Time of the Day Variability in Pit-Building Behavior of Antlion Larvae

Priyanka Chakradhari¹, Atanu Kumar Pati^1,2,3,4, Arti Parganiha^1,2,*

¹School of Studies in Life Science, Pandit Ravishankar Shukla University, Raipur, India.

²Center for Translational Chronobiology, Pandit Ravishankar Shukla University, Raipur, India.

³Emeritus Professor, Kalinga Institute of Social Sciences-Deemed to be University, Bhubaneswar, India.

⁴Executive Member, Odisha State Higher Education Council, Government of Odisha, Bhubaneswar, India.

priyankachakradhari12@gmail.com, akpati19@gmail.com, arti.parganiha@gmail.com

Abstract

Pit-building behavior in antlion larvae is a unique trait that ensures survival, growth, and metamorphosis. In the present study, we examined the 24-hour rhythm in the pit-building behavior of 16 antlion larvae collected from the PRSU campus, Raipur. We kept each larva individually in a plastic drinking cup in the laboratory. We observed the pit-building behavior of antlion larvae by monitoring two variables, i.e., the time lag for the initiation of pit reconstruction (TLIPR) and total time for pit construction (TTPC) after its demolition over three consecutive days, at four time points each day. We employed single Cosinor Rhythmometry to compute the characteristics of 24-hour rhythm in TLIPR and TTPC. We found a statistically significant 24-hour rhythm in both variables. We found the peaks of TLIPR between 14.32 h and 17.15 h, irrespective of days. Further, the factor 'time of the day' produced a statistically significant effect on the TLIPR and the maximum and minimum values were found at 14.00 h and 02.00 h, respectively. This implies that antlion larvae took time during the afternoon to initiate pit construction. This phenomenon was reversed during nighttime. TTPC exhibited a statistically significant 24-hour rhythm on day 3 and at the group level. We concluded that antlion larvae exhibit a 24-hour rhythm in pit-building behavior and are nocturnal as they initiate pit construction quickly at night.

Keywords: antlion larvae, pit-building behavior, 24-hour rhythm, time of the day, time lag for initiation of pit reconstruction, total time for pit construction

Introduction

The antlion is well known as a pit-building insect. It uses its pit for capturing ants and other prey. The antlion is an Arthropod insect and belongs to the order Neuroptera. There are three different stages in the antlion life cycle, i.e., larva, pupa, and adult stages. Further, larvae's metamorphosis passes through three instar stages (1).

Pit building is a unique behavior of an antlion. It plays a vital role in the metamorphosis of larvae to adults. This behavior is essential for the survival of the larvae. Antlion larvae are found in sandy habitats and dig a conical pit in dry and loose sand. Antlion larvae are also called sit-and-wait predators because of their unique prey-capturing behavior as they make a strange pattern in the sand (2, 3). Antlion larvae construct asymmetric conical pits with fine sand (4). The 3rd instar larvae make a larger pit than the 2nd instar larvae (5). The sand particles' sizes are vital for pit formation. Larvae preferred medium-sized sand particles for pit construction to enhance their efficiency in capturing the prey (6-8). The antlion can identify small and large particle sizes (6). Sand sizes affect the pit's shape (9).

Different environmental factors influence the pit-building activity of larvae. Many factors, namely light, temperature, rain, presence/absence of prey, habit/habitat, sand particle size, and pit density, modulate the antlion's pit-building behavior (4,6,10). Pit size negatively correlates with the relocation frequency (3). Temperature is one of the critical environmental factors for the growth, development, and survival of antlion larvae. Several behaviors, such as predation, feeding, and sand tossing frequency during pit building, increased at high temperatures (8). The larvae's pit size positively correlates with the increasing temperature (11).

There are many pieces of research on various aspects of antlion larvae, such as foraging behavior (12), habitat selection for pit-building under constant light and dark conditions (13), spatial pattern, relocation and pit-building rate, and microhabitat preference (14,15), effects of sand depth, feeding regime, density, and body mass on the foraging behavior (16), the applicability of density-dependent habitat to trap-building predators (17), effects of soil structure on the density of antlion, and the influence of specialized learning on the predatory behavior of antlion (18).

