The impact of temperature on hatching rates of diapause eggs and subsequent development in a tropical freshwater copepod, Mongolodiaptomus malaindosinensis (Copepoda: Calanoida)

Abstract

The diapause egg is an essential component of aquatic invertebrates and is a specialized type of egg that is highly resistant to harsh environmental conditions. The diapause eggs of a tropical freshwater copepod, Mongolodiaptomus malaindosinensis (Lai et Fernando, 1978), an endemic species of Southeast Asia, were collected from Kaeng Nam Ton Swamp in Khon Kaen province, Thailand. The eggs were isolated by the sugar flotation method and maintained in laboratory conditions at three different temperature levels (25, 30, and 35 °C in an incubator). The first copepod hatching occurred between 6 and 9 days. The average hatching rate per day and the total percentage of eggs hatched in M. malain-dosinensis were significantly higher (P < 0.05) at 25 °C (9.36 ± 6.7% and 55.37 ± 3.1%) and significantly lower (P < 0.05) at 35 °C (5.28 ± 3.5% and 16.67 ± 2.9%), respectively. The post-diapause developmental characteristics of M. malaindosinensis have four stages: early development embryo, intermediate development embryo, pre-nauplius, and nauplius. The results suggest that temperature levels affected hatching success in tropical copepods. There is a possibility that the reduced hatching implies that increasing temperatures over a certain threshold may be a limiting factor for its hatching and growth.

Full text

Invertebrate Zoology, 2024, 21(3): 359–368 © INVERTEBRATE ZOOLOGY, 2024
The impact of temperature on hatching rates
of diapause eggs and subsequent development
in a tropical freshwater copepod, Mongolodiaptomus
malaindosinensis (Copepoda: Calanoida)
P. Boonmak1, L. Sanoamuang2,3*
1 Department of Science and Technology, Faculty of Liberal Arts and Science, Roi Et Rajabhat
University, Roi Et 45120, Thailand.
2 Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen Uni-
versity, Khon Kaen 40002, Thailand.
3 International College, Khon Kaen University, Khon Kaen 40002, Thailand.
* Corresponding author
Phuttaphannee Boonmak: phuttaphannee@gmail.com ORCID 0000-0003-4827-5532
Laorsri Sanoamuang: la_orsri@kku.ac.th ORCID 0000-0003-0377-1225
ABSTRACT: The diapause egg is an essential component of aquatic invertebrates and is
a specialized type of egg that is highly resistant to harsh environmental conditions. The
diapause eggs of a tropical freshwater copepod, Mongolodiaptomus malaindosinensis (Lai
et Fernando, 1978), an endemic species of Southeast Asia, were collected from Kaeng Nam
Ton Swamp in Khon Kaen province, Thailand. The eggs were isolated by the sugar otation
method and maintained in laboratory conditions at three dierent temperature levels (25,
30, and 35 °C in an incubator). The rst copepod hatching occurred between 6 and 9 days.
The average hatching rate per day and the total percentage of eggs hatched in M. malain-
dosinensis were signicantly higher (P < 0.05) at 25 °C (9.36 ± 6.7% and 55.37 ± 3.1%)
and signicantly lower (P < 0.05) at 35 °C (5.28 ± 3.5% and 16.67 ± 2.9%), respectively.
The post-diapause developmental characteristics of M. malaindosinensis have four stages:
early development embryo, intermediate development embryo, pre-nauplius, and nauplius.
The results suggest that temperature levels aected hatching success in tropical copepods.
There is a possibility that the reduced hatching implies that increasing temperatures over
a certain threshold may be a limiting factor for its hatching and growth.
How to cite this article: Boonmak P., Sanoamuang L. 2024. The impact of temperature
on hatching rates of diapause eggs and subsequent development in a tropical freshwater
copepod, Mongolodiaptomus malaindosinensis (Copepoda: Calanoida) // Invert. Zool.
Vol.21. No.3. P.359–368. doi: 10.15298/invertzool.21.3.08
KEY WORDS: crustacean, dormant stage, sheries, nauplius, Southeast Asia, zooplankton.
Влияние температуры на вылупление из покоящихся
яиц и последующее развитие у тропического
пресноводного рачка Mongolodiaptomus
malaindosinensis (Copepoda: Calanoida)
П. Бунмак1, L. Саноамуанг2,3*
1 Department of Science and Technology, Faculty of Liberal Arts and Science, Roi Et Rajabhat
University, Roi Et 45120, Thailand.
