Effect of microfilaments produced by eggs of Dalbulus maidis (Hemiptera: Cicadellidae), against egg parasitoids

Abstract

Insect eggs, once oviposited, defend against abiotic and biotic factors using thickness, secretions, or other defenses. The eggs of most insect species are attacked by parasitoid wasps, which are often their most significant mortality factors. The present study is the first investigation of microfilaments as defense from parasitoids. Specifically, the effect of the corn leafhopper Dalbulus maidis DeLong (Hemiptera: Cicadellidae) egg microfilaments against parasitism by Anagrus virlai Triapitsyn (Hymenoptera: Mymaridae) and Paracentrobia subflava (Girault) (Hymenoptera: Trichogrammatidae) was evaluated. Field and laboratory experiments were conducted to evaluate several biological traits related to parasitism in D. maidis eggs with and without microfilaments. An initial field experiment found no difference in parasitism by A. virlai and P. subflava of eggs with vs. without microfilaments after 5 days of exposure of healthy D. maidis eggs to parasitism. A second field experiment then looked at exposure for 1 day, and this treatment found greater parasitism of healthy D. maidis eggs by each parasitoid species in eggs without microfilaments vs. in eggs with microfilaments. Laboratory experiments conducted separately for A. virlai and P. subflava parasitism after 1 day of exposure to healthy D. maidis eggs confirmed a higher percentage of parasitoid emergence in the eggs without microfilaments (both young and mature eggs) vs. mature eggs with microfilaments. These results suggest that eggs without microfilaments are more parasitized than eggs with microfilaments during a 1-day exposure, but that this difference disappears as egg microfilaments regrow over a 5-day period.

Resumen

Los huevos de insectos, una vez puestos, se defienden contra factores abióticos y bióticos usando su dureza, secreciones, u otras defensas. Los huevos de muchas especies de insectos son atacados por avispas parasitoides, las cuáles son un factor de mortalidad. Este estudio es el primero que investiga a los microfilamentos como defensa de parasitoides. Específicamente, el efecto de los microfilamentos producidos por los huevos de Dalbulus maidis DeLong (Hemiptera: Cicadellidae) contra el parasitismo por Anagrus virlai Triapitsyn (Hymenoptera: Mymaridae) y Paracentrobia subflava (Girault) (Hymenoptera: Trichogrammatidae) fue evaluado. Experimentos de campo y laboratorio fueron efectuados para evaluar patrones biológicos relacionados con el parasitismo en huevos de D. maidis con y sin microfilamentos. En un primer experimento efectuado en el campo no se encontraron diferencias en el parasitismo por A . virlai y P. subflava entre huevos con y sin microfilamentos cuando los huevos de D. maidis fueron expuestos a parasitismo por cinco días. En un segundo experimento efectuado en campo, los huevos fueron expuestos a parasitismo por un día, y fue encontrado mayor parasitismo por estas dos especies de parasitoides en huevos sin microfilamentos que en huevos con microfilamentos. Experimentos en laboratorio efectuados separadamente para A. virlai y P . subflava en un día de exposición a huevos de D. maidis confirmaron un mayor porcentaje de parasitoides emergidos en los huevos sin microfilamentos (ambos huevos jóvenes y maduros) que en huevos maduros con microfilamentos. Estos resultados sugieren que los huevos sin microfilamentos son más parasitados que los huevos con microfilamentos durante un día de exposición, pero estas diferencias desaparecen conforme los microfilamentos se regeneran durante un periodo de cinco días.

