Breeding behaviour of the Coromandel Marsh Dart Damselfly (Zygoptera: Coenagrionidae: Ceriagrion coromandelianum (Fabricius)) in central India

Ceriagrion coromandelianum (Fabricius) is one of the most common damselflies in the Indian subcontinent. It flies among bushes and breeds in stagnant pools, small garden tanks, tubs and ornamental cement ponds containing submerged and/or floating vegetation. The oviposition behaviour of C. coromandelianum was observed at the botanical garden of Hislop College, Nagpur, India, where small underground cement tubs are utilized to grow macrophytes. C. coromandelianum displays a refined hierarchy of preferences for oviposition and chooses floating leaves of Nymphaea nouchali (69%) over Lemna paucicostata (23%) and submerged Hydrilla verticillata (8%). In an uninterrupted oviposition bout, the female deposits 283 eggs in 16 rows (N=5) on the under surface of the N. nouchali leaf. The tiny leaves of L. paucicostata holds 7.8 eggs in 4.8 rows (N=10). In H. verticillata, the internode region of the stem can house 25.4 eggs (N=10). One or two eggs are also found neatly inserted in the thin leaf base of H. verticillata. Decaying plant material is never used for oviposition. The present investigation also clearly demonstrates that the choice of oviposition substrate not only depends upon the presence of aquatic species in the water body but also on the spatial location of the oviposition site.


INTRODUCTION
Habitat selection for oviposition is a vital and complex behavior undertaken by odonates, after the termination of copulation.Oviposition takes place either in water (aquatic), on floating plant material (epiphytic), or inside plant tissue (endophytic).For damselflies, choice of ovipositing material primarily depends upon "the initial preference" which is a suitable place both, for landing and easy deposition of egg in the plant tissue (Waage 1987;Martens 1992Martens , 1993Martens , 1994Martens , 1996Martens , 2001)).Highly stenotopic species oviposit in one or very few species of plant while eurytopic species may exhibit distinct preferences and oviposit in a wide variety of plants (Martens 1996).Some odonate species of temperate regions show preferences in selecting plant species or even parts/region of plants for oviposition (Martens 1992;Wildermuth 1993;Grunert 1995).
Previously we found that the Coromandel Marsh Dart Damselfly Ceriagrion coromandelianum (Fabricius, 1798) selects specific laminar region of the leaf of water lily, Nymphaea nouchali f. for oviposition and also reported the existence of a direct co-relation between the choice of leaf lamina region and the day of oviposition (Andrew et al. 2011a).In this damselfly, we also studied and evaluated the process of contact guarding oviposition with reference to the male in sentinel position (Andrew et al. 2011b).In continuation with the study of the reproductive biology of this species, the present paper describes the oviposition of Ceriagrion coromandelianum with reference to choice, insertion and deposition of egg in the aquatic plants Nymphaea nouchali Burm, f., Hydrilla verticillata (L.f.) Royde and Lemna paucicostata Hegelm.6746.

Site:
The observation and collection for the present work was carried out at the botanical garden of Hislop College, Nagpur, (21.16 0 N & 79.03 0 E) where small underground cement tubs/tanks are used to grow macrophytes (Image 1).There is a large circular tub (diameter 105cm) surrounded by six smaller circular tubs (diameter 42cm), followed by a row of three rectangular cement containers (84cm x 47cm).Height of the tubs varies between two to three feet.Two rectangular tubs contain floating L. paucicostata and submerged H. verticillata while the small circular tubs contain H. verticillata.The single large circular tub contains floating N. nouchali, small patches of L. paucicostata and submerged H. verticillata (Image 2).The bottom of the tubs comprise mud, debris and decaying leaves.C. coromandelianum is found ovipositing in these tanks all around the year (except from December to February).The plant material were collected after the female had completed egg deposition and taken to the laboratory for investigation under stereoscope binocular microscope.

