Oviposition behavior of the parasitoid Telenomus remus (Hymenoptera: Scelionidae) on eggs of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Abstract

Telenomus remus (Hymenoptera: Scelionidae) is an efficient parasitoid of Spodoptera eggs. However, biological control programs require taxonomic, bioecological and behavioral studies of biological agents. Although the performance of T. remus in pest control has been evaluated, little is known about its behavioral aspects that can influence IPM tactics. The aim of this study was therefore to study the parameters related to the oviposition behavior of T. remus on Spodoptera frugiperda (Lepidoptera: Noctuidae) eggs. The experiment was conducted in the laboratory, where 17 females were transferred individually to arenas containing a mass of S. frugiperda eggs. Oviposition behavior was recorded for 30 minutes. The average walking speed of the females was 0.116 mm.s^-1 and the total distance covered was 203.3 mm. The females remained on the egg masses for an average of 16.7 min, which corresponds to more than half of the total time. The average number of parasitized eggs was 15.1 per female in 30 minutes. Females with larger antennae had lower values for average speed and total distance covered. The results of the parameters evaluated show the standard oviposition behavior of T. remus females on S. frugiperda eggs and help us to better understand the species.

Key words
biological control; egg parasitoid; fall armyworm; Platygastroidea; walking behavior

INTRODUCTION

The study of insect behavior is fundamental for various practical applications, including in the areas of public health, biodiversity conservation and agricultural pest control (Matthews & Matthews 2009). These studies usually involve different aspects including movement patterns, communication, host selection, reproduction, feeding and response to environmental stimuli (Bell 1990). However, the emphasis on the behavior to be studied usually depends on the group of insects in question (Steinbeck et al. 2020).

Oviposition behavior is one of the most important when studying egg parasitoids, as it is from this moment that the first interaction between parasitoid and host occurs. This behavior is usually divided into four stages, starting with antennal drumming, positioning on the host egg, insertion of the ovipositor and post-oviposition marking (Bruce et al. 2021). The egg parasitoid Telenomus remus (Nixon, 1937) (Hymenoptera: Scelionidae) plays an important role in natural and/or applied biological control, offering a sustainable and effective approach to managing agricultural pest populations (Carneiro et al. 2010).

The efficiency of this microhymenopteran lies in its ability to parasitize high rates of Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) eggs, overcoming the defense mechanisms put in place by the female at the time of oviposition. These defense strategies include the production of small eggs, covered in a dense layer of protective filaments (egg hairs) (Kannan et al. 2021) and deposited in layers (Kasige et al. 2022), making the innermost eggs less accessible to parasitism. This parasitoid is native to Malaysia and Papua New Guinea but has been used in Central and South American countries, with reports from African and Asian continents for controlling species of the genus Spodoptera (Shen et al. 2023).

Although the performance of T. remus as an applied biological control agent is well documented (Pomari et al. 2013, Chen et al. 2023, Shen et al. 2023), its effectiveness in controlling target pest species can be improved by understanding its specific oviposition strategies. The few studies addressing this issue refer to the effect of host egg age on T. remus preference and development (Peñaflor et al. 2012), as well as the impacts of T. remus female age on parasitism capacity, development time, offspring fitness and oviposition behavior (Chen et al. 2023).

Behavioral studies also gain importance when associated with morphological analyses of insects, especially sensory organs (Hansson & Stensmyr 2011). Relationships between the size and shape of these organs can help us understand the behavior of the species, which can influence, in the case of augmentative and/or inundative biological control, greater care in the quality control of the insects produced (Roux et al. 2005, Romani et al. 2010). The antennae are the main organs that make up the primary receptor structures and can vary greatly in terms of shape and size and, to a lesser extent, the type and arrangement of the sensilla (Symonds & Elgar 2013).

Continued research into T. remus and its behavior is essential to optimize its application in biological control, since integrated management strategies, which combine the controlled release of the parasitoid associated with other agricultural practices, can be optimized with detailed knowledge of the oviposition behavior of the parasitoids. In this study, we describe the oviposition behavior of T. remus on S. frugiperda eggs. To do this, we analyzed the wasp’s movement to the egg mass, as well as its behavior before, during and after oviposition. In addition, we analyzed temporal variables regarding the movement behavior and the time the parasitoid spent on the egg mass, as well as the frequency with which the host eggs were parasitized. Finally, we analyzed the influence of female antenna size on the behavioral parameters studied.