2 Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen Uni-
versity, Khon Kaen 40002, Thailand.

P. Boonmak, L. Sanoamuang360
Introduction
Copepods play a key role in aquatic ecosys-
tems that provide considerable utility throughout
various domains, including sheries, where
cultivated copepods can serve as a vital source
of nutrition for sh and shrimp larvae. They are
used as natural foods or cultured from natural
water bodies (Williamson, Reid, 2001; Boxshall,
Defaye, 2008). Copepods provide considerable
utility throughout various domains, including
sheries, wherein cultivated copepods can serve
as a vital source of nutrition for sh and shrimp
larvae. They are used as natural foods and are
either cultured or collected from natural water
bodies. They have highly unsaturated fatty acids
and proteins, which are of great signicance
for their roles as live feeds, which are essential
for the growth and survival of reared larvae in
hatcheries (Piasecki et al., 2004; Juntarut, 2014).
Dormant stages provide a mechanism for in-
vertebrates to endure inhospitable environments
and to undertake natural long-distance dispersal
(Cáceres, 1997; Arnott et al., 1999; Folt, Burns,
1999). Various invertebrates produce dormant
stages such as diapause eggs, resting eggs, or
cysts that often sink and accumulate in sediments,
forming ‘egg banks’ (Brendonck, De Meester,
2003). Diapause eggs exhibit reduced metabolic
activity and disrupted patterns of growth and
development (Hutchinson et al., 1999; Zeller et
al., 2004). They are highly resistant to harmful
environmental conditions, such as food scarcity,
drying or freezing of the habitat, and oxygen de-
ciency, and may persist in habitats that would not
permit the survival of regular stages (Alekseev et
al., 2023). Diapause eggs are a specialized type
of egg produced by some organisms, including
calanoid copepods, in response to environmental
cues such as changes in temperature, photoperiod
(day length), and food availability. It is a period
of suspended development or reduced metabolic
activity that helps copepods survive unfavorable
conditions, such as harsh environmental factors
or low food availability (Baumgartner, Tarrant,
2017; Record et al., 2018; Hansen, 2019). Dia-
3 International College, Khon Kaen University, Khon Kaen 40002, Thailand.
* Автор для корреспонденции: la_orsri@kku.ac.th
РЕЗЮМЕ: Диапауза на стадии яйца крайне важна для водных беспозвоночных, так как
покоящиеся яйца способны выдерживать неблагопрятные условия среды. Покоящиеся
яйца тропического пресноводного веслоногого рака Mongolodiaptomus malaindosinensis
(Lai et Fernando, 1978), эндемика Юго-Восточной Азии, были собраны в болоте Кенг
Нам Тон провинции Кхон Кен Таиланда. Яйца были выделены из донных отложений
методом флотации в растворе сахара, они инкубировались в лабораторных условиях
при трех стабильных температурных режимах (25, 30 и 35 °C). Первые личинки вы-
луплялись из яиц на 6–9-й день инкубации. Средняя суточная доля развившихся яиц
и общее количество развившихся яиц M. malaindosinensis были достоверно выше (P <
0,05) при температуре 25 °C (9,36 ± 6,7% и 55,37 ± 3,1% соответственно) и достоверно
ниже (P < 0,05) при температуре 35 °C (5,28 ± 3,5% и 16,67 ± 2,9% соответственно). В
развитии M. malaindosinensis после диапаузы имеются четыре стадии: ранняя эмбри-
ональная стадия, промежуточная эмбриональная стадия, пре-науплиус и науплиус.
Полученные результаты показывают, что уровень температуры может существенно
влиять на успех вылупления из покоящихся яиц у тропических копепод. Снижение
доли развившихся яиц при повышенных температурах позволяет предположить, что
повышение температуры выше определенного уровня может оказаться лимитирующим
фактором при развитии копепод из покоящихся яиц.
Как цитировать эту статью: Boonmak P., Sanoamuang L. 2024. The impact of temperature
on hatching rates of diapause eggs and subsequent development in a tropical freshwater
copepod, Mongolodiaptomus malaindosinensis (Copepoda: Calanoida) // Invert. Zool.
