Live observation of the oviposition process in Daphnia magna.

In favorable conditions, Daphnia magna undergoes parthenogenesis to increase progeny production in a short time. However, in unfavorable conditions, Daphnia undergoes sexual reproduction instead and produces resting eggs. Here, we report live observations of the oviposition process in Daphnia magna. We observed that the cellular contents flowed irregularly through the narrow egg canal during oviposition. Amorphous ovarian eggs developed an oval shape immediately after oviposition and, eventually, a round shape. Oviposition of resting eggs occurred in a similar way. Based on the observations, we propose that, unlike Drosophila eggs, Daphnia eggs cannot maintain cytoplasmic integrity during oviposition. We also determined that the parthenogenetic eggs were activated within 20 min, as demonstrated by vitelline envelope formation. Therefore, it is plausible that the eggs of Daphnia magna may be activated by squeezing pressure during oviposition.

Introduction

Crustaceans of the genus Daphnia are used as model organisms in the fields of toxicology, ecology, and evolutionary biology [1]. Daphnia species exhibit an alternative reproductive mode. They usually undergo parthenogenesis to generate diploid female offspring without a fertilization event [2]. Parthenogenesis is maintained when surrounding conditions are favorable. However, when Daphnia are exposed to unfavorable conditions, male offspring are parthenogenetically produced, and sexual reproduction proceeds [2-4]. Various environmental factors can affect this process, such as temperature, photoperiod, food availability, and population density [2, 3, 5].

Daphnia produces different kinds of eggs in accord with its reproductive modes. In parthenogenesis, they mostly produce subitaneous eggs, or summer eggs, which can develop into organisms without the contribution of sperm [2, 3, 5]. However, in sexual reproduction, they produce resting eggs, or winter eggs, which differ greatly from subitaneous eggs, and an ephippium, which is a protective chamber for resting eggs [4, 5]. By laying resting eggs into the ephippium, they can remain in a dormant state and undergo activation when they encounter favorable conditions again. Subitaneous eggs generally outnumber resting eggs in a single oviposition event. Daphnia magna can produce up to eighty subitaneous eggs through parthenogenesis, while they produce only two resting eggs in an ephippium through sexual reproduction [2, 3, 5, 6].

Although the interval between oviposition events varies, depending on several factors, it usually takes about 3 to 4 days in favorable conditions [6]. Oocytes and nurse cells are indistinguishable in early stages, but only the oocytes accumulate yolk granules and oil droplets during maturation [7, 8]. It takes approximately 60 h for an oocyte to become a fully grown egg. When the eggs are ready to be ovulated, the animal undergoes the molting process, and oviposition follows 13 min after molting [2]. The eggs develop into neonates in the brood chamber and emerge from it before the subsequent molting process starts [1, 2].

In the present study, we recorded oviposition and egg activation in Daphnia magna. Live observations of oviposition revealed that ovarian components flowed through a very narrow egg canal. Egg activation also occurred after oviposition, as demonstrated by vitelline envelope (VE) formation.

Materials and methods

Animals

Daphnia magna was obtained from the National Institute of Environmental Research (Incheon, Korea). The animals were cultured in M4 medium and maintained under a 16 h:8 h light:dark photoperiod at 23ºC. The animals were fed with Chlorella vulgaris. Their density was controlled to 10–20 individuals in 700 ml to maintain only parthenogenetic females. To produce resting eggs, the animals were cultured in a high-density environment with up to 100 individuals in 700 ml of M4 medium.

Live imaging with microscopes

We transferred Daphnia magna that underwent molting to a petri-dish with a small amount of M4 medium. Two different microscopes were used to record the oviposition process. A stereo-microscope (Leica S8AP0) was used to observe whole animals, while a light microscope (Olympus IX81) was used to focus on the egg canal. For recording, we used Las EZ 3.3.0 for the stereo-microscope and MetaMorph 7.6.5.0 for the light microscope.

