Available via license: CC BY-NC-SA 4.0
Content may be subject to copyright.
B I O D I V E R S I T A S
ISSN: 1412-033X
Volume 25, Number 7, July 2024 E-ISSN: 2085-4722
Pages: 3106-3113 DOI: 10.13057/biodiv/d250732
Life history traits of the gastropod-associated bacterivorous nematode
Caenorhabditis brenneri (Nematoda: Rhabditida)
LOEL DALAN1,2,, VERONICA TAÑAN1,2, MICHELLE ANNE DIANO1,2, CESAR DEMAYO1,
MYLAH TABELIN1, NANETTE HOPE SUMAYA1,2,
1Department of Biological Sciences, College of Science and Mathematics, Mindanao State University-Iligan Institute of Technology. Andres Bonifacio,
Tibanga, 9200 Iligan City, Philippines. Tel.: +63-063-221-4056, email: loel.dalan@g.msuiit.edu.ph, email: nanettehope.sumaya@g.msuiit.edu.ph
2Center for Biodiversity Studies and Conservation Genomics, Premier Research Institute of Science and Mathematics, Mindanao State University-Iligan
Institute of Technology. Andres Bonifacio Avenue, Tibanga, 9200 Iligan City, Philippines.
Manuscript received: 19 May 2024. Revision accepted: 15 July 2024.
Abstract. Dalan L, Tañan V, Diano MA, Demayo C, Tabelin M, Sumaya NH. 2024. Life history traits of the gastropod-associated
bacterivorous nematode Caenorhabditis brenneri (Nematoda: Rhabditida). Biodiversitas 25: 3106-3113. Caenorhabditis species are
associated with decaying plant materials and invertebrates such as terrestrial gastropods. Few Caenorhabditis species have been studied
regarding their ecology, genetics, development, and essential life history traits (LHT). In this study, we describe the life cycle of a free-
living, wild-type, bacterivorous nematode, Caenorhabditis brenneri Sudhaus and Kiontke 2007, associated with the terrestrial slug
Philippinella moellendorffi Collinge 1899 and account for the effects of food density and temperature on its population dynamics by
employing the hanging drop technique. The bacterial food supply was sourced from the slug cadaver and identified as Alcaligenes
faecalis (MT012081). C. brenneri was fed with varying bacterial densities (109 and 5×109 cells mL-1) and incubated at 20 and 25°C. The
bacterial food density-temperature interaction was found to have no significant influence on the offspring production of C. brenneri.
Moreover, the total fertility rate (TFR) and net reproductive rate (Ro) are higher in 109 cells mL-1 in both temperatures (TFR at 20°C:
139; 25°C: 169 and Ro at 20°C: 134; 25°C: 156). Whereas the alternative generation time (To, T1, T) and population doubling time
(PDT) are faster at 25°C in both bacterial densities, the intrinsic rate of natural increase (rm) is faster at lower temperatures (20°C) in
both bacterial densities. The average lifespan of C. brenneri is seven days when fed with A. faecalis. Offspring somatic growth (body
length and width) was influenced by bacterial food density-temperature interaction with increased length and girth observed in higher
bacterial density and temperature in our knowledge. This study is the first attempt to use A. faecalis, a wild-type bacterium from the
terrestrial slug P. moellendorffi cadaver, as the bacterial food source for a Caenorhabditis LHT analysis.
Keywords: Fecundity, hanging drop, juveniles, LHT, semi-solid NGG, survivability
INTRODUCTION
Soil fauna, of which bacterivorous nematodes are part,
play critical functional and ecological roles in soil ecosystems
(van den Hoogen et al. 2019; Zhao et al. 2022). It was
documented that they impact the community composition,
including the abundance of soil microorganisms and their
direct or indirect involvement in nutrient decomposition
and recycling processes. With bacterial feeding and
facilitating organic matter mineralization, such nematodes
improve microenvironment pH (Xiao et al. 2014). Despite
this crucial role in the ecosystem, the life history of soil
fauna such as nematodes has remained understudied
(Bardgett and van der Putten 2014).
Sudhaus and Kiontke (2007) reported that Caenorhabditis
brenneri Sudhaus and Kiontke 2007 is a gonochoristic
member species of the Elegans group; it is found mainly in
different habitats, such as in decaying plant matter and soil,
and often forms an association with invertebrates such as
terrestrial gastropods with pantropical distribution (Sudhaus
and Kiontke 2007; Dalan et al. 2022; Diano et al. 2022).
Interestingly, C. brenneri has the most molecular polymorphic
synonymous sites between individuals (14.1%), making
them the most hyper-diverse eukaryote comparable to
hyper-diverse bacteria (Dey et al. 2013). Like other free-
living nematodes, Caenorhabditis species fed on bacteria
found in their surroundings formed phoretic associations
with invertebrates and reproduced in cadavers of their host
organisms (Petersen et al. 2014; Slos et al. 2017; Dalan et
al. 2022).