Graphical Abstract

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Introduction

Once laid, insect eggs are exposed to abiotic and biotic factors in the environment (Hilker et al. 2023), including exposure to attack by predators and parasitoids (Hawkins et al. 1997). When insects are attacked by idiobiont Hymenoptera parasitoids, the parasitoid needs to develop within the egg, a nongrowing stage (Jervis et al. 2008, Mills 2009). Insect eggs defend against predators and parasitoids through a number of mechanisms, including thickness of the egg chorion, egg secretions, parental care, and the formation of ootheca (Gross 1993, Fatouros et al. 2020). The thickness of the chorion has been shown to affect vulnerability to parasitoids (Gross 1993), and egg secretions produced by the mother also afford protection (Fatouros et al. 2020). Many ovipositing females provide a protective cover, such as scales or silk (Gross 1993). In other cases, females incorporate defensive compounds into their oocytes (Hilker et al. 2023). Also, eggs express immune response when attacked by parasitoids via cellular encapsulation of the egg parasitoid (Reed et al. 2007). Heady and Nault reported in 1984 that leafhopper (Hemiptera: Cicadellidae) eggs produce microfilaments from the anterior ends of the eggs. Microfilaments were found in Dalbulus genus and another 3 Deltocephalinae genera. Each filamentous tuft is composed of about 100 microfilaments. Filaments are solid, white, hydrophobic, and composed of waxes. Individual microfilaments are about 2 µm in diameter and can reach 200 µm in length. They proposed that these microfilaments provide protection from parasitoids, but did not experimentally verify this possibility.

The corn leafhopper (Dalbulus maidis DeLong) is among the species shown to produce microfilaments (Heady and Nault 1984). It is a phloem-feeding herbivore specialist on maize and its relative, teosintes (Nault and DeLong 1980, Nault 1990), and is one of the most important pests on maize throughout the American continent (Nault 1990). Nymphs and adults of D. maidis transmit plant pathogens including corn stunt spiroplasma, maize bushy stunt phytoplasma, and maize rayado fino virus (Nault 1980).

Most eggs (about 75%) are laid in clusters by the corn leafhopper on the midrib of the maize leaves (Heady et al. 1985, Moya-Raygoza 2016). Dalbulus maidis leafhoppers insert the eggs singly into plant tissue with the ovipositor (Nault 1998), and the duration of the egg stage is about 14 days at 25°C (Van Nieuwenhove et al. 2016). Eggs of the corn leafhopper produce microfilaments 72 h after oviposition on the maize leaves, and when microfilaments were removed with a paint brush 72 h after oviposition, they reformed within 24 h (Heady and Nault 1984). Heady and Nault (1984) suggest that a possible function of the microfilaments could be for defense against egg parasitoids or predators. However, no study has been performed to test the function of these microfilaments produced by D. maidis eggs (Nault 2023).

Eggs of D. maidis are parasitized by a community of egg parasitoids; however, Anagrus virlai Triapitsyn (Hymenoptera: Mymaridae) and Paracentrobia subflava (Girault) (Hymenoptera: Trichogrammatidae) are the most common and abundant parasitoids in maize fields (Moya-Raygoza 2019). These 2 species are biological control agents and have been reported throughout Tropical America, attacking eggs of D. maidis in South America (Oliveira and Spotti Lopez 2000, Virla et al. 2013), Central America (Gladstone et al. 1994), and Mexico (Virla et al. 2009, Moya-Raygoza et al. 2012, 2014, Torres-Moreno and Moya-Raygoza 2020, 2021, Moya-Raygoza and Figueroa-Bautista 2021). Anagrus virlai is a generalist parasitoid of Deltocephalinae leafhoppers and Delpacidae (Hill et al. 2023) and P. subflava is a generalist parasitoid that attack eggs of Dalbulus genus (Torres-Moreno et al. 2021). Paracentrobia subflaba disperse through small jumps, walk and short flights, whereas A. virlai have better dispersion than P. subflava since they can fly long distances (Torres-Moreno and Moya-Raygoza 2021). Anagrus virlai developed in D. maidis eggs from 1, 3, and 5 days old (Hill et al. 2019), whereas host age preference has not been investigated for P. subfalva.

The objective of the present study was to determine the parasitism of D. maidis eggs with and without removed microfilaments in field and laboratory conditions. In the field, the abundance of adult parasitoids emerged, the percentage of parasitism, and the percentage of emergence of egg parasitoids were evaluated. In the laboratory, when adult parasitoids emerged, the percentage of emergence was evaluated separately for A. virlai and P. subflava in young D. maidis eggs (without microfilaments) and mature D. maidis eggs (with and without microfilaments). To the best of my knowledge, this is the first study to investigate the role of microfilaments produced by the eggs of an insect host parasitized by hymenopterans.