Histological analysis

For histological analysis, Daphnia magna was fixed with a 4% paraformaldehyde solution for 30 min. After serial sections were obtained from paraffin-embedded blocks, we performed H&E staining. The slides were observed with a light microscope (Olympus BX51). The connection between the ovarian egg and the extruded egg was easily disrupted when the individuals struggled in the fixative. To obtain intact sample, the animals were therefore pre-exposed to ice to minimize their writhing. To observe VE formation, we fixed Daphnia magna at the indicated time points after oviposition and determined the eggs with a VE.

Results

Oviposition of parthenogenetic and resting eggs

We observed the oviposition process in Daphnia magna with stereo- and light microscopes (Fig 1A, Fig 1B, S1 Movie and S2 Movie). The ovary near the oviposition site exhibited a round shape at the bottom, and the bottom-most egg started to protrude (Fig 1A and S1 Movie). During oviposition, the egg contents were extruded through the egg canal and accumulated outside the ovary (Fig 1A, Fig 1B, S1 Movie and S2 Movie). After completing oviposition, the eggs were transferred to the brood chamber surrounded by the carapace. It takes approximately 180 seconds for the completion of oviposition [2].

Fig 1

Live images of parthenogenetic eggs undergoing oviposition in Daphnia magna.

The oviposition of parthenogenetic eggs was observed with a stereo- (A) and a light microscope (B). The dashed line indicates the outline of an ovulating egg. Arrowheads and arrows indicate the egg canal and extruded eggs, respectively. The time lapse after the initial observation is indicated in each panel. Scale bar, 200 μm. BC, Brood Chamber; Oc, Oocyte; Ov, Ovary. The movie files are deposited as and .

We performed hematoxylin and eosin (H&E) staining to confirm the oviposition process of parthenogenetic eggs in Daphnia magna. As shown in Fig 2A and 2B, we were able to observe parthenogenetic eggs at the mid-point of oviposition. The diameter of the egg canal was approximately 24 μm (Fig 2A). Since the fully matured ovarian eggs had a diameter of 240–350 μm [1], they had to be extruded forcefully to pass through the egg canal.

Fig 2

H&E staining of parthenogenetic eggs undergoing oviposition in Daphnia magna.

(A) H&E staining of an oocyte undergoing oviposition. The boxed area in the left panel is magnified to show the enlarged egg canal. (B) H&E staining of an oocyte in which approximately a half of the contents were extruded. The dashed lined indicates the ovary. BC, Brood Chamber; Ov, Ovary; Ca, Carapace; Oc, Oocyte; EC, Egg Canal. Scale bar, 50 μm.

During sexual reproduction, resting eggs are generated and placed in the ephippium. Ovaries under sexual reproduction are smaller than the case of parthenogenesis, and each ovary possesses one resting egg, total two eggs in ephippium [5]. We recorded the oviposition of resting eggs using a stereo- and a light microscope. The results showed that the contents of the resting eggs passed through a narrow egg canal and accumulated outside the ovary, as observed for parthenogenetic eggs (Fig 3A, Fig 3B, S3 Movie and S4 Movie).

Fig 3

Live images of resting eggs undergoing oviposition in Daphnia magna.

The oviposition of resting eggs was observed with a stereo- (A) and a light microscope (B). The dashed line indicates the outline of an ovulating egg. Arrowheads and arrows indicate the egg canal and extruded eggs, respectively. The time lapse after the initial observation is indicated in each panel. Scale bar, 200 μm. BC, Brood Chamber; Oc, Oocyte; Ov, Ovary. The movie files are deposited as and .

Formation of the vitelline envelope in parthenogenetic eggs

The VE formed as a result of egg activation [9, 10]. We performed H&E staining to observe VE formation in the parthenogenetic eggs of Daphnia magna. The VE was not visible in Daphnia eggs immediately after oviposition (Fig 4A①). Instead, it took time for the VE to start to appear at the egg surface (Fig 4A②). The VE eventually surrounded the eggs (Fig 4A③) and was stabilized as a firm structure (Fig 4A④). To determine when the VE formed, we fixed the eggs at various time points after oviposition and carried out H&E staining. The results showed that VE formation started in approximately 20 min after the oviposition process and took approximately 20 min for completion (Fig 4B). Our results revealed that VE formation took some time in the parthenogenetic eggs of Daphnia magna.