Bacteria as a food source are essential in their various
life strategies, influencing reproduction, growth, virulence,
and sometimes forming symbiotic relationships with
nematodes. Assessment of essential life history traits (LHT)
of nematodes using associated bacteria is essential for
understanding nematode growth and development in
controlled environments. This will provide insight into how
population growth dynamics change as various factors
influence them. LHT analysis is an important step in
measuring the species' reproductive ability. It can be used
as basic information to maximize its productivity when
commercializing the species at the industrial level (Addis et
al. 2016a). It also shows in detail and precision how an
individual nematode species performs. Thus, such research
studies are critical to natural ecosystem management, soil
bioremediation, and agriculture (Zhao et al. 2022).
One way to study the LHT is by utilizing the hanging
drop method, which provides a clear visualization of the
DALAN et al. – Life history trait of Caenorhabditis brenneri
3107
setup, giving an advantage in observing and recording the
different life cycle parameters. A culture medium of Gelrite
powder is the standard medium used in the hanging drop
method. This medium provides high transparency, which
enables researchers to facilitate convenient screening on
culture plates with minimum disturbance to the setups.
Moreover, nematode extraction is possible without the
medium's residues adhering to nematodes (Muschiol and
Traunspurger 2007). The Gelrite medium is a transparent,
semi-solid, gel-like culture medium that allows clear
examination of the test organisms. Individuals or pairs of
nematodes were introduced into a drop of semi-solid
medium containing a bacterial food source at the underside
of the lid of a cell-well plate. This method of analysis was
already established in EPNs using their respective symbiotic
bacteria (Addis et al. 2014; Addis et al. 2016a, b, c) and on
different free-living nematodes (Muschiol and Traunspurger
2007; Muschiol et al. 2009; Gilarte et al. 2015; Kreuzinger-
Janik et al. 2017).
Though C. brenneri has the potential to be a model
organism for having such hyper-diverse molecular
polymorphic synonymous sites among eukaryotes (Dey et
al. 2013), the information on the basic LHT is rather
limited. This study aimed to unravel the basic LHT of the
wild-type C. brenneri isolated from the cadaver of terrestrial
slug Philippinella moellendorffi Collinge 1899, using its
associated bacteria as a food source using the hanging drop
method. We account for food density and temperature's
effects on population dynamics. For this, the offspring
production, survivability, fecundity, fertility rate, net
reproductive rate, somatic growth of offspring produced,
and average lifespan were assessed as influenced by
bacterial density and temperature.
MATERIALS AND METHODS
Isolation and identification of the associate bacterium
Bacterial lawn from P. moellendorffi cadaver on an agar
plate was picked and streaked aseptically onto Nutrient
Agar (NA) medium M001 HIMEDIA® (0.5% peptone,
0.15% HM peptone B, 0.15% yeast extract, 0.5% NaCl,
1.5% agar; 7.4±0.2 pH at 25°C) in 90×15 mm Petri dishes
and incubated at 37°C for 24 h. The resulting pure bacterial
colony was selected as the food source for C. brenneri. The
isolated bacterium was cultured in a 500 mL Erlenmeyer
flask containing 100 mL of sterile Nutrient Broth TM
MEDIA 350 (0.5% peptic digest animal tissue, 0.5% NaCl,
0.15% beef extract, 0.15% yeast extract; 7.4±0.2 pH at
25°C) and incubated at 37°C for 24 h with constant agitation.
This served as the bacterial stock culture.
Briefly, 1 mL culture from the bacterial stock culture
was loaded into a 1.5 mL Eppendorf tube and centrifuged
at 10,000 rpm for 2 min to pellet the cells, and the
supernatant was discarded. Genomic DNA extraction
followed the manufacturer's (Dongsheng Biotech's Quick
Genomic Bacteria DNA Extraction Kit N1152) protocol.
The wash buffer (PE) solution was diluted with 96-100%
ethanol before use. Extracted DNA was stored in a 1.5 mL
Eppendorf tube and sent to Macrogen, South Korea, for
PCR amplification, purification, and sequencing. The
primer pair 27F 5’-AGAGTTTGATCCTGGCTCAG-3'’ and
1492R 5’-GGTTACCTTGTTACGACTT-3'’ (Chen et al.
2015) was used to amplify segment of 16S rDNA gene
following a standard thermal profile of 94°C for 1 min pre-
denaturation; 30 cycles of 94°C for 30 s denaturation; 55°C
for 30 s annealing; 72°C for 1 min extension and 72°C for
10 min then cooled at 4°C final extension. Generated
sequences were BLASTn searched, and all sequences were
deposited in the National Centre for Biotechnology (NCBI)
(Stover and Cavalcanti 2017). The obtained molecular
sequences were trimmed, annotated, and subsequently
submitted to NCBI Genbank for record.