Vol.21. No.3. P.359–368. doi: 10.15298/invertzool.21.3.08
КЛЮЧЕВЫЕ СЛОВА: ракообразные, покоящиеся стадии, рыболовство, науплиус,
Юго-Восточная Азия, зоопланктон.

Impact of temperatures on diapause egg hatching rates 361
pause eggs are dierent from regular eggs in
that they have adaptations that enable them to
survive adverse conditions for extended periods
of time. These adaptations may include thicker
and more resilient eggshells or protective coat-
ings. The copepod embryos inside these eggs
halt their development and metabolic activities
until the conditions become more favorable for
growth and survival (Hairston, Van Brunt, 1994;
Hairston et al., 1995).
Diapause eggs are an essential adaptive strat-
egy that allows calanoid copepods to cope with
unpredictable environmental changes and ensure
their long-term survival. This phenomenon has
been extensively studied in various species of
copepods and contributes to our understanding
of the ecological dynamics of aquatic ecosystems
(Grice, Marcus, 1981; Dahms, 1995; Sichlau et
al., 2011). Embryonic diapause is divided into
two types, which are induced by physiological
conditions and by a season of the year. Some
freshwater copepods, such as Onychodiaptomus
sanguineus (Forbes, 1876), avoid periods of
intense sh predation by producing diapausing
eggs 1.3 generations before the major onset of
sh-induced mortality (Hairston, Munns, 1984).
Many populations of marine Calanoida in tem-
perate waters vary in abundance on a seasonal
basis, and in coastal areas, the production of
resting eggs is common. Embryonic diapause
within the egg envelope is common among the
Centropagoidea, whether freshwater or marine.
In contrast to freshwater species, many marine
Centropagoidea produce diapause eggs that are
morphologically distinguishable from the regular
ones, such as Paracartia latisetosa (Krichagin,
1873), a seasonally dominant copepod species
in Mediterranean coastal waters. This species
is well known for the production of diapause
eggs, which allow it to overwinter (Belmonte,
Pati, 2007).
Temperature is an important inuencing
parameter in egg production (Ban et al., 2000;
Bonnet et al., 2009; Begum et al., 2012; Liu et
al., 2015), embryonic development time (Ban,
1994; Liu et al., 2014), and hatching success
(Benni et al., 2010; Tordesillas et al., 2016).
The egg production of copepods depends on
clutch size and the rate of clutch production and
is also related to the cube of the body length.
Both of these variables are potentially complex
functions of environmental and physiological
conditions (Hopkins, 1977; Watras, 1983).
Copepod diapause eggs evolved to synchronize
hatching with optimal environmental conditions
for larval survival and growth. As temperatures
rise, diapause eggs receive cues that indicate
suitable conditions for hatching. This tempera-
ture-dependent response ensures that copepod
larvae emerge when food availability is higher
and environmental stressors are minimized (Ban,
1994; Beyrend-Dur, 2010).
A tropical freshwater calanoid copepod,
Mongolodiaptomus malaindosinensis (Lai
et Fernando, 1978), is an endemic species of
Southeast Asia and is one of the most common
copepods in northeast Thailand. It can be found in
many lakes, reservoirs, and ponds (Sanoamuang,
Dabseepai, 2021; Boonmak, Sanoamuang, 2022;
Watiroyram, Sanoamuang, 2017). This species
has to date been found also in Malaysia (Lai,
Fernando, 1978a), Singapore (Lai, Fernando,
1978b), Cambodia (Chaicharoen, Sanoamuang,
2022), and Vietnam (Boonmak, Sanoamuang,
2022). According to our previous study, the life
history traits of M. malaindosinensis simply
depended on food quantity (Boonmak et al.,
2018). The main purpose of this study was to
determine the responses of the hatching success
and development characteristics of diapause eggs
of M. malaindosinensis tested under dierent
temperature conditions in order to clarify how
this tropical copepod responds to temperature
changes in the laboratory.
Materials and methods
SEDIMENT COLLECTIONS. Nine samples of
sediments were collected by a hand net with a 30
μm mesh size from the littoral zone (depth: 2 m) of
three stations located in the north of Kaeng Nam Ton
Swamp (16°24′29.97″N, 102°45′39.08″E, altitude
151–153 m), Khon Kaen province, Thailand, on
November 28, 2021. The environmental conditions
of sampling sites were measured using a PCD650
EUTECH multi-parameter, as follows: Water tem-
peratures ranged from 28.8 to 31.4 ºC, pH ranged
from 7.62 to 7.85, conductivity ranged from 342 to
420 μS cm1, and dissolved oxygen ranged from 4.1
to 4.8 mg L1. In the original habitats of M. malain-
dosinensis, water temperatures throughout the year
ranged from 25 to 30 ºC (Boonmak et al., 2018).