MATERIALS AND METHODS

Rearing the host Spodoptera frugiperda

The laboratory population used in the bioassays was established from individuals of S. frugiperda collected in 2022 in a non-Bt corn field located in Mogi Mirim, SP, Brazil (22°28ʹ3ʹS and 46°54ʹ21ʹW). To maintain the population under controlled conditions (25±1°C; 70±10% RH; photophase: 14 h), the larvae were kept on a natural diet consisting of leaves of hybrid corn P2501 (Pioneer Sementes Ltda., Santa Cruz do Sul, RS, Brazil). The leaves were placed in bioassay plates with 16 wells, each 5.5 cm long × 4.0 cm deep × 3.0 cm high (Advento do Brasil Indústria e Comércio de Plásticos Ltda., São Paulo, SP, Brazil). One larvae was placed in each well and remained confined until pupation.

The pupae were placed in Gerbox® boxes (12 × 12 × 4 cm), with filter paper at the bottom to maintain humidity, in air-conditioned B.O.D. chambers (25±1°C; 70±10% RH; photophase: 14 h), and remained there until the adults emerged. After emergence, the adults were kept in cylindrical PVC cages (20 cm × 20 cm), lined internally with filter paper (oviposition substrate), closed on the upper surface with fine fabric (voile), and fed with a 10% aqueous solution of honey (p v-1). The host eggs were removed from the oviposition substrate every two days and placed in plastic containers with a natural diet until the larvae hatched.

Rearing the parasitoid Telenomus remus

The parasitoids came from the population established at Embrapa Soja (Londrina, PR, Brazil) and were reared and multiplied following the method proposed by Stecca et al. (2016) under controlled conditions (25±1°C; 70±10% RH; 14 h photophase). The parasitoids were reared in cages, with around 200 females per cage and a sex ratio of 0.8, made with glass tubes (30 × 10 cm) closed at their ends by cork plugs and covered with black fabric. At each end, a 5 cm piece was covered with black tape to make it easier to handle the insects and to provide the wasps with shelter from the light.

The parasitoids were fed a fillet of honey on pieces of aluminum foil and the females were provided S. frugiperda eggs for oviposition every day from the laboratory. Approximately 1,000 eggs were provided for every 40 parasitoid females more than two days old, to ensure that the females had copulated, for an exposure period of 6 hours. Part of the parasitized eggs was used for behavioral tests, while the other part was used for rearing maintenance.

Behavioral analysis

The T. remus couples were kept for 24 hours in glass jars measuring 2.4 cm in diameter and 8.0 cm in height, containing pure honey as food. A sterile polypropylene arena (8mm diameter × 5mm height) was used to conduct this bioassay. In the central area of each arena, a mass containing 20 eggs, deposited less than 12 hours previously, was inserted. The egg masses used were not manipulated, i.e. we selected those with only one layer, making it possible to see the eggs in their entirety and filming them properly.

In order to introduce the females individually into the arenas without them escaping, it was necessary to anesthetize them in a cold room at -5°C for 1 min. After introducing the female, the arena was covered with a glass coverslip. From then on, all the female’s movements were filmed for 30 min using a Stereomicroscope (Stereo Discovery V20 Zeiss), with a 20x magnification lens.

The footage files were named with the code of the individuals and analyzed in a free multimedia player VLC media player (free software) at different playback speeds to describe the oviposition behavior of T. remus on S. frugiperda eggs, including pre-oviposition, oviposition and post-oviposition behavior. The duration of these events was obtained as follows: (1) pre-oviposition, from the moment the female positioned herself on the chosen egg until the ovipositor was inserted; (2) oviposition, from the insertion of the ovipositor until its removal; (3) post-oviposition, from the removal of the ovipositor until the female abandoned the host egg. The time between ovipositions was calculated from the time the ovipositor was removed from the first egg until it was inserted into the next. By analyzing the videos, we described the sequence in which the eggs were parasitized, taking into account the shape of the mass, as well as the number of times the females repeated eggs that had already been parasitized.

