Research Article |
Corresponding author: Seiji Tanaka ( stanaka117@yahoo.co.jp ) Academic editor: Michel Lecoq
© 2021 Seiji Tanaka.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Tanaka S (2021) Embryo-to-embryo communication facilitates synchronous hatching in grasshoppers. Journal of Orthoptera Research 30(2): 107-115. https://doi.org/10.3897/jor.30.63405
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Synchronous hatching within single egg clutches is moderately common in locusts and other insects and can be mediated by vibrational stimuli generated by adjacent embryos. However, in non-locust grasshoppers, there has been little research on the patterns of egg hatching and the mechanisms controlling the time of hatching. In this study, the hatching patterns of six grasshoppers (Atractomorpha lata, Oxya yezoensis, Acrida cinerea, Chorthippus biguttulus, Gastrimargus marmoratus, and Oedaleus infernalis) were observed under various laboratory treatments. Under continuous illumination and a 25/30°C thermocycle, the eggs of these grasshoppers tended to hatch during the first half of the daily warm period. Eggs removed from egg pods and cultured at 30°C tended to hatch significantly earlier and more synchronously when kept in groups vs. singly. In general, eggs hatched earlier when egg group size was increased. Egg hatching was stimulated by hatched nymphs in some species, but not in others. In all species, two eggs separated by several millimeters on sand hatched less synchronously than those kept in contact with one another, but the hatching synchrony of similarly separated eggs was restored if they were connected by a piece of wire, suggesting that a physical signal transmitted through the wire facilitated synchronized hatching. In contrast, hatching times in the Emma field cricket, Teleogryllus emma, which lays single, isolated eggs, were not influenced by artificial clumping in laboratory experiments. These results are discussed and compared with the characteristics of other insects.
egg hatching, egg pod, hatching synchrony, Orthoptera, vibration
Most grasshopper species deposit their eggs a few centimeters underground in a foamy egg pod that can contain as many as 200 clumped eggs, depending on species (
The daily hatching time in grasshoppers is thought to be controlled by environmental factors such as daily photoperiod and temperature cycles, as observed in other insects (
Synchronous hatching within a single egg pod was originally hypothesized to be triggered by a thermal threshold mechanism, whereby the eggs are ready to hatch but require a certain temperature to do so. In this scenario, rising temperatures in the spring heat the soil, and synchronous hatching is induced on the day when the soil at the level of the buried eggs finally exceeds the species-specific threshold temperature (
Recently, a new mechanism controlling synchronized hatching was discovered. In S. gregaria, L. migratoria, and the Bombay locust Nomadacris (also known as Patanga) succincta (Johannson, 1763), eggs kept in contact with one another hatched synchronously, while those kept separately hatched asynchronously (
In the present study, I document the hatching behavior of six grasshopper species in response to thermocycles, number of eggs in the group, presence of early hatched nymphs, and vibrations transferred through a wire. To explore the taxonomic breadth of the vibration response, I also tested to see if the eggs of a cricket that lays eggs singly would hatch synchronously if artificially placed in a group. This paper describes the results of these observations and compares them with those previously reported for other insects.
Insects.—Five species of grasshopper – the longheaded grasshopper Atractomorpha lata (Motschilsky, 1866), the Oriental longheaded grasshopper Acrida cinerea (Thunberg, 1815) the bow-winged grasshopper Chorthippus biguttulus (Linaeus, 1758), the band winged grasshopper Gastrimargus marmoratus (Thunberg, 1815) and Oedaleus infernalis Saussure, 1884 – were collected in Tsukuba, Ibaraki, Japan from August to October of 2017 and 2018. Egg pods of the rice grasshopper Oxya yezoensis Shiraki, 1910 were collected in Tsukuba in September 2017 and in paddy fields in Kuroishi, Aomori, Japan in May 2018 and sent to Tsukuba, where experiments were performed. All species are of the family Acrididae, except for A. lata, which is of the family Pyrgomorphidae. Adults of each species were reared under outdoor conditions on various host plants, such as Bromus catharticus, Artemisia indica var. maximowiczii, and Miscanthus sinensis in nylon-screened cages (22 × 39 × 43 cm) in which a plastic cup (volume: 340 ml) filled with moist sand (10–15% water by wt) was placed as the oviposition substrate. L. migratoria and C. biguttulus are bivoltine and produce non-diapause eggs in early summer but diapausing eggs in the fall. For the five species, I used overwintering, diapausing eggs, which, in nature, remain in the egg stage for several months. Laid egg pods were kept outdoors until December and then stored in a refrigerator (7°C) for 2–5 months until used. The eggs of all five species appeared to have entered diapause at the end of the anatrepsis stage, by the arrival of winter, and were ready to hatch upon transfer to warm conditions in late January. In contrast, eggs of A. lata are known to have no diapause and overwinter in a state of quiescence (Y. Ando, pers. comm.), but were maintained as above. All of these species occur in grasslands in Japan and hatch in the spring when semimonthly mean soil temperatures measured every 60 min at a depth of approximately 3 cm at an exposed site in Tsukuba ranged from 12.8 to 28.8°C from early April to late July in 2020 (Tanaka, S. pers. obs.).