Then, the sediments were spooned into a 180-mL
plastic bottle containing a small amount of natural
water and placed in an incubator (MLR-350, Sanyo,
Japan) at 25 ºC, and they were stored in the labora-

P. Boonmak, L. Sanoamuang362
Hatching rate
(%)
Temperature (°C)
25 30 35
Mean SD nMean SD NMean SD n
HR per day 9.36a6.70 3 7.22a3.47 3 5.28b3.51 3
Total HR 55.37 a 3.06 3 31.48 b 1.16 3 16.67 c 2.91 3
Table 1. The mean values and standard deviations (SD) of the hatching rate (HR) per day and the total
percentage of eggs hatched in Mongolodiaptomus malaindosinensis (n, number of replicates in each
treatment).
Таблица 1. Средние значения и стандартное отклонение (SD) ежедневной доли развившихся яиц
(HR) и общий процент развившихся яиц у Mongolodiaptomus malaindosinensis (n, количество
повторностей в каждой серии).
Values with the same superscript characters in each test indicate no signicant dierence among temperatures (post-
hoc Duncan test, df = 2, P < 0.05 for both).
relation to the initial number of eggs introduced in
each replicate well. The post-diapause development
characteristic of M. malaindosinensis was checked at
40x magnication under a light microscope (Optika
B-1000FL-HBO, Italy) with a camera lens exfocus
at 0.66x magnication (Optika, Italy).
STATISTICAL ANALYSIS. A one-way ANOVA
was used to determine the dierences in the eect
of various temperatures on egg hatching success.
Standardized data were explored to detect outliers,
then log10 [x + 1] was transformed before performing
statistical analyses to decrease the variance of the data
set and to avoid violating assumptions of normality.
All statistical analyses were performed with SPSS
19.0 software (IBM Inc., 2011). The signicance
level was set at P < 0.05.
Results
HATCHING SUCCESS OF DIAPAUSE
EGGS. The rst embryos of M. malaindosi-
nensis emerged from the diapause eggs on day
6 at 30 °C, followed by days 7 and 9 at 25 and
35 °C, respectively. The total percentage of eggs
hatched varied signicantly (df = 2, F = 101.653,
P < 0.05) at dierent temperatures and was high-
est at 25 °C (averages: 55.37 ± 3.1%) and lowest
at 35 °C (averages: 16.67 ± 2.9%) (Table 1).
Egg hatching rates per day varied signi-
cantly (df = 2, F = 4.987, P < 0.05) at dierent
temperatures, and the lowest hatching rates aver-
aged 5.28 ± 3.5% at 35 °C. At other temperatures,
the mean hatching rates varied between 9.36 ±
6.7% and 7.22 ± 3.5% at 25 and 30 °C, respec-
tively (Table 1). The egg incubation period at
25 °C was between 7 and 14 days, and at other
temperatures, the range of egg incubation periods
varied between 6 and 11 and 9 and 12 days at
tory for 120 days before being used in the hatching
experiments.
EXPERIMENTAL CONDITIONS. Diapause
eggs of calanoid copepods were separated from the
sediments by the sugar otation method (Ban, Minoda,
1992). The unsterilized sediments were sieved through
a 100-μm mesh and then a 30-μm mesh screen. After
that, the sediments on the 30-μm mesh screen were
placed in a centrifuge tube containing a dense sugar
solution (1 kg L–1). The solution was stirred and then
centrifuged at 3,000 rpm for 5 minutes. The superna-
tant was immediately placed in a 10-μm mesh sieve
and rinsed with distilled water to remove the sugar.
The material remaining on the 30-μm mesh sieve was
washed with ltered (Whatman GF/C) tap water into
a Petri dish. Diapause eggs of calanoid copepods were
counted under a compound microscope and transferred
with a ne glass capillary tube from the dish to another
one containing ltered tap water.