The videos obtained were also analyzed using the automated computer tracking software EthoVision® XT (Noldus Information Technology Corp., Wageningen, Netherlands), to obtain the speed and distance of the wasp walking both in the arena and on the egg mass, as well as the time the female spent on the egg mass.

Morphometric analysis

After the videos were taken, the females were fixed in 70% alcohol, and then the antennae were measured using a 20x magnifying glass to analyze the influence of their length on the behavioral parameters studied. The insects were divided into three groups according to the size of their antennae: (1) Antennae ≤ 0.380 mm; (2) Antennae > 0.380 - < 0.400 mm; (3) Antennae ≥ 0.400mm.

Statistical analysis

A generalized linear model (GLM) (Nelder & Wedderburn 1972) with a Gaussian distribution was used to analyse average walking speed, total distance travelled, dwelling time, pre-oviposition time, post-oviposition time and time between ovipositions. Similarly, oviposition time data was also analyzed using GLM with a quasi-Poisson distribution and the number of parasitized eggs with a Poisson distribution.

For all variables, the quality of fit of the data to the models was assessed using the half-normal probability plot with simulation envelope, using the “hnp” package (Moral et al. 2017). In the event of significant differences between treatments, multiple comparisons with Tukey’s post hoc test (p < 0.05) were carried out using the “glht” function of the “multcomp” package, with p-value adjustments (Hothorn et al. 2008). All analyses were carried out using the “R” statistical software, version 4.2.2 (R Core Team 2024).

RESULTS

Oviposition behavior

After being released into the arena, the T. remus female walked around the space, drumming the substrate with its antennae. When it encountered the S. frugiperda egg mass, the drumming process changed pace and became subtly slower (Figure 1a). Two of the females (11.8%) oviposited in the first egg they came into contact with, one of which touched the egg, left the area and, after walking around the arena, returned and oviposited in the same egg.

Figure 1
Stages in the oviposition behavior of Telenomus remus on Spodoptera frugiperda eggs. a - Antennal drumming. b - Pre-oviposition positioning. c - Ovipositor insertion. d - Post-oviposition marking.

When they found the egg mass, the females showed well-defined pre-oviposition, oviposition and post-oviposition behaviors:

(1) Pre-oviposition: the female drummed the entire surface of the egg to be parasitized with her antennae (Figure 1a). The female then moved to the neighboring egg, positioned herself with her back to the egg to be parasitized and placed her hind legs on it. At this point, the female remained motionless (Figure 1b). The average pre-oviposition time was 29.6 s, ranging from 20.6 to 40.3 s (Table I);

Table I
Number of parasitized eggs and duration of each stage in the oviposition behavior of Telenomus remus on Spodoptera frugiperda egg masses, during 30 minutes of observation.

Female	Parasitized eggs	Time (s)
		Pre-oviposition	Oviposition	Post-oviposition	Between ovipositions
1	9	20.56	33.11	11.89	54.17
2	17	22.53	46.00	11.00	47.93
3	19	29.37	40.32	10.32	43.50
4	13	25.85	41.38	13.08	44.67
5	11	35.00	47.27	9.45	54.71
6	18	25.78	28.50	9.89	44.87
7	17	30.35	46.29	10.88	42.69
8	16	27.31	41.13	9.50	45.00
9	14	25.86	50.57	11.07	41.40
10	17	28.12	39.35	15.35	45.50
11	19	27.63	36.16	12.47	48.94
12	15	40.27	39.00	11.53	62.57
13	15	39.27	34.57	13.50	62.67
14	17	30.76	40.29	13.13	49.25
15	13	28.69	35.23	9.92	40.80
16	15	32.33	40.47	8.87	44.55
17	12	33.58	37.17	11.25	57.89
Mean	15.1	29.60	39.81	11.36	48.89
SE	0.68	1.28	1.35	0.42	1.70

(2) Oviposition: the female contracted her abdomen and inserted the ovipositor into the upper lateral region of the host egg (Figure 1c) and remained motionless, except for subtle movements with the antennae and also back and forth movements of the head. Immediately before removing the ovipositor, the female drummed her antennae more intensely, without touching them to the substrate. The average oviposition time was 39.8 s, ranging from 28.5 to 47.3 s (Table I);

(3) Post-oviposition: immediately after removing the ovipositor, the female rubbed the end of the ovipositor around the egg puncture site, in circular and zigzag movements (Figure 1d). After this behavior, the female immediately showed three different behaviors: (1) she went in search of other eggs to oviposit (n = 210), (2) she moved to the edge of the arena (n = 6), or (3) she cleaned her body (n = 36), where “n” represents the number of times a given behavior occurred. Cleaning occurred mainly on the antennae, abdomen and wings. No host-feeding behavior was observed. The average post-oviposition time was 11.4 s, ranging from 8.9 to 15.3 s (Table I). The average time between ovipositions was 48.9 s, ranging from 40.8 to 62.7 s (Table I).