Fig 4

Vitelline envelope formation after oviposition in Daphnia magna.

(A) H&E staining of parthenogenetic eggs forming a VE. ① Before VE formation; ② VE starting to form; ③ VE completely formed and inflated; ④ stabilized VE. Arrowheads indicate the VE. Scale bar, 50 μm. (B) The number of eggs with a VE was counted at the indicated time points after the initiation of oviposition.

Discussion

In this study, we used video microscopes to observe the oviposition process in Daphnia magna. During oviposition, Daphnia oocytes were forced through a narrow egg canal, and the egg contents flowed through the canal (Fig 5). After the completion of oviposition, the parthenogenetic eggs were activated after approximately 20 min, as evidenced by VE formation (Fig 5). We believe that this is the first report to provide live video recording of the oviposition process in Daphnia magna.

Fig 5

Schematic illustration of the oviposition process and vitelline envelope formation in Daphnia magna.

An ovarian egg is extruded into the egg canal, and the egg contents accumulate outside the ovary. The egg undergoing extrusion continues to elongate until oviposition is completed. Thereafter, the VE forms within 20 min.

The body plan resides within the ovarian oocytes in Drosophila melanogaster. For example, mRNAs of bicoid and nanos are already located at the anterior and posterior ends, respectively, of pre-ovulatory oocytes [11]. Drosophila eggs maintain cellular integrity without significant disturbance when they pass through the narrow egg canal for ovulation [12]. However, our observations in Daphnia magna revealed that when the ovarian eggs passed through the narrow egg canal, their cellular contents flowed through the canal irregularly. The diameter of the egg canal is less than one-tenth of that of the pre-ovulatory eggs [1]. Amorphous ovarian eggs developed an oval shape immediately after oviposition and, eventually, a round shape (Fig 5). Therefore, we propose that, unlike Drosophila eggs, Daphnia eggs cannot maintain cytoplasmic integrity during oviposition. A few arthropods are known to have very narrow egg canals. For example, Pimpla turionellae, a haplodiploid wasp, has a narrow egg canal whose diameter is approximately one-third the width of the eggs [13]. The eggs of these wasps are extruded when they ovulate and undergo distortion to pass through the egg canal [14]. However, it remains to be investigated whether Pimpla oocytes maintain cellular integrity or not during oviposition.

In vertebrates and echinoderms, egg activation is caused by a fertilization event. However, in some case of arthropods, the fertilization event is not a precondition for their eggs to be activated [15]. As a result, arthropods can reproduce by using asexual methods such as parthenogenesis [16]. In the case of Drosophila, eggs are activated by pressure when they pass through the female reproductive tract [12, 17–19]. In the current study on Daphnia magna, we observed VE formation as a sign of egg activation during parthenogenesis. The VE of subitaneous egg was formed within approximately 20 min after oviposition, suggesting that activation of the parthenogenetic eggs occurred within this time period. The timing of VE formation has been reported in several arthropods, particularly in shrimps, in which the eggs are activated as soon as they are laid and exposed to seawater [16]. The eggs of Penaeus monodon, the black tiger shrimp, produce a complete hatching envelope within 15 min post-spawning [20, 21]. In addition, the eggs of Sicyonia ingentis, a prawn, start to form the hatching envelope approximately 25–30 min after spawning, and the process is complete in 40–45 min [22]. These reports indicate that it takes time for arthropod eggs to form the VE after the activation event. Therefore, it is likely that the parthenogenetic eggs of Daphnia are also activated prior to the VE formation. We speculate that the oviposition event in which Daphnid eggs pass through a narrow egg canal may be critical for egg activation. An increase in intracellular calcium levels triggers egg activation throughout the animal kingdom [16]. In the case of Drosophila, changes in intracellular calcium levels are induced by the action of the mechanosensitive ion channel during ovulation [15]. It remains to be determined when the change in intracellular calcium levels is induced in the parthenogenetic eggs of Daphnia magna.