RESULTS
Ceriagrion coromandelianum is one of the most common damselflies in the Indian subconti nent.The male is bright yellow with olivaceous and pale greenish yellow eyes.The females are initially as bright as the males but with maturity turn dull yellow to light brown.The intensity of dullness increases with maturity.It flies among bushes and breeds in stagnant pools and small garden tanks, tubs and ornamental cement ponds containing floating and/or submerged vegetation.Females visit the waters only for reproduction but the males can be spotted at all times around the shrubs near the ovipositing site.
For oviposition, the tandem pair of C. coromandelianum flies towards the tubs and alights on a floating substrate (N.nouchali, H. verticillata, L. paucicostata).The female starts probing the underside with the ovipositor located at the terminal tip of the abdomen.Initially, the female evaluates the undersurface of the substrate by feeling and testing the plant material with the stylus of her ovipositor.If she finds that the substrate is not suitable for oviposition, she repositions herself by moving a few inches along the rim of the leaf lamina (in case of N. nouchali) or relocates to a different site, not more than 5cm (in case of H. verticillata and L. paucicostata) away.Once settled, she begins to perforate the plant tissue with her saber shaped sharp ovipositor valvules and starts laying eggs in it.The female never uses decaying plant material for oviposition.
Experiments were conducted by placing plastic leaf models of Nymphaea in a plastic water tub to understand the behaviour of oviposition.The tandem pair readily settled on the plastic leaf and the female started probing the under-surface of the artificial leaf with the ovipositor, but after hectic probing (168 Sec, Max-225; Min.-15, N=5), the tandem pair left the site (Image 2).To understand the order of preference for oviposition substrate by C. coromandelianum the data of 20 days (when maximum number of oviposition was noticed) was evaluated.C. coromandelianum displays a refined hierarchy of preference for oviposition substrate and chooses floating leaves of N. nouchali (69%) over L. paucicostata (23%) and submerged H. verticillata (8%) (Table 1, Fig. 1).

Oviposition in Nymphaea nouchali
Leaves of Nymphaea nouchali form a perfect landing site for C. coromandelianum and provide a large surface area for oviposition (Image 3).In an uninterrupted oviposition bout, the female deposits 288 eggs (Max 322; Min 243; N=5) in 19 rows along the undersurface of the leaf (Image 3).The eggs are mostly arranged in slightly concentric rows and the distance between two eggs is about 689.4±26.5 µm (Max 1230µm; Min 230µm; N=100).The distance between two rows is 2.4±0.3 mm (Max 3.32mm; Min 1.6mm; N=5).Graphic representation by plotting the trend line of second order polynomial shows that maximum number of eggs are laid in the middle row (Table 2, Fig. 2).

Oviposition in Lemna paucicostata
The puncture mark of the ovipositor of C. coromandelianum can be easily observed on the  reddish-brown undersurface of L. paucicostata (Image 4).Carefully peeling the leaf under the microscope reveals that the tiny leaf of L. paucicostata holds 37.8 eggs in 4.8 rows (Max 47; Min 30; N=10) (Table 3).The distance between two eggs is 182±20.6µm and the distance between the two rows is 1610±70 µm (Max 1800µm; Min 1610µm; N=20).The statistical analysis of the data by plotting the trend line of second order polynomial reveals that the maximum numbers of eggs are laid in the middle rows (Fig. 3).

Oviposition in Hydrilla verticillata
In H. verticillata, the internode region of the stem is used for oviposition.Each internode region can house 25.4 eggs of C. coromandelianum (Max 33; Min 14; N=10) (Table 4).The eggs are arranged mostly in two to four longitudinal rows.There is a gap of 71.6±12.7 µm between the eggs.The rows are at a distance of 847.6±52 µm (Max 980µm; Min 670µm).Sometimes oblique or overlapping rows of eggs are also found in    the internode region.Rarely, one or two eggs are found neatly inserted in the very thin leaf base of the plant (Image 5).At times, the female sitting on N. nouchali dips the abdomen in water and the ovipositor encounters the underlying H. verticillata.She ensures the suitability of H. verticillata plant by probing it with the ovipositor and starts depositing eggs in it.This has been tested by deliberately placing or removing H. verticillata below N. nouchali during oviposition.If H. verticillata is removed, the female explores the surrounding areas with the ovipositor without moving from her position and begins ovipositing in N. nouchali, and, if H. verticillata is placed between the ovipositor and N. nouchali leaf, the female easily switches and starts ovipositing in H. verticillata.
After a complete duration of oviposition, the female dislodges herself from the tandem and flies away into the surrounding shrubs or darts away up to a different site.At times, the tandem couple exhibits the ritual of 'water-touching' after completing oviposition, i.e., the pair in tandem flies over the water body, and then the female dips her abdominal tip in the water and then the pair flies away among the surrounding bushes.The male is reluctant to leave the female and tries to either recopulate or force her to re-oviposit by leading her back to water.Sometimes, the female releases herself from the male after resting in tandem for 2-7 minutes.The released female is at times followed by other males but she quickly darts away from the ovipositing site.