The average number of parasitized eggs was 15.1 per female over the 30-minute period, which represents 75.5% of the eggs provided (n = 20). The number of parasitized eggs per female varied between 9 and 19 S. frugiperda eggs (Table I).

The females that repeated previously parasitized eggs represented 76.5% of the total number of individuals tested, while only 23.5% did not repeat these eggs (Figures 2 and 3).

Figure 2
Movement of Telenomus remus females (n=17) (A), duration of stay (B), and position of the Spodoptera frugiperda egg mass in the arena (C). The repeated eggs are represented in gray.

Figure 3
Number of times each Telenomus remus female repeated previously parasitized Spodoptera frugiperda eggs.

The number of times each female went to the edge of the arena after the first oviposition was counted. Although the majority of females did not go to the edge after the first oviposition (58.8%), some of them did. The highest number of times a female had contact with the edge of the arena after the first oviposition was 10 times (Figure 4).

Figure 4
Number of times each Telenomus remus female went to the edge of the arena after parasitizing the first Spodoptera frugiperda egg.

Walking behavior

The average walking speed of the females in the arena was 0.116 mm.s^-1, with an average distance covered of 203.3 mm (Table II). At the extremes, we highlight female 1, with a walking speed of 0.219 mm.s^-1 and a distance covered of 393.6 mm. However, female 14 showed a speed of 0.042 mm.s^-1 and a distance covered of 74.0 mm.

Table II
Oviposition behavior parameters of Telenomus remus females on Spodoptera frugiperda egg masses, quantified by EthoVision® XT software during 30 minutes of observation.

Female	Velocity mean (mm/s)	Distance moved total (mm)	Eggs Spodoptera cumulative duration (min)
1	0.219	393.6	5.893
2	0.059	99.2	19.825
3	0.065	116.6	21.327
4	0.064	114.1	17.391
5	0.141	254.0	15.614
6	0.166	298.0	16.972
7	0.068	122.6	15.946
8	0.164	294.3	18.061
9	0.129	221.7	14.950
10	0.178	313.2	22.403
11	0.168	302.4	17.672
12	0.084	149.0	17.687
13	0.166	251.6	18.893
14	0.042	74.0	16.921
15	0.089	159.8	12.133
16	0.073	130.9	18.315
17	0.099	161.8	13.368
Mean	0.116	203.3	16.669
SE	0.013	22.8	0.918

The females remained on the egg mass for an average of 16.7 min, which represents 55.7% of the total evaluation time. The longest time spent on the egg mass was 22.4 min, while the shortest was 5.9 min (Table II). Graphically, the parameters analyzed can be seen in Figure 2.

Antenna morphometrics

Significant differences were found in the parameters of average speed (F=6.08, p=0.015) and distance walked (F=7.10, p=0.009) (Table III). For these parameters, females with an antenna length >0.400 showed significantly lower average walking speed and distance walked compared to females with an antenna length >0.380 - <0.400 (Table III). On the other hand, the antenna size range of females did not significantly affect the other parameters studied (Length of stay: F=0.25, p=0.777; Parasitized eggs: F=0.50, p=0.606; Pre-oviposition time: F=0.20, p=0.816; Oviposition time: F=0.04, p=0.957; Post-oviposition time: F=0.33, p=0.725; Time between ovipositions: F=0.33, p=0.724) (Table III).

Table III
Antenna size of females and oviposition behavior parameters of the egg parasitoid Telenomus remus on Spodoptera frugiperda egg masses, during 30 minutes of observation.

