---
title: "Lit Review: Experimental design questions"
author: "Kathleen Durkin"
date: "2024-06-20"
categories: ["pacific_oyster"]
format:
  html:
    toc: true
engine: knitr
bibliography: ../../../references.bib
---

Addressing some questions/notes raised during preliminary project planning

### Gametogenisis/spawning conditions

We'd need to make sure they don't spawn, so need to do some digging into what conditions might induce spawning. Would be important during priming stage.

In hatchery settings:

-   "In the Pacific Northwest, **the Pacific oyster may be induced to spawn and viable gametes may be obtained at any time during the year**. Year-round spawning is possible because the Pacific oyster normally does not completely spawn out in all Pacific Northwest estuarine areas, generally too cool to induce complete spawning."

    Conditioning: Oyster with sufficient glycogen stores held at **18-20C for 4 weeks** to develop mature gametes. These conditioned oysters will be prime for induced spawning for the next 2-4 weeks.

    Initiating spawning: Raise water to 25C, then to 30C over 30min period. Then **fluctuate between 25C and 30C**. **Addition of gametes** to the water can also induce spawning, presumably because of some hormone signaling.

    [@breese1975]

In natural/outplant conditions:

-   In France's Thau Lagoon, which is characterized by no tidal amplitude (oysters remain submerged), high summer temps and salinities (\>26C and 36 PSU), low phytoplankton concentration, and high oyster growth rates.

    "**gametogenesis intensity (GSI, fecundity) was correlated to the abundance of diatoms**"

    "spawning in C. gigas does not occur below a daily seawater temperature of **17-18°C"**, but also noted that mass spawning in the warm Thau Lagoon occurred at a much higher temp of 22C, likely because of the water's higher average temperature and/or the absence of a tide cycle that would expose the oysters to dessication or aerial temperature stress. So there's some **room for thermal acclimatization/adaptation in the spawning trigger**!

    Other possible spawning triggers include phytoplankton concentration, lunar cycle, and presence of thunder storms.

    [@ubertini_gametogenesis_2017]

Experimental testing:

-   Experimental testing of environmental conditions impact on gametogenisis (gonadal development/broodstock conditioning)

    **Fastest gonadal development (oocyte maturation) at 22C (first mature oocytes in 19 days)**

    ![](https://ars.els-cdn.com/content/image/1-s2.0-S0044848601008985-gr4.jpg)

    [@chavez-villalba_broodstock_2002]

Summary: Generally speaking, gametogenisis is prompted by a minimum temperature of 17C - 18C in the presence of sufficient energy storage. Fastest maturation happens around 22C, with spawning triggered by warmer temperatures than used during conditioning (e.g., 25C to 30C). If we're acclimatizing in the 25C range there will likely be resultant gametogenisis, but conditioning for \~2 weeks is sufficiently short to prevent full gonadal maturation ("ripening") and subsequent spawning. Not entirely clear though what happens if you hold at 20C for 2 weeks and then drop back down in temp -- would gametogenisis continue? Holding at higher temps after ripening can trigger spawning, so we'd need to be mindful of the hardening -\> stress test timeline. Then again, if 25-30 is ideal to trigger spawning, maybe higher temps (35-40C) would be too hot for it...

### Hardening regime

Should look at all the hardening papers to try to assess best timeline and conditions (e.g. how long to harden, how long to wait between hardening and stress)