Cell density and quantification of the bacterium
Quantification was done by distributing 100 mL stock
bacterial culture to 20 mL centrifuge tubes. The tubes were
centrifuged at 4,500 rpm for 10 min. The supernatant was
discarded, and pellets were washed with sterile K-Medium
(3.1 g NaCl and 2.4 KCl in 1 L), homogenized by vortex,
and centrifuged for 5 min and decanted. The pellets were
pooled into one 50 mL Falcon tube. Ten milliliters (10 mL)
of semi-solid Nematode Growth Gelrite (NGG) medium
(2.5 g peptone, 3 g NaCl, 3 g Gelrite and adjusted to 1 L;
autoclaved and cooled to 55°C, 1 mL of 147 g L-1
suspension of CaCl2×2 H2O, 1 mL of 246. 6 gL-1 MgSO4×7
H2O and 25 mL of 136 g L-1 KH2PO4 at pH 6 and 1 mL of
1 g L-1 cholesterol suspended in alcohol) (Dancheng Caixin
Sugar Industry Co., LTD) were added to pellets and
homogenized through the vortex. Two milliliters of
homogenized semi-solid NGG with bacterial cells were
loaded into the spectrophotometer, and the absorbance
values at 600 nm were obtained and calculated. Cell
densities of 109 and 5×109 cells mL-1 were obtained by
dilution. The semi-solid NGG containing the appropriate
bacterial density was distributed to several 1.5 mL
Eppendorf tubes, stored in the refrigerator, and served as
the food stock for LHT.
Maintenance and acclimatization of the nematode
The nematode C. brenneri was previously isolated from
the same cadaver of P. moellendorffi, a terrestrial slug in
the Philippines (Dalan et al. 2022). This was also the same
cadaver where the pure culture of the associate bacterium
used as stock culture was isolated. Before starting the LHT
analysis, the nematodes were maintained by culturing them
in NA plates seeded with the bacterial stock culture. Sub-
cultures were obtained and maintained by transferring a
block of NA containing all stages of nematode into fresh
NA plates every five days. Before starting LHT analysis, C.
brenneri cultures were acclimatized for one week on 90×15
mm Petri dishes with solid NGG medium seeded with 500
µL of bacterial stock culture and incubated at room
temperature for 24 h.
Life stage synchronization of the nematode
Life stage synchronization using the hanging drop
method was based on Muschiol and Traunspurger (2007)
with slight modifications. Briefly, 9 drops of 15 µL semi-
solid NGG (with 109 cells mL-1) were distributed in the
B I O D I V E R S I T A S
25 (7): 3106-3113, July 2024
3108
inner lid of the top cover of 90×15 mm Petri dishes. One
gravid female from the exponentially growing C. brenneri
culture in solid NGG was picked and transferred into each
drop to allow egg laying and hatching. Next, to avoid
desiccation, the bottom pair of the Petri dishes were
covered with tissue paper soaked with 3 mL of distilled
water, sealed, and incubated at room temperature for 24 h.
After 24 h, the added gravid females were removed, and
the hatched juveniles were selected for the LHT experiment.
LHT experimental setup
The experiment started by picking nine pre-adults/J4
stage female C. brenneri (visible vulva) with a "nematode
picker" (one bristle of toothbrush glued to the tip of a
chopstick) from the previous hanging drops. It was
individually introduced into fresh drops of semi-solid
NGG. This was followed by picking one male C. brenneri
to complete the pair (one male and female C. brenneri per
drop) in semi-solid NGG drops of varying bacterial
densities (109 and 5×109 cells mL-1) and incubated in 20
and 25°C. The nematode pairs were transferred into new
drops every 24 hours. Offspring were checked and quantified
every 24 hours. If the male C. brenneri died, they were
removed and replaced with live males from the nematode
stock culture. The experiment ended with the death of the
last adult female C. brenneri.
Determination of LHT parameters
A life table was constructed to have an accessible
overview of the reproductive timetable of C. brenneri and
to acquire the necessary parameters for analyzing LHT.
Total fertility rate (TFR) is the total number of juveniles
an adult produces during its maximum lifespan
TFR = (∑ mx)
Where:
mx : age-specific fecundity
Net reproductive rate (Ro) is the average number of
offspring an adult produces during its lifespan
Where:
lx : age-specific survival probability
mx : age-specific fecundity
The intrinsic rate of natural increase (rm) is the rate at
which an individual in a stable population increases under
optimal conditions, calculated using Euler's equation
Where:
lx : age-specific survival probability
mx : age-specific fecundity
Population doubling time (PDT) is the time needed for
an increasing population to double its number of individuals.
PDT = ln (2)/rm (4)
Where:
Ln : natural logarithm
rm : intrinsic rate of natural increase
Alternative measures of generation time (T0/T1/Th) were
To or cohort generation time; T1, which is the time necessary
for the increasing population to grow by a factor of Ro; and
T, the mean parental age at which a new generation is
produced.