Materials and Methods

Field Experiments

Two experiments were conducted under natural field conditions during the maize-growing wet season of 2023, in the same maize field. The first experiment was performed in August and September, and the second experiment was performed in November. Both experiments were conducted in the Zapopan region, in Jalisco State, Mexico. The experimental region was located at 20° 44ʹ 40.6″ N, 103° 30ʹ 57.7ʹ W, 1,663 m elevation.

In the first experiment, reared D. maidis adults collected from the Zapopan region were allowed to oviposit on maize plants to attract egg parasitoids (sentinel maize plants). For oviposition, D. maidis adults (2 wks old) were confined in a single-leaf cage (size 4.0 by 5.5 by 2.0 cm) attached to a leaf of a potted maize plant of the landrace ‘maize ancho’. Each potted maize plant was at the 5-leaf stage at the moment of D. maidis oviposition. D. maidis adults were allowed to oviposit in a rearing room at 25 ± 2°C, 50% relative humidity, and a photoperiod of 12:12 (L:D) h for 72 h. After 72 h, D. maidis females were removed.

Samples were collected 3 times during each month. In August, 7–8 D. maidis adults were confined in each single-leaf cage, and a total of 24 potted maize plants with microfilaments and 24 potted maize plants without microfilaments were exposed to attract egg parasitoids. In September, 8–9 D. maidis adults were confined in each single-leaf cage, and a total of 30 potted maize plants with leaves with microfilaments and 30 potted maize plants with leaves without microfilaments were exposed to egg parasitoids. After the D. maidis adults were removed from the August and September maize plants, the plants with the D. maidis eggs were divided into 2 groups: one with microfilaments and one without. In the ‘without microfilaments’ group, the microfilaments of the D. maidis eggs were mechanically removed with a paint brush, as done by Heady and Nault (1984). In the other group, the microfilaments remained on the leaf.

Pots with sentinel eggs (with and without microfilaments) were transported immediately to the maize field in the Zapopan region. The pots were placed about 2 m from the edge of the maize crop along a transect, with a distance of 5 m between each pot. Two days after pots were placed in the field, microfilaments were removed again with a paint brush in the ‘without microfilaments’ treatment. Sentinel maize plants with and without microfilaments were left in the maize field for 5 days to attract parasitoids.

After 5 days, the sentinel plants were returned to the laboratory, where the plants were kept in the controlled conditions described above. Three or 4 days later, the total number of parasitized and nonparasitized D. maidis eggs were counted using a stereomicroscope (Stemi DV4; Carl Zeiss, Germany). Parasitized eggs show a red or black color a few days after being parasitized. Each maize leaf with D. maidis eggs was placed in a Petri dish with wet absorbent paper at the bottom to prevent drying out of the maize leaf. The Petri dishes were sealed with transparent plastic and placed in the controlled conditions described above. Petri dishes were examined every other day for 40 days. Adult parasitoids that emerged were used for laboratory experiments, and some adults were placed in 95% ethanol for subsequent identification. In this experiment, the number of D. maidis eggs exposed to parasitism, the percentage of parasitism, the number of adult parasitoids that emerged, and the percentage of emergence were quantified. To obtain the percentage of parasitism, the total number of eggs with coloration from each maize leaf was divided by the number of eggs laid on each leaf, multiplied by 100. Also, to obtain the percentage of emergence, the total number of adult parasitoids that emerged from each maize leaf was divided by the number of eggs laid on each leaf, multiplied by 100.

In the second experiment, conducted in November, sentinel maize plants with and without microfilaments were used to attract egg parasitoids. The same protocol was performed as in the first experiment, but in this second experiment, the oviposition period for D. maidis adults was 48 h. After the oviposition period, D. maidis adults were removed, and maize plants remained in the laboratory rearing room for 4 days. After these 4 days, microfilaments were removed from the ‘without filaments’ potted maize plants, while the other potted maize plants were allowed to retain the microfilaments. Maize plants with and without microfilaments were transported to the maize field in the Zapopan region to be exposed to natural parasitism for only 24 h. After 24 h, the sentinel plants were returned to the laboratory to keep the plants in the controlled conditions mentioned above, and 3 or 4 days later, the total number of D. maidis eggs for each leaf was counted. Each maize leaf with D. maidis eggs was then placed in a Petri dish with wet absorbent paper at the bottom to prevent drying out of the maize leaf. The Petri dishes were sealed with transparent plastic and placed in the controlled conditions described above. Petri dishes were examined every other day for 40 days. Samples were collected 4 times. In total, 27 maize pots containing leaves with microfilaments and 27 maize pots containing leaves without microfilaments were included in this experiment. Adult parasitoids that emerged were used for laboratory experiments, and some were placed in 95% ethanol for subsequent identification. In this experiment, the number of D. maidis eggs exposed to parasitism, the number of adult parasitoids that emerged, the number of individuals of each species, the number of females and males of each species, and the percentage of emergence were quantified. The percentage of parasitism was not obtained because nymphs of D. maidis emerged before the coloration appeared in parasitized eggs. The percentage of emergence was obtained as in the first experiment.