| Species                                 | Age      | Source      | Hardening regime                                                                                                                                   | Outcome                                                                                                           | Paper                      |
|-----------------------------------------|----------|-------------|----------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------|----------------------------|
| Manila clam (R. philippinarum)          | adult    | wild caught | 28C or 30C for 2 hrs, recover for 12 hrs, stress test of 30C or 32C 1wk                                                                            | 30C hardening improved survival at 30C stress                                                                     | [@zhang_metabolomic_2023]  |
| Olympia oyster (O. lurida)              | juvenile | hatchery    | 15C or 21C 2wks, plus tide treatment (submerged & exposed for 6hr each, 2x/day). MHW 15C, 18C, 21C, and 24C for 72hr. Survivors outplanted 9months | no effects on short-term mortality, but hardening improved outplanting survival. Temp hardening inhibited growth. | [@shukla_resisting_2023]   |
| Kumamoto oyster (C. sikamea)            | juvenile | hatchery    | 15C or 21C 2wks, plus tide treatment (submerged & exposed for 6hr each, 2x/day). MHW 15C, 18C, 21C, and 24C for 72hr.                              | no effects on mortality, temp hardening inhibited growth                                                          | [@shukla_resisting_2023]   |
| Pacific oyster (C. gigas)               | juvenile | hatchery    | 15C or 21C 2wks, plus tide treatment (submerged & exposed for 6hr each, 2x/day). MHW 15C, 18C, 21C, and 24C for 72hr.                              | no effects on mortality, tidal hardening improved growth.                                                         | [@shukla_resisting_2023]   |
| California mussel (M. californianus)    | adult    | wild caught | hardened at 25C, 30C, or 35C for 2hr, recovered at 14C for 1-28 days, then stress test 40C for 2hr                                                 | 30C and 35C improved survival for 2-3 weeks, but effect gone by day 28                                            | [@moyen_single_2020]       |
| Sydney rock oyster (S. glomerata)       | adult    | hatchery    | Four \~2hr emmersion hardening at 30C and 40C, each separated by 1wk, then MHW of emmersed at 50C for 2hr                                          | 40C hardening improved survival and growth, as did control+antibiotics                                            | [@scanes_acclimation_2023] |
| Pearl oyster (P. maxima)                | adult    | wild caught | two iterations of 28C or 32C for 3 days, with 3 day recovery for each                                                                              | Changes in enzyme activity for antioxidents, biomineralization, metabolism                                        | [@he_repeated_2021]        |
| species of tropical oyster (I. nucleus) |          | wild caught | reciprocal cross of air (26C and 40C, 4hrs) X water (26C and 35C, 4hrs)                                                                            | Hardening increased thermal tolerance and lethal range, respiration, hsp70 expression                             | [@giomi_importance_2016]   |

[@zhang_metabolomic_2023]:

Wild-caught adult Manila clams acclimated at 13.5C for 1 week, increased by 1C per day to 18C, then maintained for 1 week. Then hardened at 28C or 30C for 2 hours, then left to recover at 18C for 12 hours. Then subjected to extreme stress test (increase 2C/hr), either 30C or 32C for one week. Found that 30C hardening significantly improved survival time at 30C stress, but 28C did not (in comparison to non-hardened). Also, hardening made no difference at the 32C stress test. Maybe this means that it's best to harden at temperature of stress test (i.e. harden at temperature expected to experience in MHW conditions)?

[@shukla_resisting_2023]:

Juvenile O. lurida, juvenile C. sikamea, and juvenile C. gigas from hatchery. Acclimated at 15C, treatment groups hardened at 21C (0.5C/hr) for 2 weeks. In both control and temperature treatment, also included a tide treatment, in which oysters were intermittently submerged and exposed for 6hr each, twice a day. After hardening, recovered at immerged at 15C for 24hr. MHW treatments (reached at 0.5C/hr) were 15C, 18C, 1C, and 24C, no tidal component, for 72hr. Surviving O. lurida were outplanted in Sacramento for 9 months (March 2022 - Dec. 2022).

Very little mortality (\<10%) in all species/treatments during hardening/MHW test, and no significant effect of temp or tidal hardening on MHW survival. In O. lurida and C. sikamea, temp hardening had a negative impact on growth (i.e. 15C control was largest). However, in C. gigas, temp hardening had no effect on growth and tidal hardening improved growth. In outplanting, all O. lurida with tidal hardening died, but those hardened at 21C (fully submerged) had much higher survival than control (15C submerged).

[@moyen_single_2020]:

Adult, wild-caught M. californianus hardened at 25C, 30C, or 35C for 2hr, then recovered at 14C for 1-28 days, then underwent stress test at 40C for 2hr. Following stress test, back in 14C and monitered for 4 weeks. 30C hardening conferred improved heat tolerance for 14 days, and the 35C hardening improved survival for 21 days after extreme stress, but effect gone by day 28.

This experimental design has a great component of waiting **4 weeks after extreme stress to evaluate mortality**. I really like this idea, since stress could kill after the actual event is over. Also, test temperatures selected because 25C was lowest temp at which HSPs are upregulated in species, 35C is \~1C below species' critical temperature of cardiac function, and 40C is avg lethal temp in species. Also has a cool graph of \# days between heat stress bouts.