Live observation of parthenogenetic egg oviposition by stereo-microscope.

The oviposition process of a parthenogenetic egg in Daphnia magna was recorded using a stereo-microscope. The timing indicated in Fig 1A is matched with the timeline of this movie.

(MP4)

Click here for additional data file.

Snapshots of parthenogenetic egg oviposition by light microscope.

The oviposition process of a parthenogenetic egg in Daphnia magna was recorded using a light microscope. The timing indicated in Fig 1B is matched with the timeline of this movie.

(AVI)

Click here for additional data file.

Live observation of resting egg oviposition by stereo-microscope.

The oviposition process of a resting egg in Daphnia magna was recorded using a stereo-microscope. The timing indicated in Fig 3A is matched with the timeline of this movie.

(MP4)

Click here for additional data file.

Snapshots of resting egg oviposition by light microscope.

The oviposition process of a resting egg in Daphnia magna was recorded using a light microscope. The timing indicated in Fig 3B is matched with the timeline of this movie.

(AVI)

Click here for additional data file.

21 Aug 2019

PONE-D-19-15062

Live observation of the oviposition process in Daphnia magna

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Reviewer #1: While the scope is narrow, this descriptive morphological study revealed some interesting details concerning the process of egg deposition in daphnia. The following are detailed comments.

1. Some statements regarding basic daphnia biology are not accurate. Second paragraph of introduction, during parthenogenic reproduction, Daphnia magna can produce up to ~80 neonates depending on availability of food, ambient temperatures, age, etc. The range of 20-30 is too narrow. The authors are suggested to check with the classic review by Hebert (1978). Third paragraph of introduction, the brood interval is not fixed at 3 days but varies depending on several factors.

2. Third paragraph of results, each ephippium is known to contain two resting eggs. Why one resting egg?

3. The first sentence of the 4th paragraph needs to be backed up by references. So, according to this statement egg activation occurs before vitelline envelope formation? Also, what did the authors exactly mean by egg activation?

4. Second last sentence of the 4th paragraph in the results section, “to form after approximately 20 min” is not clear. It meant to be “approximately 20 min after egg deposition”?

5. Second sentence of the third paragraph in discussion, saying “arthropod eggs” is too general. Consider “the eggs of some arthropods”. Same paragraph, “The VE formed within…….”.

6. There does not appear to be good logic in the last 4 sentences of the 3rd paragraph in discussion. There is a lack of causal relationship between the three sentences concerning shrimp and prawn and the last sentence. What does a delay in VE formation observed in a shrimp and a prawn have anything to do with the speculation that egg activation in daphnia is triggered by squeezing in the oviduct?

7. References are not standardized. Each item should be in the same format and the Latin names italicized.

Reviewer #2: This article studied the oviposition process and egg activation of Daphnia magna from the perspective of morphology. This study is not suitable for submission to this journal and is recommended for submission to professional morphology journals.

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4 Oct 2019

Reviewer #1: While the scope is narrow, this descriptive morphological study revealed some interesting details concerning the process of egg deposition in daphnia. The following are detailed comments.

1 Some statements regarding basic daphnia biology are not accurate. Second paragraph of introduction, during parthenogenic reproduction, Daphnia magna can produce up to ~80 neonates depending on availability of food, ambient temperatures, age, etc. The range of 20-30 is too narrow. The authors are suggested to check with the classic review by Hebert (1978). Third paragraph of introduction, the brood interval is not fixed at 3 days but varies depending on several factors.

We are grateful of helpful comments of the reviewer. We carefully took a look at the review by Hebert (1978) and corrected as suggested. Ebert (2005) also indicated that an adult female may produce a clutch of eggs every 3 to 4 days until her death.