(5)
T1 = (lnRo)/rm (6)
T =
Where :
x : time in days
lx : age-specific survival probability
mx : age-specific fecundity
Ro : net reproductive rate
rm : intrinsic rate of natural increase
Somatic growth assessment of juveniles
Offspring from every experimental setup were pooled
into each designated 5 mL tube and fixed. Ten random
offspring from every setup were picked into embryo dishes
and processed into anhydrous glycerin (Ryss 2017). Solution
1 (8 parts formalin, 2 parts glycerol, and 90 parts water)
was heated and dropped into the dish with the nematodes,
covered for 24 h at room temperature. Solution 2 (5 parts
glycerol and 95 parts 96% ethanol) was added, one drop of
the solution every two hours, five times. Nematodes were
fixed into slides by a paraffin wax ring with a drop of
Solution 3 (glycerol) in the middle and sealed by heating.
Nematodes were photographed and measured using an
Olympus light microscope BX53 equipped with an Olympus
DP27 microscope digital camera and Cellsens imaging
software, and they were measured using ImageJ Software.
Data analysis
Life history parameters (TFR, Ro, rm, PDT, alternative
generation times, and average lifespan) of C. brenneri LHT
were computed based on the life tables obtained. The
What-if-Analysis function and its goal seek feature in
Microsoft Excel were used to compute the rm values. The
mean number of offspring produced was analyzed using the
Kruskal-Wallis test to determine the influence of bacterial
density and temperature in offspring production. The best-
fitted ANOVA model was used to determine the influence
of bacterial density and temperature and the somatic growth
(length and width) of C. brenneri. Statistical analysis was
done using the RStudio program (R Core Team 2023).
RESULTS AND DISCUSSION
Identification of the associated bacterium
Molecular analysis of the 16S rDNA using BLASTn
software showed that the associated bacterium from P.
moellendorffi cadaver was Alcaligenes faecalis Castellani and
Chalmers 1919, a Gram-negative bacillus aerobic bacterium,
which was annotated as A. faecalis (MT012081).
DALAN et al. – Life history trait of Caenorhabditis brenneri
3109
Life history parameters of the nematode
Moreover, C. brenneri produced more offspring per
adult during its maximum lifespan (TFR) and a high mean
number of offspring produced during its entire lifespan (Ro)
when cultured in lower bacterial density (109 A. faecalis
cells mL-1). In both bacterial densities, the faster generation
time and population doubling time of C. brenneri were
observed in higher temperatures (25°C). However, both
bacterial densities observed high rm at lower temperatures
(20°C). The average lifespan of adult female C. brenneri
was seven days (Table 1)
Influence of bacterial density and temperature on the
nematode offspring production
Bacterial density did not significantly influence offspring
production of the nematode (χ2 (1)=0.303, p=0.5817).
Likewise, the temperature did not significantly influence
the offspring production of C. brenneri (χ2 (1)=0.044,
p=0.8345). The mean number of offspring produced did not
differ significantly between treatments (Figure 1).
Fecundity and survivability of the nematode
C. brenneri is a fast-reproducing species with peaked
fecundity observed 48 h after introduction to hanging drops.
Fecundity also gradually declined as assessment continued
in hanging drops. Regarding their survivability, adult C.
brenneri had a stable population at 5×109 A. faecalis cells
mL-1 until day five of assessment compared to day four at
109 A. faecalis cells mL-1 in both temperatures (Figure 2).
Somatic growth analysis of the nematode offspring
Morphometric analysis on the somatic growth of C.
brenneri offspring showed that interaction between bacterial
food density and temperature had a significant influence on
the body length (F 1=33.983, p<0.001) and body width (F
1=41.530, p<0.001). The increased body length of hatched
juveniles was observed in higher bacterial density (5×109
A. faecalis cells mL-1) and higher temperature (25°C)
(p<0.05). The same outcome was also observed in the body
width of hatched juveniles (p<0.05), as shown in Figure 3.
Figure 1. The mean number of Caenorhabditis brenneri offspring
in varying bacterial density and temperature was assessed in
hanging drop. Black points indicate mean, and letters signify
significant differences
Figure 2. A-B. Fecundity; and C-D. survivability of Caenorhabditis brenneri as influenced by varying bacterial densities and
temperatures assessed in hanging drop
A
B
C
D
B I O D I V E R S I T A S
25 (7): 3106-3113, July 2024
3110
Discussion
The bacterium isolated and identified from the terrestrial
slug P. moellendorffi cadaver was A. faecalis, an aerobic,
bacillus, Gram-negative bacterium commonly isolated from
soil and water environments (Kong et al. 2014). This species
is unable to degrade urea to ammonia, can produce catalase
through aerobic respiration, cannot utilize citrate as a
carbon source, and is not efficient in fermenting sugars. It
is also sometimes found in human bodily fluids, the
alimentary canal, and feces. It is generally considered non-
pathogenic, though opportunistic infection does occur in
the form of urinary tract infection, infection in the
bloodstream, skin and soft tissue, and middle ear (Mordi et
al. 2015; Tena et al. 2015; Huang 2020). Most LHT studies
in hanging drops of free-living nematode utilized
Escherichia coli Escherich, 1885 strain OP50 as the common
bacterial food source and at 20°C incubation (Muschiol and
Traunspurger 2007; Muschiol et al. 2009; Gilarte et al.
2015; Kreuzinger-Janik et al. 2017; Mondejar et al. 2023).