DISCUSSION
In Ceriagrion coromandelianum, the selection of the landing site for oviposition is visual.This was demonstrated by the experiment of using plastic models of Nymphaea leaves as site for oviposition, where the female readily alighted on the artificial leaf and started to feel the under-surface of this leaf for oviposition.Since the plastic was harder (than aquatic vegetation) the female could not penetrate the ovipositor and lay eggs.Once the female dips the abdomen inside water, it is the ovipositor which determines the choice of the insertion site, which can either be the landing substrate (N.nouchali), or the underlying H. verticillata as demonstrated in the experiment of removing or placing H. verticillata below the landing substance.Matushkina & Gorb (2007) found that coenagrionids and platycnemidid damselflies predominantly choose tissues of Nymphaeaceae for oviposition, while lestids mostly laid eggs in coastal plants.Lestes temporalis and Chalcolestes spp.oviposit into woody branches of trees and bushes.Aeshna viridis used leaves of Stratiotes aloides while Coenagrion mercuriale, mostly used the aquatic Berula erecta (Matushkina & Gorb 2002).Matushkina & Lambret (2011) observed that before inserting her ovipositor in the plant, the damselfly Lestes macrostigma, touches the plant tissue six times before ovipositing.These touches may provide information leading to a choice of insertion site.The damselfly Platycnemis pennipes oviposit in at least 25 species of plants but while ovipositing into the flowering stem of Nuphar lutea, it also exhibits similar hierarchy of preferences in which the age of stems, colour, size and association with a floating leaf are taken into consideration (Martens 1996;Corbet 1999).
In India, Srivastava & Babu (1985) found that C. coromandelianum prefers the aquatic plant Salvinia over Azolla and never oviposits in Spirodela polyrhiza, Trapa natans, Eichhornia crassipes, Lemna microphylla and Nelumbo sp. or dead or decaying leaves at Sagar, Madhya Pradesh.Prasad (1990) found that it oviposits in all aquatic plants at Kolkata, West Bengal while  Sharma (2009) reported that it prefers H. verticillata and Polygonum barbatum over other aquatic plants at Hoshiarpur, Punjab.In the present study we have noticeably demonstrated the C. coromandelianum displays a refined hierarchy of preferences for oviposition and chooses floating leaves of N. nouchali over L. paucicostata and submerged H. verticillata.The present investigation clearly demonstrates that the choice of oviposition substrate not only depends upon the presence of aquatic species in the water body but also on the spatial location of the oviposition site.

Image 1 .
The study site: water tubs at Botanical garden of Hislop College, Nagpur.

Figure 2 .
Figure 2. Ceriagrion coromandelianum -the average number of eggs per row laid in the leaf lamina of Nymphaea nouchali.

Figure 3 .
Figure 3. Ceriagrion coromandelianum -the average number of eggs per row laid in the leaf lamina of Lemna paucicostata.

Image 2 .
Ceriagrion coromandelianum trying to settle and oviposit in artificial plastic leaf of Nymphaea.The experimental tub (a).Tandem pair landing on the leaf (b) and female trying to insert eggs along the under-surface of the plastic leaf (c).Image 3. Ceriagrion coromandelianum ovipositing on leaf of Nymphaea nouchali (a,b).Rows of eggs inserted in the leaves (c) (photographed under dissecting microscope).Ceriagrion coromandelianum ovipositing in Lemna paucicostata (a).Puncture oviposition marks on the reddish-brown under-surface of the leaf (b).Eggs placed in tiers in the leaf lamina (c) Image 5. Ceriagrion coromandelianum ovipositing in Hydrilla verticillata (a) Oviposited eggs in the internode region of the stem of H. verticillata (b) (the apical brown region of the egg can be clearly demarcated while a semi transparent first instar larva clings to the plant).Single egg neatly placed in the leaf base (c).