The average water temperature in freshwater
resources in northeast Thailand throughout the year
ranged from 25 to 30 ºC (Department of Irrigation,
2022). In Kaeng Nam Ton Swamp, the animals ex-
perience water temperatures ranging between 26 and
31 °C (Phugonkchana et al., 2017). The experimental
temperatures were therefore selected at three dier-
ent temperatures (25, 30, and 35 °C), providing a
range between the lowest and highest temperatures
in Kaeng Nam Ton Swamp. At each temperature, 150
eggs were used with three replicates. Thus, a total of
1350 eggs were placed individually in 5-mL 48-well
polystyrene tissue-culture plates (TR5000; Trueline,
Romeoville, Illinois, U.S.A.). Eggs were lled with
ltered tap water and maintained at the three tem-
peratures in an incubator (MIR-254, Panasonic). For
each temperature, the hatching rate of the eggs was
checked twice per day under a dissecting microscope
(Olympus, SZX12, Japan) at ca. 10–40× magnica-
tion. The observed time in 30 days was considered
the endpoint. The egg hatching rate (%) for each
replicate well was then calculated as a percentage in

Impact of temperatures on diapause egg hatching rates 363
30 and 35 °C, respectively (Fig. 1). One-way
ANOVA demonstrated that there were signi-
cant dierences among dierent temperatures
on days 8, 9, 10, and 11, but on days 7 and 12,
there were no signicant dierences in hatching
rates at each temperature. Statistically, a linear
relationship between the temperature and the
hatching rate was evident during the study; the
values are expressed in the following equation:
Hatching rate = –3.87x + 150.61 (R2 = 0.982,
P < 0.05).
POST-DIAPAUSE DEVELOPMENT.
Developmental patterning of the embryo was
observed under a light microscope at 40x mag-
nication with a camera lens exfocus at 0.66x
magnication (Fig. 2). The majority of embryos
of M. malaindosinensis were isolated from sedi-
ments in a pond (Fig. 1A). These embryos were
presumed to be in the diapauses stage because
they did not develop, hatch, or deteriorate when
isolated from the sediment and incubated at 25 °C
under aerobic conditions with constant light or
a 12:12 light: dark cycle to mimic conditions of
the natural weather in Thailand. Live embryos
in the diapause stage could not be dierentiated
from embryos in an early stage of post-diapause
development with light microscopy, so these were
grouped together and designated as the Early
Development (ED) embryonic stage (Fig. 1A).
Development in progress rst became apparent
when bilaterally symmetrical spaces appeared
between the cyst wall and the inner embryo mass
at one end of the embryo in a stage designated as
the Intermediate Development (ID) embryonic
stage (Fig. 1B–C). There was some variability in
the timing of individual developments. Reaching
the ID embryonic stage within 24–48 hours, the
individual progressed to the pre-nauplius stage,
characterized by an oval shape with well-dened
bilateral symmetry and body axis formation (Fig.
1D). The jerking of the body was visible late in
the pre-nauplius stage. Hatching of the nauplius
larva started with the rupture of the outer cyst
wall (Fig. 1E) and the complete emergence of the
nauplius inside a exible layer structure that is
shed in two stages as the space surrounding the
Fig. 1. The hatching percentages of diapause eggs of Mongolodiaptomus malaindosinensis in cultures for 14
days at three dierent temperatures.
Рис. 1. Процент развившихся покоящихся яиц Mongolodiaptomus malaindosinensis в культуре за 14
дней при трех разных температурах.

P. Boonmak, L. Sanoamuang364
Fig. 2. Morphological characteristics of Mongolodiaptomus malaindosinensis during post-diapause devel-
opment, emergence, and hatching. A — early stages of development; B–C — intermediate stages of devel-
opment; D — pre-nauplius with an ocellus (black arrowhead); E — emergent pre-nauplius and outer wall
(black arrow) of cyst shed; F — inner membrane (white arrow); G–H — inner membrane fully expanded;
nauplius begins to burst swimming; I — free-swimming nauplius larva. Abbreviations: A1 — antennule;
A2 — antenna; Md — mandible. Scale bars ― 100 µm.
Рис. 2. Морфология Mongolodiaptomus malaindosinensis во время развития из покоящегося яйца и вылу-
пления: A ― ранняя стадия эмбрионального развития; B–C ― промежуточные стадии эмбрионального
развития; D ― пре-науплиус с науплиалоьным глазом (показан стрелкой); E ― вылупившийся пре-
науплиус внешняя стенка сброшенной цисты (показана стрелкой); F ― внутренняя мембрана (показана
стрелкой); G–H ― внутренняя мембрана полностью расправлена, науплиус начинает спорадические
плавательные движения; I ― свободно плавающий науплиус. Обозначения: A1 — антеннула; A2 ―
антенна; Md — мандибула. Масштаб 100 µм.
negatively aected by the highest temperature.