Antenna size (mm)	Average speed (cm.s -1 ) 1	Distance walked (cm) 1	Lenght of stay (min) 1	Parasitized eggs 2	Pre-oviposition time (s) 1	Oviposition time (s) 3	Post-oviposition time (s) 1	Time between ovipositions (s) 1
≤0.380	0.16 ± 0.05 a	280.82 ± 14.33 a	15.93 ± 2.74ns	13.60 ± 1.54ns	30.25 ± 3.39ns	39.97 ± 2.27ns	11.54 ± 1.07ns	52.39 ± 3.27ns
>0.380 - <0.400	0.12 ± 0.05 a	204.96 ± 34.19 a	17.01 ± 0.73	15.80 ± 0.73	30.72 ± 2.49	40.08 ± 3.96	10.84 ± 0.77	47.23 ± 3.90
≥0.400	0.06 ± 0.02 b	113.14 ± 14.32 b	17.77 ± 1.36	15.60 ± 1.33	28.42 ± 1.93	41.03 ± 1.43	11.76 ± 0.57	48.65 ± 2.53
F	6.08	7.10	0.25	0.50	0.20	0.04	0.33	0.33
p-value	0.015	0.009	0.777	0.606	0.816	0.957	0.725	0.724

• ¹Data (mean ± SE) followed by the same letter in a column do not differ significantly (GLM with Gaussian distribution, followed by Tukey post hoc test; p>0.05).
  ²Data (mean ± SE) followed by the same letter in a column do not differ significantly (GLM with Poisson distribution, followed by Tukey post hoc test; p>0.05).
  ³Data (mean ± SE) followed by the same letter in a column do not differ significantly (GLM with Quasi-Poisson distribution, followed by Tukey post hoc test; p>0.05).

DISCUSSION

In our study, the oviposition behavior of T. remus was divided into four stages, even though some previous studies with species of the same genus or family show a fifth stage (Bruce et al. 2021). This additional phase is described as “resting” by several authors and occurs shortly after the female marks the egg (Wiedemann et al. 2003). Here, we prioritize behavioral aspects from the time the female arrives at the laying site until she marks the egg, which we call post-oviposition.

In general, host habitat recognition is a well-known aspect among parasitoid insects, especially within the Platygastroidea (Austin et al. 2005). Females are attracted by numerous chemical cues provided by the host, the plant species of the target pest or even a combination of the two factors (Greenberg et al. 2023). Walking in the area provides the females with information about the egg mass, causing the insect to remain in the area or abandon it (Austin et al. 2005). Parasitoid females need to provide their offspring with a minimally suitable host for the embryo to reach the adult stage (Varshney et al. 2022) and this may explain, in hypothesis, their great selectivity at this stage of oviposition behavior (Bruce et al. 2021, Chen et al. 2023).

The antennae are the main olfactory organs of parasitoids and each species has a certain number of sensilla capable of identifying different compounds (Zhang et al. 2015). According to Chen et al. (2013), T. remus has 11 types of sensilla on its antennae, which is more than the other species reported by Zhang et al. (2015). Antennal drumming occurs to evaluate the area and is a determining factor in choosing the host egg (Cave & Gaylor 1987). This is even more evident in T. remus, as the females of this species do not parasitize any eggs before performing the inspection with their antennae. Although the antennae are fundamental and specialized organs for odor recognition, they may not be able to perceive certain information. This is why the tarsal, abdominal, and ovipositor sensilla are also important in this process. (Greany et al. 1977, Bruce et al. 2021).

In our evaluations, the insertion of the ovipositor was observed in the upper lateral region of the host egg. However, it should be noted that our experimental design consisted of a single-layered egg mass. As this study is pioneering in its evaluation of specific parameters, we recognize that, for proper execution and the establishment of a baseline, the egg mass should not exhibit layers and should maintain an average scale pattern, even though these parameters may differ in the field (Li et al. 2023).

The ovipositor of some Telenomus species contains sensory structures capable of detecting the quality of the host egg (Bruce et al. 2021, Wikantyoso & Putra 2023). This may explain why when the female returns to a previously parasitized egg and inserts the ovipositor, the time taken for the “oviposition” process is drastically reduced. What probably happens in this case is that she realizes that the egg has already been parasitized and, often, this is not perceived by the antennae (Larocca et al. 2007).