For the experiments, eggs of all species were handled in the same way: each egg pod was washed with chlorinated tap water; the eggs were separated from the pod and individually placed on wet tissue paper in a 9-cm plastic Petri dish until used. They were maintained at 30 ± 1°C under continuous illumination in incubators. The compound eyes could be seen through the chorion several days before hatching. The number of eggs per pod varied from ~ 10 in C. biguttulus to more than 100 in A. cinerea.
Hatching under thermocycles
.—Eggs of each species were kept either singly or in a group in pits on moist non-sterilized white sand (~ 15% moisture content by wt; Brisbane White Sand, Hario Co. Ltd., Japan) in a 9-cm Petri dish with a transparent lid and exposed to a thermocycle of 25/30°C under continuous illumination at least 5 days before hatching, unless otherwise mentioned. The eggs were incompletely covered with sand. The time required for the hatching rhythm of each species to be entrained by the thermocycle is unknown, but it was assumed that 5 days was sufficient based on previous studies with other grasshopper species (
Effect of egg group size on hatching time.—Eggs from each pod were divided into treatments that differed in the number of grouped eggs: 1 vs. 2, 2 vs. 4, or 4 vs. 10, except for C. biguttulus, in which only two treatments (1 vs. 15 eggs) were prepared because fewer eggs were available for this species. The eggs in a group were held in a sand pit in a plastic Petri dish, and singly kept eggs were held in sand pits in another dish, as described earlier. The hatching time of eggs was recorded under continuous illumination and temperature (30 ± 1°C). Mean hatching times of the various treatment groups were calculated and compared. For each species, a value of 5 h was assigned to the mean hatching time of the largest group to standardize comparisons between different group sizes.
Effect of hatched nymphs on the hatching times of late-hatching eggs.—Whether the hatching time of an egg was influenced by the presence of an early-hatching nymph was determined under 30 ± 1°C and continuous illumination by treating pairs of eggs from the same pod in three different ways: 1) two eggs placed horizontally and in contact with one another on moist sand in a well of plastic 24-well plates (Thermo Fisher Scientific KK, Tokyo, Japan), 2) eggs separated by 2–3 mm on sand, and 3) eggs separated by a stainless steel wire screen that kept hatchlings from physically touching unhatched eggs. The hatching times were determined as described earlier, and the hatching intervals of eggs in pairs were calculated. Because photographs were taken every half-hour, 0.5 h was added to the hatching interval of two eggs and, thus, the minimum hatching interval was 0.5 h.
Stimuli inducing synchronized hatching.—To determine the stimuli responsible for synchronized hatching when two eggs are kept in contact, pairs of eggs from the same pod in each species were treated in three different ways: (1) eggs horizontally placed in contact with one another on sand in the same well, (2) those separated by 2–5 mm, and (3) those similarly separated but connected by a piece of stainless steel wire (diameter, 0.1 mm; length, 0.7 cm). All treatments were done for all grasshoppers except for the two species in which the separation of eggs did not show a marked effect on the hatching intervals. In the last two species, the eggs in (2) were separated by a wire screen, and those in (3) were separated by a screen but connected by a piece of stainless steel wire placed through the screen separator. Connecting wires were laid on top of the two eggs (Fig.