In contrast, a recent study utilized an unidentified bacterium
that the free-living bacterivorous nematode, Diplolaimella
stagnosa Lorenzen 1966 carried as a food source to assess
the life cycle and effects of temperature and food
availability on its population dynamics (Zhao et al. 2022).
The present study, however, is the first attempt to use A.
faecalis, a wild-type bacterium isolated from the terrestrial
slug P. moellendorffi cadaver, as the bacterial food source
for C. brenneri LHT analysis.
The onset of reproductive maturity in C. brenneri is
early (24 h in hanging drop) and gradually increases until
48 h. However, such a peak in the production of juveniles
declines after 48 h and continues to drop until the last day.
This development contrasts with other Caenorhabditis
LHTs, such as Caenorhabditis elegans Maupas 1900 in
hanging drop. It is observed in several studies that C.
elegans started laying eggs and hatching within 72 h and
exponentially increased to 120 h. This discrepancy could
result from the difference in the utilization of cohort
juveniles for the study. The cohort of juveniles used in this
study has a mean age of 24 h compared with 4 h used by
Muschiol et al. (2009). In this case, we started with J4
compared to their J1/J2, thus contributing to the early
reproductive maturity and aging of C. brenneri.
Previous LHT studies implied that bacterial food
density of 5×109 cells mL-1 was determined to be ideal for
most of the Caenorhabditis species LHT as it precludes
food limitation. Lower than this, such as bacterial density
at 109 E. coli strain OP50 cells mL-1, reportedly reduced
fecundity in Caenorhabditis briggsae Schiemer 1982.
Moreover, high bacterial density, such as 1010 E. coli strain
OP50 cells mL-1, resulted in reduced life expectancy in C.
elegans (Johnson et al. 1990). In this study, offspring
production, as measured in TFR and Ro of C. brenneri, was
not influenced by the combined interaction of bacterial
density and temperature even independently. The reduced
fecundity observed at 109 bacterial cells mL-1 seems to vary
from species to species, even from the same taxon. The
fecundity of C. brenneri in the current study is higher (139
and 169 offspring) at lower bacterial density (109 bacterial
cell mL-1) in both temperatures. Although statistically,
fecundity between the bacterial densities does not differ
significantly from each other (Table 1).
A
B
Figure 3. Somatic growth (A. Body length; B. Body width) of
Caenorhabditis brenneri juveniles in varying bacterial density
and temperature assessed in hanging drop. Black points indicate
the mean, and letters indicate significant differences
Table 1. LHT parameters of Caenorhabditis brenneri in varying bacterial densities and temperatures were assessed in hanging drops
109 A. faecalis cells mL-1
5×109 A. faecalis cells mL-1
20°C
25°C
20°C
25°C
n
9
9
9
9
TFR
139±23.37
169.26±29.97
106.28±22.84
133.28±23.54
Ro
134±23.53
156±27.59
95.33±23.21
122.89±21.63
To (d)
2.6
1.84
2.6
1.88
T1 (d)
4.6
2.40
3.9
2.58
T (d)
13.87
9.24
15.61
9.55
PDT (d)
0.51
0.33
0.59
0.37
rm (d-1)
1.37
2.09
1.18
1.87
Average lifespan (d)
7.13±2.03
6.75±1.48
7.63±2.34
7±2.06
Notes: TFR: total fertility rate; Ro: net reproductive rate; To, T1, T: alternative mean generation time; rm: intrinsic rate of natural increase;
PDT: population doubling time; d: days
DALAN et al. – Life history trait of Caenorhabditis brenneri
3111
Table 2. LHT parameters of different nematode species were assessed in the hanging drop method
Species
C. brenneri
C. elegans
Poikilolaimus
sp.
Panagrolaimus
sp.
Pristionchus
pacificus
Steinernema riobrave
Steinernema feltiae
Strain/origin
Slug cadaver
N2
MY6
Movile Cave, Romania
PS312
Sr-12
Sr-HYB 19
EN02
Bacteria
A. faecalis
E. coli strain OP50
Xenorhabdus cabanillasii
Xenorhabdus
bovienii
Density
109 cells mL-1
5×109 cells mL-1
Temperature
20°C
25°C
20°C
25°C
20°C
25°C
TFR
139
169.26
106.28
133.28
295
290
187
77
115
975
822
493
Ro
134
156
95.33
122.89
291
289
108
64
109
792
683
359
To (d)
2.6
1.84
2.6
1.88
4.79
4.42
13.36
33.71
5.71
4.04
5.46
5.61
T1 (d)
4.6
2.40
3.9
2.58
4.13
3.86
13.5
28.36
4.08
5.92
5.33
5.35
T (d)
13.87
9.24
15.61
9.55
3.75
3.5
13.79
26.21
3.33
4.79
5.25
5.12
PDT (d)
0.51
0.33
0.59
0.37
0.5
0.46
2.25
4.21
0.62
0.58
0.58
0.64
rm (d-1)
1.37
2.09
1.18
1.87
1.37
1.46
0.165
0.309
1.125
1.13
1.23
1.1
Average lifespan (d)
7.13
6.75
7.63
7
16.7
14.7
-
-
22.5
7.4
6.9
6.7
References
This study
(Muschiol et al.