While the ovipositor of T. remus is inserted into the host egg, the insect remains motionless. This behavior, as well as the pumping movement with the head, has been described in some species of Scelionidae (Bruce et al. 2021). Many species depend partially or totally on changes in hydrostatic pressure in the body cavity to carry out the extension and retraction of the ovipositor, and egg release occurs during this rocking movement (Field 1998).

Marking the parasitized egg is a stage that demonstrates successful oviposition (Rosi et al. 2001). The female of T. remus rubs the ovipositor over the egg, suggesting the deposition of a marking pheromone (Carneiro & Fernandes 2012). Many scelionids present chemical clues to facilitate the recognition of previously parasitized eggs, such as Trissolcus basalis (Wollaston, 1858) (Hymenoptera: Scelionidae), which releases a lipid substance produced by Dufour’s gland (Rosi et al. 2001). Traces of hydrocarbons left by the female previously can also be an indication of the status of a particular egg (Darrouzet et al. 2010).

It is important to note that in field conditions, T. remus females probably do not suffer from a shortage of host eggs, as they prefer to lay Spodoptera species, which normally lay a large number of eggs en masse. In this case, marking with pheromones would not be necessary (Gerling & Schwartz 1974).

The number of parasitized eggs is a fundamental parameter to be studied in natural enemies, as it can be decisive in concluding that a species is a good biological control agent. In this study, the average number of parasitized eggs per female during 30 min of exposure was 15.1. This represents 75.5% of the eggs provided to the females in the bioassay. According to Pomari et al. (2013), T. remus can achieve 80-100% parasitism in mass releases in field crops. Therefore, we believe that if the evaluation time were a little longer, the females would probably parasitize all the host eggs. However, our results are in line with other authors who highlight high parasitism rates of T. remus on S. frugiperda eggs (Kenis et al. 2019, Veena et al. 2023).

With regard to the duration of the stages of oviposition behavior, it is necessary to define the start and end time of each stage objectively so that possible comparisons can be made fairly. In oviposition behavior, the time of each stage is a very variable parameter, even within the same species. Even so, it is possible to find a pattern. The time can vary depending on numerous factors, such as the age and thickness of the host egg, which can make it difficult for the ovipositor to penetrate the chorion, as well as the ambient temperature (Becchi et al. 2020).

The average pre-oviposition time for T. remus was 29.6 s, similar to the results for Cleruchoides noackae (Lin & Huber) (Hymenoptera: Mymaridae), which has an average time of 35 s (Haas et al. 2018). Some studies with scelionids have only evaluated the time taken for the antennae to drum (Wiedemann et al. 2003), while in this study we timed the time from the female’s choice of host egg to the insertion of the ovipositor.

The average oviposition time for T. remus was 39.8 s, similar to that obtained for Telenomus heliothidis (Ashmead, 1893) (Hymenoptera: Scelionidae), which varied between 31.8 and 37.1 s (Strand & Vinson 1983), and for Eumicrosoma blissae (Maki, 1937) (Hymenoptera: Scelionidae) with an average duration of 42 s for this phase (Sadoyama 1998). Although this may indicate that the duration of this period is common for scelionids, other previous studies have shown much longer times in relation to this parameter. For example, for Telenomus triptus (Nixon, 1937) (Hymenoptera: Scelionidae) the average oviposition time was 3 min (Higuchi & Suzuki 1996) and for Gryon gallardoi (Brèthes, 1914) (Hymenoptera: Scelionidae) it was 3.9 min (Wiedemann et al. 2003). On the contrary, for Telenomus busseolae (Gahan, 1922) and Telenomus isis (Polaszek, 1993) (Hymenoptera: Scelionidae) the oviposition time was 138 and 201 s, respectively (Agboka et al. 2002).

According to other studies, it is possible to infer that the time taken for each stage of oviposition behavior is a parameter that needs to be analyzed for each parasitoid species on each host pest species. Even so, variations can occur according to the sexual condition of the female (virgin or mated), the age of the female and the eggs of the host (Peñaflor et al. 2012, Becchi et al. 2020).

The average post-oviposition or marking time for T. remus was 11.4 s, a value very close to that obtained for T. heliothidis, which varied between 7.2 and 8.1 s (Strand & Vinson 1983). However, it differed from that of E. blissae, which had an average duration of 34 s (Sadoyama 1998). The duration of marking by the female is highly variable, as it also depends on the success of the oviposition period. If this stage has not been carried out properly, the female may simply abandon the egg without marking it (Larocca et al. 2007, Bruce et al. 2021).