2. Third paragraph of results, each ephippium is known to contain two resting eggs. Why one resting egg?

We understand that each ovary produce one resting egg. Since an ephippium originate from two ovaries, it contains two resting eggs. The text was corrected as suggested.

3. The first sentence of the 4th paragraph needs to be backed up by references. So, according to this statement egg activation occurs before vitelline envelope formation? Also, what did the authors exactly mean by egg activation?

As suggested, we backed up the sentence with two references (Anderson, 1967; Masuda et al., 1991). The most critical event of egg activation may be the elevation of intracellular calcium levels (Horner and Wolfner, Dev Dyn 237:527-544, 2008). Egg activation in Drosophila is known to occur during ovulation (Kaneuchi et al., PNAS 112:791-796, 2015). Egg activation of shrimp was also reported during spawning and formation of vitelline envelope follows after egg activation (Pongtippatee-Taweepreda et al, 2004; Pongtippatee et al., 2012). VE formation is followed after egg activation (Anderson, 1967; Masuda et al., 1991).

4. Second last sentence of the 4th paragraph in the results section, “to form after approximately 20 min” is not clear. It meant to be “approximately 20 min after egg deposition”?

I appreciate for your correction. We changed the sentence as suggested.

5. Second sentence of the third paragraph in discussion, saying “arthropod eggs” is too general. Consider “the eggs of some arthropods”. Same paragraph, “The VE formed within…….”.

I appreciate for your careful reading. We changed the sentence as suggested.

6. There does not appear to be good logic in the last 4 sentences of the 3rd paragraph in discussion. There is a lack of causal relationship between the three sentences concerning shrimp and prawn and the last sentence. What does a delay in VE formation observed in a shrimp and a prawn have anything to do with the speculation that egg activation in daphnia is triggered by squeezing in the oviduct?

It is likely that egg activation in Daphnia also precedes VE formation. We speculate that Daphnid eggs are activated during oviposition which is the most visible event prior to VE formation. In fact, Drosophila eggs are activated by pressure when they pass through the reproductive tract (Horner and Wolfner, 2008). We carefully rewrote the sentences to emphasize our points.

7. References are not standardized. Each item should be in the same format and the Latin names italicized.

We formatted the whole manuscript, following strictly the instruction of Plos One. We also standardized the list of references as suggested.

Reviewer #2: This article studied the oviposition process and egg activation of Daphnia magna from the perspective of morphology. This study is not suitable for submission to this journal and is recommended for submission to professional morphology journals.

We admit that this manuscript is descriptive. However, our works provide an insight how a parthenogenic egg starts development after oviposition. It is known that developmental patterns of embryos in many species are already fixed in oocytes. For example, the anterior-posterior and dorsal-ventral axes of Drosophila embryos are determined during oogenesis. Even if Drosophila eggs pass through a tight reproductive track for ovulation, their cytoplasmic arrangement should not be disturbed. However, the oviposition pattern of the parthenogenic Daphnia egg does not allow us to imagine that the developmental axis are maintained during oviposition. What we observed is that the egg contents flow through a narrow egg canal. After oviposition, Daphnia eggs are known to follow a strict developmental pattern. Significance of this work may be that we raise a possibility that developmental axis of Daphnia may not be fixed during oogenesis but be established after oviposition. Since our observations provide an insight on a new way for developmental pattern formation, we believe that this manuscript brings a general interest in the field of animal development as well as Daphnia research.

Submitted filename: response (2, Plos One).docx

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14 Oct 2019

Live observation of the oviposition process in Daphnia magna

PONE-D-19-15062R1

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Reviewer #1: Yes

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Reviewer #1: The authors responded to reviewers' comments and concerns pretty well.

Couple of typos:

1. Page line 9 from top, "smaller than the case of" to "smaller than those for".

2. Page line 4 from bottom, "Daphnid" to "daphnid".

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22 Oct 2019

PONE-D-19-15062R1

Live observation of the oviposition process in Daphnia magna

Dear Dr. Rhee:

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