The hatching success of eggs in tropical copepods
can indeed be aected by high temperatures.
Elevated temperatures can have a signicant
impact on the reproductive and developmental
processes of many aquatic organisms. The sud-
den decline in hatching success at the highest
temperature tested can be attributed to severe
thermal stress, as has been observed for other
calanoid copepods (Lee et al., 2003; Rhyne et
al., 2009; Yoshida et al., 2012). Eects of high
temperatures can often lead to a decrease in the
nauplius expands and a thin hatching membrane
is stretched (Fig. 1F–H). The nauplius rushed
until the thin hatching membrane ruptured,
releasing the free-swimming nauplius (Fig. 1I).
Discussion
Both the egg hatching rates per day and the
total percentage of eggs hatched in Mongolodi-
aptomus malaindosinensis were considerably
higher at 25 °C, in contrast to the increasing
temperature at 35 °C, where hatching rates were

Impact of temperatures on diapause egg hatching rates 365
percentage of copepod eggs that successfully
hatch because copepod embryos are sensitive
to temperature changes, and temperatures above
their optimal range can disrupt their develop-
ment (Jiménez-Melero et al., 2005, 2012; Liu
et al., 2014, 2015). The signicantly high rates
of hatching success of diapause eggs in M.
malaindosinensis at 25 °C suggested that this
is the optimal temperature for hatching, and an
increase in temperature beyond this range may
limit the population growth and reproduction of
M. malaindosinensis (Boonmak et al., 2018).
While copepods are ectothermic organisms,
excessively high temperatures can accelerate
their metabolic rates and shorten the time they
spend in the egg stage. It can also lead to pre-
mature hatching, which reduces the chances of
successful survival and recruitment of copepod
populations. Moreover, elevated temperatures
can induce stress in copepod embryos, aecting
their metabolic processes and overall develop-
ment. This stress can lead to a higher rate of egg
mortality and lower hatching success (Patterson
et al., 2021; Reed et al., 2021). Many studies
have shown that varying temperature regimes
can inuence the timing and synchronization of
diapause egg hatching, impacting the success of
copepod generations. Warmer temperatures can
accelerate hatching rates, potentially leading to
earlier emergence and altered population dynam-
ics (Monchenko, 1996). However, the impact of
high temperatures on copepod hatching success
is not uniform across all species and populations.
Dierent species and populations have vary-
ing temperature tolerances and thresholds. For
example, the diapause egg-hatching success of
the copepod Acartia tonsa Dana, 1849, increased
with increasing temperature up to a certain point
and then declined, and the optimal temperature
range for hatching was between 18 and 22 °C
(Kiorboe, Hirst, 2008). For temperate species,
the diapause egg hatching of Calanus glacialis
Jaschnov, 1955 and C. ponticus Krichagin, 1873
in the Arctic Ocean found that hatching success
was higher at lower temperatures (between 0 and
4 °C) and decreased at higher temperatures; this
may be due to the copepods being adapted to the
cold Arctic environment (Häfker et al., 2018;
Kosobokova et al., 2011).
Diapause eggs of M. malaindosinensis used a
longer hatching time (day 7 at 25 °C) than regu-
lar eggs, which are one to three days (authors’
unpublished data indicate that in a preliminary
study, the regular eggs of M. malaindosinensis
hatching emerged between 24 and 60 hours and
the mean hatching success was 85% at 25 °C).
Generally, depending on the temperature of the
water, copepod embryogenesis can take between
8 and 48 hours, after which a nauplius larva will
emerge from the egg (Roman, Pierson, 2022). The
lower hatching percentage of copepod diapause
eggs compared to regular eggs can be attributed
to several factors related to diapause, which is a
period of dormancy or suspended development
commonly observed in many organisms. Dia-
pause eggs are designed to remain dormant for
an extended period, often until environmental
conditions become more favorable for the sur-
vival and growth of the ospring. That can lead
to a delay in hatching, reducing the percentage
of diapause eggs that successfully hatch com-
pared to regular eggs (Dussart, Defaye, 2001).