"Heat hardening has typically been defined as a transient response that confers improved heat tolerance immediately after the initial heat-stress bout for up to 32 h \[9,12,13,39,40\], while longer-lasting improvements in heat tolerance are termed as heat acclimation."

[@scanes_acclimation_2023]:

Australian adult, hatchery-reared S. glomerata acclimated for 1 week at 23C. Two emmersion heat treatments, 30C and 40C. First hardening 1.5hr, back to ambient 1 wk, then three more hardening treatments of 2 hrs each, separated by 1 wk each time (total of 4 hardening treatments over 4 weeks). Simulated MHW 1wk after final hardening, kept at emmersed 50C for 2hr. Monitered for 48 hr, then checked at day 8 and 16. Also note there's an antibiotic group, since study investigates pathogen load.

40C hardening improved survival and growth, but 30C did not. Also, control+antibiotic oysters had improved survival. Suggests hardening may be bolstering capacity to repel pathogenic bacteria as one mechanism of improving post-HW survival.

[@he_repeated_2021]:

Adult, wild-caught P. maxima oysters acclimated at 24C for 1 day, then exposed to MHW conditions at 28C and 32C for 3 days, 3 days recovery, followed by another exposure and recovery. Note that 36C treatment also attempted but saw 100% mortality. Observed significant changes in activities of antioxidant enzymes, biomineralization-related enzymes, and energy-metabolizing enzymes. Note this isn't actually a hardening experiment, it's a repeat MHW exposure experiment. It's energetics-focused though, so should probably read in more depth at some point.

[@giomi_importance_2016]:

Wild-caught tropical oyster I. nucleus, acclimated at 26C for 1month. Emmersed at 26C (control) and 40C for 4 hrs, then immersed again in 26C and 35C (reciprocal exposure setup, 4 treatment combos total). Temperature-induced mortality rate and 50% lethal temp then measured, plus respirometry and hsp70 mRNA expression. Thermal tolerance, lethal temperature threshold, and oxygen consumption rates significantly improved in heat-stressed group (40C-26c and 40C-35C), and hsp70 expression increased. Interestingly though, **feeding rates following stress were much lower in the heat-stressed groups**.

Similar resilience evaluation approach to what we're thinking in own project (quantifying temperature induced mortality and 50% lethal temp as a biomarker)

[@pereira_can_2020]:

[@nancollas_influence_2022]:

I really like the [@moyen_single_2020] approach of basing hardening conditions off of known thresholds for physiological response (e.g. hsp70 upregulation, cardiac function, mortality). What would these thresholds be for Pacific oysters?

### Defining an MHW

MHW definition is pretty distinct, would need to decide what type of scenario to simulate

Marine heatwaves (MHWs) are defined as "a discrete period of prolonged anomalously warm water at a particular location, and quantitative definitions are based on ocean temperatures exceeding a fixed (Frölicher et al. 2018), seasonally varying (Hobday et al. 2016), or cumulative (Eakin et al. 2010) threshold." [@oliver_marine_2021]

### Ploidy

When selecting which assays to use, it's useful to know which physiological parameters show ploidy-specific stress response.

### Physiological assays

![Assays suggested in Ariana's Sea Grant biomarkers proposal](images/oyster_assays.png)

[Glycogen stores:]{.underline} @hwang_involvement_2023 found that Pacific oysters store glycogen in many tissues, but glycogen content over a year only varies significantly in the **mantel edge tissue**. This suggests to me that mantel edge is the tissue whose glycogen content would be most dramatically impacted by changes to energy requirements/allocation, and is the best tissue to sample for a glycogen assay.

### Thermal tolerance

Understanding the known thermotolerance of Pacific oysters will inform what temperatures/durations we choose for the hardening regime and extreme stress test.

1.  Need to figure out sample sizes for whatever tests I want to do. Would be benefited by running some test assays before starting experiment. A bunch of these assays have been used with Pacific oysters already, so could find those and make a table or something. 
2.  Apparently heartbeat is potentially a better metric for metabolic rate than respiration, and is nondestructive
3.  Should also keep in mind that genetic composition can completely change the true sample size (e.g., if triploids and diploids are siblings)

-   can measure clearance rate (feeding) as an easy non-destructive way. Would be pretty straightforward to measure (can talk to larkin abut it, he did it)

-   ploidy -- should look into differences in ploidy, can then maybe try to manipulate it (e.g. hsps are different, try to harden in a way that would affect hsp expression)