2009)
(Muschiol and Traunspurger 2007)
(Gilarte et al.
2015)
(Addis et al. 2014)
(Addis et al. 2016a)
Note: TFR: total fertility rate; Ro: net reproductive rate; To, T1, T: alternative mean generation time, rm: intrinsic rate of natural increase; PDT: population doubling time; d: day
B I O D I V E R S I T A S
25 (7): 3106-3113, July 2024
3112
This initial increase in fecundity could be attributed to
the bacteria used to feed the nematodes. Commonly used
bacteria in the hanging drop method are E. coli strain OP50
or their symbiotic bacteria (in the case of EPNs). Escherichia
coli strain OP50 is a modified uracil auxotroph that cannot
synthesize the essential nutrient uracil, resulting in the
production of a thin bacterial lawn, and a biofilm
formation-deficient mutant which is suitable for culturing
nematodes while providing easy observation (Arata et al.
2020). The deficiency of uracil in E. coli strain OP50 could
explain why C. briggsae had a reduced fecundity at 109
bacterial cells mL-1 (Schiemer 1982), unlike in C. brenneri,
fed with a wild-type bacterium, A. faecalis. Therefore, as a
wild-type strain bacterium, A. faecalis could have synthesized
uracil and, in turn, provided an additional nutrient source
that resulted in the high fecundity of C. brenneri in this
study. This showed that C. brenneri can proliferate in food
sources and temperature gradients with a considerable
difference and still produce a similarly substantial number
of offspring (Figure 1).
Life history parameter values in this study with conditions
like those of the previous Caenorhabditis species LHT
studies showed some differences (Table 2). Compared to
the LHT of C. elegans (N2 and MY6), C. brenneri has a
lower TFR and Ro (at 20°C with 5×109 cells mL-1), which is
only half the value that of C. elegans. This could indicate
that not all Caenorhabditis species have the same reproductive
capability. The fast population doubling time (PDT) was
also observed in C. brenneri, the same as those of C.
elegans (0.5 days). The intrinsic rate of natural increase
(rm) is one life history parameter widely considered the best
option in showing the population dynamics of a nematode
population.
Compared to lifespan and fecundity alone, rm incorporates
all survival and fecundity into a single measure. In this
study, C. brenneri had a comparable rm (1.18) compared to
C. elegans (N2=1.38 and MY6=1.5) in the same condition.
This suggests that the C. brenneri is also a fast-producing
Caenorhabditis species. Furthermore, C. brenneri exhibits
a boom-and-bust lifestyle as it has a short lifespan of only
seven days compared to >10 days of C. elegans. Instead, C.
brenneri has the same lifespan as EPNs, with an average
lifespan of seven days.
A long lifespan does not always signify a favorable
growth condition, as it has been shown that dietary restrictions
affect fecundity and growth but increase longevity with the
tradeoff of offspring fitness (Moatt et al. 2016; Mautz et al.
2020). A previous study recorded that about 50% of
Caenorhabditis remanei Sudhaus 1974 and C. brenneri
females survived for more than 20 days upon feeding E.
coli strain OP50 in culture media at 25⁰C (Amrit et al.
2010). Feeding nematodes with other bacteria aside from
the common E. coli strain OP50 has also been shown to
significantly alter some of the life history parameters, such
as an increase in lifespan in C. elegans due to the bacteria's
unique nutritional composition (Stuhr and Curran 2020). A
study on C. elegans revealed that metabolically active
bacterium such as E. coli strain OP50 significantly attract
nematodes, leading to high fecundity and shorter lifespan
than inactive bacteria strains (Yu et al. 2015). However, the
present study's short lifespan of C. brenneri could also be
attributed to the dietary nutritional value of A. faecalis,
which is uncommonly used as a standard food source for
lab-grown nematodes in studying growth and reproduction.
Besides offspring production, survivability, fecundity,
and other population parameters, somatic growth in terms
of body length and width of the resulting offspring was also
assessed. It was found that bacterial food density-temperature
interaction significantly affects the development of C.
brenneri body length and width. Such increases in length
and girth were all observed in higher bacterial density and
temperature. These could be attributed to the nutritional
composition of A. faecalis, a change from the usually
utilized E. coli OP50 strain. Such high bacterial food sources
and temperatures help promote an ideal nutrition and
environment to produce a healthy brood of progenies.
Studies on Caenorhabditis LHT using the hanging drop
method are few. This study showed that bacterial food
density and temperature do not significantly influence the
offspring production of C. brenneri. However, the interaction
between bacterial density and temperature significantly
affects the size of offspring in terms of body length and
width. C. brenneri is a fast-producing, short-lived
Caenorhabditis species exemplified by the higher rm, lower
PDT values, and measures of alternative generation time.
