure 3 Effects of the marine heatwave (MHW) and fish predator cues (FPC) on

the survival of males (a) and females (b) in F1 Pseudodiaptomus incisus Data are

visualized as mean ± SEs

The reproductive success and grazing of P incisus were overall affected

negatively by MHW (Fig 4, Fig 5) The hatched nauplii per clutch dropped by62% at 34°C (Fig 4c) The cumulative nauplii and faecal pellets were 27% and28% lower in MHW than in the control temperature (Fig 5) The FPC effect on thepercentage of females with hatched eggs was insignificant (Fig 4b) The number of

hatched nauplii per clutch, and cumulative faecal pellets of P incicus increased in

the presence of FPC (Fig 4c, Fig 5), but the FPC effect on the latter was several

Figure 4 Effects of the marine heatwave (MHW) and fish predator cues (FPC) on

the number of eggs per clutch (a), percentage of females produced hatched eggs (b),

hatched nauplii hatched from a clutch (c) of F1 Pseudodiaptomus incisus Data are

visualized as mean ± SEs

For the clutch size, the effect of FPC was statistically insignificant in both

temperatures (Fig 4a) P incisus produced approximately 15% more cumulative

nauplii in the presence of FPC only in control temperature, but not in MHW (Fig.5a) The correlation of cumulative nauplii production and cumulative faecal pelletswas insignificant (F1, 36= 2.65, P = 0.11, slope ± 1 SE = 0.38 ± 0.24, R2= 0.89)

Figure 5 Effects of the marine heatwave (MHW) and fish predator cues (FPC) on

cumulative nauplii per female (a) and faecal pellets per individual (b) of F1

Pseudodiaptomus incisus Data are visualized as mean ± SEs.

Effects of MHW, FPC, and their interactions on F2 generation of

P x incisus

Survival rates of F2 P incisus females and males was reduced by 8-10%

under MHWs (Fig 6) FPC did not affect F2 female and male survival as the FPC effect was insignificant (Fig 6) F2-FPC did not alter the lethal effect of F2-MHW, as indicated by an insignificant interaction of F2-MHW × F2-FPC (Fig 6).Strikingly, TGP to MHW (F1-MHW × F2-MHW) resulted in a similar female andmale survival compared to the control temperature; all had ~10% higher survivalthan F2-MHW exposed females and males (Fig 6), suggesting that lethal F2-MHWeffect was ameliorated This pattern was independent of F1-FPC and F2-FPC (Fig.6) The lethal MHW effect in the parental generation was no longer present in F2generation when F2 copepods were reared in the control temperature (Fig 6) Therewere no lethal effects of PFC on the survival of F2 males and females and it wasindependent of F1-FPC

F2-Figure 6 Immediate and transgenerational effects of the marine heatwave (MHW)

and fish predator cues (FPC) on the survival rate (mean ± SE) of F2

Pseudodiaptomus incisus males (a) and females (b) *CT: control temperature.

The clutch size, percentage of females with hatched eggs and hatched nauplii

from a clutch of F2 P incicsus were 42%, 5%, and 22% reduced in F2-MHW (Fig.

7a-c) The F2-MHW effect was independent of the F2-FPC as F2-MHW × F2-FPCwas insignificant Importantly, F2-MHW was generally less intense when F1 wasalso exposed to MHW (Fig 7a-c) Parental exposure to MHW (F1-MHW) alsoresulted in 22% and 33% reductions in the size of clutches and hatched nauplii from

a clutch of F2 P incisus (Fig 7a,c) No F1-MHW effect on percentage of F2

females with hatched eggs occurred, as it was similar to the control level (Fig 7b).Overall, F2-FPC caused increased clutch size of F2 females by 4%, but this patternwas mainly driven by large clutches of control F2-females that their parentalgeneration was also exposed to both MHW and FPC (F1-MHW × F1-FPC),resulting in two-, three- and four-way interactions (Fig 7a) The F2-FPC effect onother reproductive parameters was absent or minimal (Fig 7b,c)