However, we did not find a single research article on the 24-hour rhythm in antlion larvae. The antlion larvae show complex behavior concerning pit-building behavior, maintenance of those pits, and prey capture mechanisms. It is therefore important to understand if these behaviors have underlying clock-controlled mechanisms. Most of the species of antlion larvae are sit-and-wait predators and the circadian rhythm in its physiology and behavior must be in harmony with the prey’s 24-hour rhythm. The study of rhythmic mechanisms in such an interesting insect species is very important to shed light on how it measures and responds to space and time in its natural habitat.

In the current study, therefore, we attempted to examine the 24-hour rhythm in the pit-building behavior of antlion larvae by monitoring two variables, i.e., time lag for initiation of pit reconstruction (TLIPR) and total time for pit construction (TTPC) after its demolition. Our study is the first original report on antlion larvae's rhythmic behavior. Further, we assessed the effects of the factor 'time of the day' on TLIPR and TTPC.

Materials and methods

Subjects

There are about 5000 species of order Neuroptera distributed worldwide among which about 335 species are present in India belonging to 125 genera and 13 families. Out of 5000 species, 2000 species of antlion belong to the family, Myrmeleontidae. 17 species out of 33 species of Neuroptera belonging to the family Myrmeleontidae are present in Madhya Pradesh and Chhattisgarh (19).

We collected antlion larvae from their natural habitat from Pandit Ravishankar Shukla University campus, Raipur, India. We excavated the larva from its pit with the help of the forceps. We randomly selected sixteen (n = 16) live antlion larvae for the study and maintained them under laboratory conditions. We kept these larvae individually in plastic cups (diameter: 5.8 cm, height: 5.8 cm, and filled with sand up to a depth of 3.5 cm) to control the cannibalism. We sent all three larval instars, pupa, and adult specimens of antlion to India's Zoological Survey (ZSI), Kolkata, to identify the species. The ZSI (Lot No. 53/2019) identified the species as:

Phylum:        Arthropoda

Class:            Insecta

Order:           Neuroptera

Family:         Myrmeleontidae

Subfamily:   Myrmeleontinae

Tribe:           Myrmeleontini

Genus:         Myrmeleon

Species:       tenuipennis

 Experimental design

We conducted the study in an isolated room. We fed them with ants every day for seven consecutive days during the acclimation period. We exposed the larvae to LD 12:12 photoperiod throughout the acclimation and experimental periods. Light intensities were 250 Lux during the daytime and 12-15 Lux during the nighttime (Figure 1). We used a Lux meter (Lutron LX-1102, Made in Taiwan) to measure the light intensity.

The experimental design was approved by the RDC (Research Degree Committee) before experimenting (Approval No. (Reg. No.; PhD/18/ZOO/01). We did not harm/ sacrifice any animal for any study rationale. After completion of the study, we released all the experimental larvae to their natural habitat.

Observations of the variables

We studied the pit-building behavior of antlion larvae by observing two variables, namely (1) time lag for initiation of pit reconstruction (TLIPR) and (2) total time for pit construction (TTPC) after its demolition. TLIPR is the larvae's time for initiating pit reconstruction after its demolition. TTPC is the larvae's pit-building time for pit construction from the beginning till its completion. We monitored these two variables at four equidistant time intervals for two hours each in a day, i.e., 08:00-10:00 h, 14:00-16:00 h, 20:00-22:00 h, and 02:00-04:00 h after the demolition of the pits every day over three consecutive days. We recorded the pit-building behavior using a digital video camera (Panasonic HC-V180) and a still camera (Nikon D-3400). We played the video in slow motion and computed the pit-building behavior, namely TLIPR, and TTPC. Our study follows the principles of the Declaration of Helsinki.

Statistical analysis

We employed the single Cosinor rhythmometry (20,21) and analyzed the time series data at a fixed time window (t = 24 h) to determine the 24-hour rhythm in the TLIPR and TTPC. We obtained 24-hour rhythm parameters, such as the Mesor (rhythm-adjusted mean), the amplitude (half of the difference between the minimum and the maximum in the fitted cosine function), and the acrophase (Ø, the timing of the maximum value of the rhythmic function with reference to local midnight) for TLIPR and TTPC. We employed one-way ANOVA followed by the post hoc test (Duncan's multiple range test) to determine the effects of the factor, ‘time of the day' on the TLIPR and TTPC. We used SPSS (version 20) for data analysis. We set the significance level at p ≤ 0.05.