Effect of clumping of cricket eggs on the hatching time.—More than 20 adults of the Emma field cricket, Teleogryllus emma (Ohmachi & Matsuura, 1951), were collected in Tsukuba in August and September 2018, and allowed to lay eggs in moist sand in plastic cups at room temperature. The cups containing the eggs were then kept outdoors until February, when the eggs were ready to hatch when transferred to warm conditions (Tanaka, S. pers. obs.). The eggs were separated from the sand by washing with cold tap water and divided into two batches; 5 groups of 10 eggs were placed either as groups or singly in sand pits in 9-cm plastic Petri dishes. The dishes were then incubated at 30 ± 1°C under continuous illumination with 10 days required for the eggs to hatch. The hatching times of the eggs were recorded.
Statistical analyses.—The hatching times were compared using ANOVA, Tukey’s multiple comparison test, or t-test. The proportions of eggs that hatched synchronously were compared with the χ2-test. The comparisons of hatching intervals were made with the Steel-Dwass test or the Mann-Whitney’s U-test. These analyses were performed using a statistics service available at http://www.gen-info.osaka-u.ac.jp/MEPHAS/kaiseki.html. Descriptive Statistics were presented in Excel (Microsoft Office 365) or StatView (SAS Institute Inc., NC, USA). Differences were judged as significant when p < 0.05.
Hatching under thermocycles.—The hatchlings of each species had a characteristic body shape, size, and color (Fig.
Hatching activity of eggs kept in a group (A–F.) and those kept singly (G–L.) in a thermocycle of 30 (orange) and 25°C (blue) under continuous illumination in the six indicated grasshopper species. The numbers of eggs that hatched over 2–5 days were pooled and plotted against the time of day. Black arrows indicate the medians. Asterisks indicate a significant difference between the two treatments by the Mann Whitney U-test at the 5% level. The photographs on the right show hatchlings of respective species. Scale bars: 5 mm.
Effect of egg group sizes on hatching time.—The relationship between number of eggs in a treatment and hatching time varied depending on the species. Eggs hatched earlier as the number of eggs in the group increased from 1 to 4 or 10 in A. lata, A. cinerea, and O. infernalis (Fig.
Relationship between number of eggs in a group and mean hatching times in six grasshopper species under continuous illumination and 30°C temperature. For each species, hatching times were normalized by assigning a value of 5 h to the mean hatching time of the largest group. n (number of eggs in each treatment) is given above each histogram. Different letters indicate significant differences in mean values at the 5% level using the Tukey’s multiple test (A–E.) or the t- test (F.). ns indicates no significant difference.
Effect of hatched nymphs on the hatching times of later-hatching eggs.—The mean hatching interval of two eggs was significantly larger in eggs separated by a few millimeters than those kept in contact with one another, but it was further increased when the eggs were separated by a screen in A. lata, A. cinerea, O. infernalis, and O. yezoensis (Fig.
Hatching intervals of two eggs kept in contact with one another (top panel), separated by 3–5 mm (middle panel), or separated by a screen (bottom panel) in the six indicated grasshopper species. Eggs were maintained under continuous illumination and 30°C temperature (A–F.). Different letters indicate significant differences in mean values at the 5% level using the Steel-Dwass test. Diagram on the right shows how the eggs were arranged in wells.
Stimuli inducing synchronized hatching.—The hatching interval of two eggs that were kept in contact with one another was significantly shorter than those that were separated by a few millimeters (Fig.
Hatching intervals of two eggs kept in contact with one another (top panel), separated by ~5 mm (middle panel) with or without a screen,or connected by a piece of wire (bottom panel) at 30°C under continuous illumination in the six indicated grasshopper species (A–F.). Different letters indicate significant differences in mean values at the 5% level by the Steel-Dwass test. Diagrams in panels show how the eggs were arranged in wells.
Effect of clumping of cricket eggs on their hatching time.—The Emma field cricket showed no significant difference in the mean hatching time (t-test; p = 0.12) and its variance (F-test; p = 0.07) between the eggs kept in a group of 10 eggs and those kept singly (Fig.
Hatching activity of the eggs of Teleogryllus emma kept in a group (A.) and those kept singly (a distance of approximately 5 mm) (B.) at 30°C under continuous illumination. The hatching times for 5 groups of 10 eggs were pooled and calculated by designating the time of the first hatching egg as 1 h. The mean hatching time ± SD (sample size) is given in each panel. s2 indicates the variance. Diagrams on the right show the experimental setup.