ACKNOWLEDGEMENTS
The authors would also like to acknowledge the
contribution of MEP-PRISM Laboratory for their assistance
on the microscopy works and Dr. Temesgen Addis in
providing the template for computing the life history
parameters of the nematode.
REFERENCES
Addis T, Demissie S, Strauch O, Ehlers RU. 2016b. Influence of bacterial
density and mating on life history traits of Heterorhabditis bacteriophora.
Nematology 18 (8): 963-972. DOI: 10.1163/15685411-00003008.
Addis T, Mijuškovic N, Strauch O, Ehlers RU. 2016c. Life history traits,
liquid culture production and storage temperatures of Steinernema
yirgalemense. Nematology 18 (3): 367-376. DOI: 10.1163/15685411-
00002966.
Addis T, Teshome A, Strauch O, Ehlers RU. 2014. Life history trait
analysis of the entomopathogenic nematode Steinernema riobrave.
Nematology 16 (8): 929-936. DOI: 10.1163/15685411-00002819.
Addis T, Teshome A, Strauch O, Ehlers RU. 2016a. Life history trait
analysis of the entomopathogenic nematode Steinernema feltiae provides
the basis for prediction of dauer juvenile yields in monoxenic liquid
culture. Appl Microbiol Biotechnol 100: 4357-4366. DOI:
10.1007/s00253-015-7220-y.
Amrit FR, Boehnisch CM, May RC. 2010. Phenotypic covariance of
longevity, immunity and stress resistance in the Caenorhabditis
nematodes. PLoS One 5: e9978. DOI: 10.1371/journal.pone.0009978.
Arata Y, Oshima T, Ikeda Y, Kimura H, Sako Y. 2020. OP50, a bacterial
strain conventionally used as food for laboratory maintenance of C.
elegans, is a biofilm formation defective mutant. MicroPubl Bio 5:
2020. DOI: 10.17912/micropub.biology.000216.
Bardgett RD, Van Der Putten WH. 2014. Belowground biodiversity and
ecosystem functioning. Nature 515: 505-511. DOI: 10.1038/nature13855.
Chen Y, Lee C, Lin Y, Yin K, Ho C, Liu T. 2015. Obtaining long 16S
rDNA sequences using multiple primers and its application on dioxin-
containing samples. BMC Bioinformatics 18: S13. DOI:
10.1186/11471-2105-16-S18-S13.
DALAN et al. – Life history trait of Caenorhabditis brenneri
3113
Dalan LB, Diano MAB, Tandingan De Ley I, Sumaya NH. 2022. First
report of Caenorhabditis brenneri (Nematoda: Rhabditida) isolated
from the cadaver of Philippinella moellendorffi (Stylommatophora:
Ariophantidae), a terrestrial slug in the Philippines. J Helminthol 96:
1-5. DOI: 10.1017/ S0022149X22000475.
Dey A, Chan CKW, Thomas CG, Cutter AD. 2013. Molecular hyperdiversity
defines populations of the nematode Caenorhabditis brenneri. Proceed
Natl Acad Sci 110: 11056-11060. DOI: 10.1073/pnas.1303057110.
Diano MA, Dalan L, Rolish P, Sumaya, NH. 2022. First report,
morphological and molecular characterization of Caenorhabditis
brenneri (Nematoda: Rhabditidae) isolated from the giant African
land snail Achatina fulica (Gastropoda: Achatinidae). Biologia 469-
478. DOI: 10.1007/s11756-021-00972-x.
Gilarte P, Kreuzinger-Janik B, Majdi N, Traunspurger W. 2015. Life-
history traits of the model organism Pristionchus pacificus recorded
using the hanging drop method: Comparison with Caenorhabditis
elegans. PLoS One 10: e0134105. DOI: 10.1371/journal.pone.0134105.
Huang C. 2020. Extensively drug-resistant Alcaligenes faecalis infection.
BMC Infect Dis 20: 833. DOI: 10.1186/s12879-020-05557-8.
Johnson TE, Friedman DB, Foltz N, Fitzpatrick P, Shoemaker JE. 1990.
Genetic variants and mutations of Caenorhabditis elegans provide
tools for dissecting the aging process. In: Harrison DE (eds). Genetic
Effects on Aging II. Telford, Inc, Caldwell, New Jersey
Kong L, Zhu S, Zhu L, Xie H, Wei K, Yan T, Wang J, Wang J, Wang F,
Sun F. 2014. Colonization of Alcaligenes faecalis strain JBW4 in
natural soils and its detoxification of endosulfan. Appl Microbiol
Biotechnol 98: 1407-1416. DOI: 10.1007/s00253-013-5033-4.
Kreuzinger-Janik B, Brinke M, Traunspurger W, Majdi N. 2017. Life
history traits of the free-living nematode, Plectus acuminatus Bastian,
1865, and responses to cadmium exposure. Nematology 19 (6): 645-
654. DOI: 10.1163/15685411-00003077.
Mautz BS, Lind MI, Maklakov, AA. 2020. Dietary restriction improves
fitness of aging parents but reduces fitness of their offspring in
nematodes. J Gerontol Ser A 75: 843-848. DOI: 10.1093/gerona/glz276.