Including, diapause eggs may have evolved to
resist hatching cues until they have experienced
a sucient duration of the cues simultaneously
(Belmonte, Pati, 2007). Moreover, metabolic pro-
cesses in copepod eggs are typically suppressed
to conserve energy (Liu, Ban, 2017). Diapause
eggs often require specic environmental cues to
trigger hatching, such as changes in temperature,
salinity, or light conditions (Epp, Lewis, 1980;
Hirche et al., 1997). However, it may be found
that the hatching success of diapause eggs of
some marine copepods, Centropages hamatus
(Lilljeborg, 1853), was higher than 80% for
eggs stored for 4–17 months at 25 °C (Marcus,
Murray, 2001).
Previous studies on M. malaindosinensis de-
scribe the eects of food variables on life history
traits, embryo development time, and survival in
live copepods and their regular eggs (Boonmak
et al., 2018), but none provide a description of
diapause developmental events that can be used
to characterize the impacts of environmental vari-
ables on development. In the present work, the
diapause development of M. malaindosinensis
was described based on the Antarctic freshwa-
ter copepod Boeckella poppei (Mrazek, 1901),
which fully describes encysted post-diapause
development in a member of the genus Boeckella
in which emergence and hatching depend on the
osmotic expansion of a space between a hatching
membrane and the fully formed nauplius larva
(Reed et al., 2018, 2021). In M. malaindosinensis,

P. Boonmak, L. Sanoamuang366
the number of membrane layers was not clear, it
has been observed thatthe outer layers matching
the dimensions of the diapause embryo are shed
sequentially during expansion of this space, and
the hatching membrane eventually ruptures as a
consequence of burst swimming by the nauplius.
This event described previously for B. poppei
(Burns, 1980; Jamieson, 1988; Hall, Burns, 2001;
Reed et al., 2018) and the brine shrimp, Artemia
franciscana Kellogg, 1906 (Covi et al., 2016) is
similar to that of M. malaindosinensis, but the
proportional expansion of the space between
the hatching membrane and nauplius larva is
larger in M. malaindosinensis. Previous stud-
ies in Artemia sp. reported that emergence and
hatching may depend on the action of a chitinase
working in concert with osmotic swelling, but the
role of chitinases in the emergence and hatching
of copepods remains unexplored (Rosowski et
al., 1997). The physiological mechanisms that
regulate the transition from diapause to post-
diapause in many copepod species are still
unknown. At present, transcriptional analysis
is used to nd hypotheses related to potential
mechanisms that terminate diapause before an
organism can resume its development (Lenz et
al., 2021; Roncalli et al., 2021).
Conclusions
The results of this study indicate that the
temperature of 25 °C is suitable for hatching in
Mongolodiaptomus malaindosinensis, as shown
by the relatively high rates of hatching success ob-
served. Additionally, it is plausible that elevated
temperatures beyond a certain threshold may
signicantly impact the population dynamics
and reproductive capabilities of tropical cope-
pods, as shown by the reduced hatching success
observed. This suggests that high temperatures
could potentially act as a limiting factor for popu-
lation growth and reproduction in this species.
The eect of temperature on the diapause egg
hatching of copepods appears to vary depend-
ing on the species and the temperature range
examined. Optimal hatching temperatures have
been found to range from 2 to 25 °C, with some
species showing higher hatching success at lower
temperatures. The timing of diapause onset may
also play a role in determining hatching success at
dierent temperatures. Overall, further research
is needed to better understand the mechanisms
underlying the eect of temperature on diapause
egg hatching in copepods.
Funding
This work was funded by a grant from the Thai-
land Science Research and Innovation (TSRI) in the
Fundamental Fund 2022 category.
Author contribution statement
PB was responsible for the literature review, sedi-
ment collection, methodology, photographs, prepara-
tion of gures, and the rst draft of the manuscript.
LS was responsible for conceptualization, writing and
editing of the nal manuscript, and general revisions.
Compliance with ethical standards
CONFLICTS OF INTEREST: The authors declare
that they have no conicts of interest.
Acknowledgements. The authors thank Dr. Xin
Liu for his technical training and consultation in
the sugar otation method. The authors gratefully
acknowledge Russell J. Shiel for his critical review
and linguistic corrections to the manuscript. The
authors thank Weerathan Maskasem and Prapatsorn
Dabseepai for eld assistance.
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Responsible editor A.Yu. Sinev