Although the egg pods of many grasshopper species hatch more or less synchronously (
In nature, each grasshopper species tends to hatch at a specific time of day, depending in part on local habitat and current weather (
Summary of hatching behavior and responses to external stimuli in grasshopper species and some other insects.
Species | Hatching time under thermocycles | More eggs | Stimuli from hatched nymph | Vibration from wire | References | |
Increased synchrony | Shorter hatching time | Shorter hatching time | Increased synchrony | |||
Orthoptera : Acrididae | ||||||
Locusta migratoria L. 1758 | Warm period | + | + | + | + | ( |
Schistocerca gregaria Forskål, 1775 | Cool period | + | + | + | + | ( |
Nomadacris succincta Johannson, 1763 | Both periods | + | + | + | + | ( |
Atractomorpha lata Mochulsky, 1866 | Warm period | + | + | + | + | This study |
Oxya yezoensis Shiraki, 1910 | Warm period | + | - | + | + | This study |
Acrida cinerea Thunberg, 1815 | Warm period | + | + | + | + | This study |
Oedaleus infernalis Saussure, 1884 | Warm period | + | + | + | + | This study |
Gastrimargus marmoratus Thunberg, 1815 | Warm period | + | △ | - | + | This study |
Chorthippus biguttulus L. 1758 | Warm period | + | +* | + | + | This study |
Orthoptera Romaleidae | ||||||
Romalea microptera Palisot de Beauvois, 1817 | Warm period | n.d. | n.d. | n.d. | n.d. | ( |
Orthoptera : Gryllidae | ||||||
Teleogryllus emma Ohmachi & Matsuura, 1951 | n.d. | - | - | n.d. | n.d. | This study |
Hemiptera : Pentatomidae | ||||||
Nezara viridula L. 1758 | n.d. | + | + | n.d. | n.d. | ( |
Halyomorpha halys Stål, 1855 | n.d. | + | + | - | + | ( |
Lepidoptera : Crambidae | ||||||
Chilo suppressalis Walker, 1863 | n.d. | + | + | - | n.d. | ( |
Hatching
time is thought to have evolved to maximize hatchling survival against predators and weather extremes. Hatching at the wrong time of day can be lethal. For example, mid-day hatching would be lethal for grasshoppers living in hot deserts because desert soil temperatures can exceed 65°C (
Cuticle physiology may have also influenced grasshopper hatching times. This is because hatchlings require time for their integument to harden before they can actively move or feed (
Although synchronous hatching in grasshoppers has long been known (
Table
In addition to those mentioned earlier, other insects, such as a cockroach (
This paper confirms previous studies suggesting that vibrational signals from siblings can induce synchronous hatching in some insects (
The specific time at which the signals are produced by the six grasshoppers tested in this study is currently unknown. In addition to the vibrational signals generated by an embryo, other physical signals from hatching eggs, egg shell cracking, vermiform nymphs wiggling through the egg mass to reach the surface, or new hatchlings walking on the surface could also be involved.
In the present study, the eggs of the Emma field cricket failed to hatch synchronously when artificially kept in a group. This cricket requires a total of ~15 days of incubation at 30°C (excluding diapause) and does not lay eggs as a group. This result is reasonable in view of the fact that in nature, the eggs of this cricket are laid individually in soil and do not hatch synchronously, and neither nymphs nor adults aggregate. Perhaps vibration-induced hatching synchrony has been selected for only in species that lay grouped eggs and benefit from synchronous hatching. Two lady beetles, Epilachna sparsa orientalis and E. vigintioctomaculata, lay eggs as groups. The former lays eggs so they touch and the latter lays eggs that do not touch one another.
Many grasshoppers and other insects form tight aggregations in the 1st instar (Fig.
To understand the mechanism underlying synchronized hatching and its evolution, more species of insects that produce eggs in a group with different lengths of embryonic stage should be examined. Grasshopper species would be ideal insects to use to explore this subject.
I thank Mr. Toshiyuki Kimura for sending me the Oxya yezoensis eggs, Dr. Takumi Kayukawa for his cooperation with experiments, and Mr. Masanari Aizawa for providing me with information on the Bombay locusts on Minami-Daito Island and permission to use the photograph in Fig.