Results

24-hour rhythm in TLIPR and TTPC

Table 1 depicts the Cosinor summary of rhythms in TLIPR and TTPC of the antlion. The Cosinor analysis revealed a statistically significant 24-hour rhythm in TLIPR on day 1, day 2, and group level. TTPC exhibited a statistically significant 24-hour rhythm on day 3 and at the group level.

Peak

The peaks of TLIPR were very consistent and occurred between 14.32 h and 17.15 h in all three days. Further, the peak of pooled data (15.04 h) occurred between these two time points. We noticed a narrow peak spread of about 2.83 h for the rhythm in TLIPR. We observed TTPC rhythm peaks at 3.30 h, 6.96 h, and 2.31 h during day 1, day 2, and day 3, respectively. The peak of TTPC occurred in the late night. At the group level, it occurred at 2.63 h (Table 1).

 Effects of the factor 'time of the day' on TLIPR and TTPC

One-way ANOVA results revealed that the factor 'time of the day' produced a statistically significant effect on the TLIPR (F_{3, 188} = 23.34, p < 0.001). Further, Duncan's multiple range test showed that the antlion larvae took the least time to initiate pit reconstruction after its demolition at 02:00 hour (18.02 ± 3.41 minute) and the maximum at 14:00 hour (65.33 ± 5.44 minute). Both of these mean values were statistically significantly different from each other. Further, The TLIPR at 20:00 hour was statistically significantly higher than the time taken at 02:00 hour. The TLIPR at 08:00 hour (22.94 ± 3.77 minutes) did not differ significantly from that of the 20:00 hour (34.54 ± 4.68 minutes) and 02:00 hour (18.02 ± 3.41 minutes) but significantly differed from that of the 14:00 hour. On average, antlion larvae took 36.96 ± 4.33 minutes to initiate pit reconstruction after its demolition.

In addition, the factor 'time of the day' produced a statistically significant effect on the TTPC (F_{3, 148} = 2.948; p < 0.05). On average, the antlion larvae took 25.13 ± 2.90 minutes to complete the pit construction after its demolition, irrespective of the time. The minimum time taken for pit construction was 17.14 ± 3.54 minutes at 14:00 hour, and the maximum time taken was 29.25 ± 3.12 minutes at 08:00 hour. The TTPC at 14:00 hour was statistically significantly different from that of the other three data points. Further, the TTPC at 08:00 hour (29.25 ± 3.12 minute), 20:00 hour (26.60 ± 2.40 minute), and 02:00 hour (27.52 ± 2.52 minute) did not differ statistically significantly from each other (Figure 2).

Out of 16 larvae, we eliminated one larva (larva ID 7) from the experiment. It did not construct the pit on day 2 and day 3 due to cocoon formation. The scattered graph represents the 15 antlion larvae's original time for TLIPR and TTPC (Figure 3) for three successive days. The mean value is described in a line graph with the respective SE. There was a consistent fluctuation in the mean value of 15 larvae in TLIPR based on raw data. However, in the variable TTPC, we did not find a regular change over the time scale.

Discussion

In the current investigation, we examined the 24-hour rhythm in the pit-building behavior of antlion larvae using two variables: time lag for initiation of pit reconstruction (TLIPR) after the demolition of their pits and total time for pit construction (TTPC). Perhaps this is the first study that focused on evaluating the 24-hour rhythmic pattern in pit-building behavior in antlion.

We detected a statistically significant 24-hour rhythm in pit-building behavior in TLIPR. The antlion took the least time to initiate pit reconstruction during night hours and the highest time during day hours. ANOVA results also support the above findings that antlion larvae took the least time during the nighttime compared to the other three time points for the pit reconstruction initiation. We found that the peaks of the rhythm of TLIPR always occurred in the afternoon hours (around 14:00 h) for all three experimental days and at the group level. It means a longer lag phase was recorded during the afternoon time. The bathyphase (lowest value) of TLIPR was about 12 hours apart and occurred at around 02:00. However, we did not find any peer research for comparing our results. Therefore, it is the first report that documented a 24-hour rhythm in the pit-building behavior of antlion larvae.