Moatt JP, Nakagawa S, Lagisz M, Walling C. 2016. The effect of dietary
restriction on reproduction: A meta-analytic perspective. BMC Evol
Biol 16: 199. DOI: 10.1186/s12862-016-0768-z.
Mondejar AJ, Paglinawan F, Tabelin C, Aguilos M, Aguilos R, Opiso E,
Martinez JG, Metillo EB, Sumaya NH, Villacorte-Tabelin M. 2023.
Survival, reproduction, and life history traits evaluation of
Heterocephalobellus sp. and Cephalobus sp. from an artisanal and
small-scale gold mine site, Davao de Oro, Philippines as bioindicators
of heavy metal contamination. Philippine J Sci 152 (6A): 2031-2048.
Mordi RM, Burke ME, Odjadjare EE, Enabulele SA, Umeh OJ. 2015.
Prevalence of urinary tract infections (UTI) among pregnant women
in university of Benin teaching hospital (UBTH) Benin City, Nigeria.
J Asian Sci Res 5: 198-204. DOI: 10.18488/journal.2/2015.5.4/2.4.198.204.
Muschiol D, Schroeder F, Traunspurger W. 2009. Life cycle and
population growth rate of Caenorhabditis elegans studied by a new
method. BMC Ecology 9: 14. DOI: 10.1186/1472-6785-9-14.
Muschiol D, Traunspurger W. 2007. Life cycle and calculation of the
intrinsic rate of natural increase of two bacterivorous nematodes,
Panagrolaimus sp. and Poikilolaimus sp. from chemoautotrophic
Movile Cave, Romania. Nematology 9 (2): 271-284. DOI:
10.1163/156854107780739117.
Petersen C, Dirksen P, Prahl S, Strathmann EA, Schulenburg H. 2014.
The prevalence of Caenorhabditis elegans across 1.5 years in selected
North German locations: The importance of substrate type, abiotic
parameters, and Caenorhabditis competitors. BMC Ecol 14: 4. DOI:
10.1186/1472-6785-14-4.
R Core Team. 2023. R: A language and environment for statistical
computing. R Foundation for Statistical Computing. Vienna, Austria.
https://www.R-project.org/
Ryss AY. 2017. A simple express technique to process nematodes for
collection slide mounts. J Nematol 49 (1): 27-32. DOI:
10.21307/jofnem-2017-043.
Schiemer F. 1982. Food dependence and energetics of free-living
nematodes. Oecologia 54: 108-121. DOI: 10.1007/BF00541117.
Slos D, Sudhaus W, Stevens L, Bert W, Blaxter M. 2017. Caenorhabditis
monadelphous sp. n.: Defining the stem morphology and genomics of
the genus Caenorhabditis. BMC Zool 2: 4. DOI: 10.1186/s40850-
017-0013-2.
Stover NA, Cavalcanti ARO. 2017. Using NCBI BLAST. Curr Protoc
Essential Lab Tech 14 (1): 11-34. DOI: 10.1002/cpet.8.
Stuhr NL, Curran SP. 2020. Bacterial diets differentially alter lifespan and
healthspan trajectories in C. elegans. Commun Biol 3 (1): 653. DOI:
10.1038/s42003-020-01379-1.
Sudhaus W, Kiontke K. 2007. Comparison of the cryptic nematode
species Caenorhabditis brenneri sp. n. and C. remanei (Nematoda:
Rhabditidae) with the stem species pattern of the Caenorhabditis
elegans group. Zootaxa 1456: 45-62. DOI: 10.11646/zootaxa.1456.1.2.
Tena D, Fernández C, Lago M.R. 2015. Alcaligenes faecalis: An unusual
cause of skin and soft tissue infection. Jpn J Infect Dis 68 (2): 128 -
130. DOI: 10.7883/yoken.JJID.2014.164.
van den Hoogen J, Geisen S, Routh D, et al. 2019. Soil nematode
abundance and functional group composition at a global
scale. Nature 572:194-198. DOI: 10.1038/s41586-019-1418-6.
Xiao HF, Li G, Ming D, Hu F, Li HX. 2014. Effect of different bacterial-
feeding nematode species on soil bacterial numbers, activity, and
community composition, Pedosphere 24 (1): 116-124. DOI:
10.1016/S1002-0160(13)60086-7.
Yu L, Yan X, Ye C, Zhao H, Chen X, Hu F, Li H. 2015. Bacterial
respiration and growth rates affect the feeding preferences, brood size
and lifespan of Caenorhabditis elegans. PLoS One 10 (7): e0134401.
DOI: 10.1371/journal.pone.0134401.
Zhao J, Zhang J, Zhu X, Lu J, Jin B, Chen H. 2022. The life cycle of the
bacterial-feeding nematode Diplolaimella stagnosa and its population
growth in response to temperature and food availability. Front Ecol
Evol 10: 953608. DOI: 10.3389/fevo.2022.9536.