The time between ovipositions makes it possible to better understand the behavior of a species, establishing patterns that can be used in future tests. Our results show that the average time between ovipositions was only 9 s longer than the average oviposition time, so that the time the female spends in the interval between two ovipositions is almost equivalent to the time of one oviposition.

Our results also showed that 76% of the females returned to eggs that they themselves had oviposited, even though in this case the oviposition time was drastically reduced. This suggests that at some point the female realizes the situation of the host egg and removes her ovipositor (Gerling & Schwartz 1974). It is worth noting that in this experiment the females were given a limit of 20 eggs and this may have influenced this parameter (Cave 2000).

According to Gerling & Schwartz (1974), a possible explanation for this lies in the evolutionary adaptation of the species. In general, scelionids have marking pheromones, which help them to use resources efficiently. However, T. remus may have lost or reduced its ability to produce pheromones, as the number of host eggs is usually abundantly available. Additionally, the authors do not rule out the possibility of a simple cleaning movement of the ovipositor after it has been removed from the host.

With regard to the number of times the female went to the edge of the arena, we believe that this is essential to indicate the insect’s interest in the egg mass. The number of times was counted immediately after the first oviposition, and our results showed that 58.8% of the females did not go to the edge, and only 11.8% of them went more than twice. This may indicate that T. remus females in the field, after the first oviposition, tend to remain in the egg mass and consequently continue parasitizing. This behavior makes the species even more relevant for use as a biological pest control agent.

The three behavioral parameters that we analyzed using automated computer tracking (EthoVision® XT system) are interconnected. The higher the average walking speed, the greater the distance covered and the shorter the time the female spends on the egg mass. This relationship can be observed, for example, in female 1, but cannot be treated as a rule. This female differed the most from the others in all parameters. As this is a behavioral study, this case is considered normal and treated as intraspecific variation (Costi et al. 2020).

The average walking speed of T. remus females is considered an essential parameter for them to find their host. The higher the speed, the more agile and capable the female. On the contrary, the lower the walking speed in oviposition behavior, the greater the probability of success in choosing the host (Boyle et al. 2020). Speed can also be influenced by the surface on which the hosts are located and the ambient temperature (Manzano et al. 2002).

The average distance traveled by T. remus females is also a parameter that shows the insect’s ability to move in relation to the host’s resources. Although the result presented by female 1 that the longest distance showed the shortest time spent on the egg mass is true, this cannot be considered normal for the species. When we look at female 10, which covered an above-average distance, we notice that she had the longest time on the egg mass. The relationship between these two parameters needs to be made with caution, as the female can travel great distances over the host eggs and, in this case, there would not be an inversely proportional relationship.

The length of time the T. remus female remains on the egg mass is an important parameter, as it indicates its interest in the host. From this, we can infer the behavior of the female of this species in the field, although there are other factors to consider. However, a longer time on the host does not always result in a greater number of parasitized eggs, as the female may be on the site but take longer to oviposit. This was observed with females 11 and 12, who spent 17 minutes on the eggs; however, the former parasitized four more eggs than the latter.

In this study we also reported, for the first time, that the size of the antennae of T. remus females influences the behavioral parameters of the oviposition process. The walking speed and distance covered by the females were affected according to the size of the antennae without, however, affecting parasitism efficiency. In any case, future studies could focus on more detailed analyses of the morphology of parasitoid antennae and how this finding might influence other behavioral parameters, in addition to the possibility of affecting parasitism efficiency.

By carefully analyzing each parameter related to the oviposition behavior of T. remus, we provide relevant information about this species, which stands out for its remarkable potential in the biological control of Lepidoptera, especially S. frugiperda. It should be noted that our study presents the most comprehensive compilation of data on the oviposition behavior of T. remus to date, consolidating it as a valuable contribution to the understanding and effective use of this species of egg parasitoid in applied biological control programs.

ACKNOWLEDGMENTS

The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the grants to the first and last authors. They would also like to thank the Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC, grant EPA2022601000016) for financial support.

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Publication Dates

• Publication in this collection
  07 Oct 2024
• Date of issue
  2024

History

• Received
  17 June 2024
• Accepted
  11 Aug 2024