The TLIPR was lower (larvae took the least time for initiation of pit building) during nighttime which means larvae quickly become active and initiated pit construction. This phenomenon indicates the 'nocturnality' of the antlion larvae. Although the antlion larvae took the least time for initiation of pit reconstruction during the nighttime, they were active for a longer duration at the same time. On the contrary, they took a long time to initiate pit reconstruction during the afternoon and became inactive immediately after completing the pit building. These two facts reveal the nocturnality of the antlion larvae. During night hours, they were continuously active in reconstructing their pit repeatedly whenever their pits were disturbed by falling sand particles from neighboring antlion larvae pits.

The behavior of larvae has been examined by Scharf et al. (13) under constant light (LL) versus constant dark (DD) conditions. However, they did not investigate antlion behavior by exposing them to LD 12:12 photoperiod. Scharf et al. (13) observed a dichotomous behavior in that a majority of antlions preferred the LL condition and those that preferred the DD condition were larger in size. The authors found a higher activity for pit construction of antlion larvae under constant light conditions. In contrast, in the present study antlion larvae preferred low-intensity light (10-15 lux) during nighttime to construct pits compared to high-intensity light (250 lux) during daytime. Therefore, at this moment, it is difficult to elaborate and compare our findings with others, given the scarcity of literature. Could the nocturnal behavior of antlion larvae be attributed to the availability of more prey (food) during nighttime? However, we have not tested this conjecture in our study. In-depth studies on transverse and longitudinal time scales are necessary to answer this question. One possible explanation for the least activity during the daytime could be energy conservation. The results of Griffiths (23) support this speculation. The author reported that pit construction is an energy-consuming process so larvae avoid pit construction during day hours as prey (food) availability is usually low at this time. The time of prey availability might be one of the crucial zeitgebers (24) that could impact the antlion larvae's pit-building behavior.

Concerning the effect of temperature, Arnett and Gotelli (11) (2001) reported that the pit-building behavior of antlion larvae was more at high temperatures than the low temperatures. Conversely, our results revealed that antlion larvae were more active in building their pits during nighttime. The ambient temperature at night is relatively lower (≈ 10°C) than during daytime (≈ 30°C) in the winter season at the study location. The contradictory results might be due to the species-specific temperature preference for their activity (25,26). About 90% of the species prefer shady areas/ lower temperatures to build their pits (25), while some species build their pits in higher temperature conditions (26).

Conclusion

Concludingly, antlion larvae exhibit a 24-hour rhythm in pit-building behavior. Antlion larvae took a longer time during the afternoon to initiate pit construction while the phenomenon was reversed during nighttime. These findings confirm that antlion larvae are nocturnal. Circadian rhythm studies in antlion are extremely important as the findings of those studies might elucidate the intricate temporal mechanisms that might have evolved to optimize complex behavior concerning pit-building behavior, maintenance of those pits, and prey capture strategies.

Future direction

We recommend that in future studies under laboratory conditions the circadian rhythm in pit-building behavior should be examined in greater detail exposing the antlion larvae to LL and DD photoperiodic schedules. We also suggest that the effects of various light regimes combined with high/low-temperature conditions on the circadian rhythm associated with the pit-building behavior of antlion larvae should be investigated. Further, the effects of timed feeding should be examined on circadian rhythm in pit-building and other behaviors. These recommended studies might throw light on intricate circadian clock mechanisms underlying pit-building and other associated behaviors in antlion species.

Funding

This work was supported by the University Grants Commission, New Delhi, India, under the scheme of Joint CSIR-UGC NET for Junior Research Fellowship [F. No. 16-9 (June 2017)/2018(NET/CSIR), UGC-Ref. No.: 883/ (CSIR-UGC NET JUNE 2017) dated 26/12/2018] to PC.

Acknowledgments

This work is a part of the Ph.D. program of one of the authors (PC). We are grateful to the University Grants Commission, New Delhi, for financial support through its DRS-SAP scheme sanctioned in the thrust area - Chronobiology to the School of Studies in Life Science, Pandit Ravishankar Shukla University (PRSU), Raipur. The Head of the Department, School of Studies in Life Science obliged us to provide logistics support. We are grateful to Mr. Bhupendra Kumar Sahu for helping us with useful comments in the manuscript.

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