Identification of the parasitoids that emerged in the first and second experiments was based on Triapitsyn et al. (2019) for Anagrus sp. and Torres-Moreno et al. (2022) for Paracentrobia sp.

Laboratory Experiments

Laboratory-controlled experiments were performed with living A. virlai and P. subflava adult parasitoids found on sentinel maize plants placed in the Zapopan region during 2022 and 2023. Before starting the laboratory experiments, the conditions of the maize leaf after oviposition by D. maidis were described. D. maidis females caused damage during a 48 h oviposition period, during which eggs were laid on the maize leaf and microfilaments were not yet formed (Fig. 1A), and after 6 days of oviposition, the D. maidis eggs showed microfilaments (Fig. 1B).

Parasitism of young eggs.

For the experiment looking at young eggs (48 h after oviposition by D. maidis; at this age, eggs do not yet produce microfilaments), reared D. maidis adults were used in controlled conditions of 25°C, 50% relative humidity, and a photoperiod of 12:12 h L:D. For oviposition, D. maidis adults (2 wks old) were confined in a single-leaf cage (size 4.0 by 5.5 by 2.0 cm) attached to a leaf of a potted maize plant of the landrace ‘maize ancho’. Each potted maize plant was at the 5-leaf stage at the moment of D. maidis oviposition. In each single-leaf cage, 7–8 D. maidis adults were confined. The oviposition period was for 48 h, and after 48 h, the females were removed. After immediately removing the confined leafhopper adults, the maize leaf with D. maidis eggs was placed in a test tube, into which one live A. virlai or P. subflava female (obtained from the field experiments) was introduced. Once the parasitoid female was placed, the test tube was sealed with cotton to avoid the parasitoid scaping. Each parasitoid was left in the test tube for 24 h under the controlled conditions described above, and then was removed. Six or 7 days after removal of the parasitoid, the total number of D. maidis eggs was counted. Each maize leaf with D. maidis eggs was placed in a Petri dish with wet absorbent paper at the bottom to prevent drying out of the maize leaf. The Petri dishes were sealed with transparent plastic and placed in the controlled conditions described above. Petri dishes were examined every other day for 40 days. For each species of parasitoid (A. virlai and P. subflava), the number of D. maidis eggs exposed, the number of parasitoids that emerged, and the percentage of emergence were quantified. In total, for A. virlai, the procedure was performed 30 times (30 leaves), and for P. subflava was performed 36 times (36 leaves).

Parasitism of mature eggs.

For the experiment looking at mature eggs (6 days after oviposition by D. maidis), the experimental controlled conditions, oviposition period, and the number of D. maidis adults confined in each single-leaf cage were the same as in the previous treatment (48 h oviposition period). After the oviposition period of 48 h, D. maidis adults were removed, and maize plants remained in the rearing room for 4 days, during which microfilaments emerged from the eggs. After 4 days, the microfilaments were mechanically removed from the maize plants with a paint brush in the ‘without microfilaments’ treatment, whereas maize plants in the ‘with microfilaments’ treatment remained unchanged. As with the young eggs experiment, to test parasitism, one maize leaf with 6-day-old D. maidis eggs was placed in a test tube, into which one live A. virlai or P. subflava female (obtained from the field experiments) was introduced. Once the parasitoid was introduced, the test tube was sealed with cotton to avoid the parasitoid escaping. The parasitoid was kept in the tube for 24 h under the controlled conditions described above and was then removed. Three or 4 days after the parasitoids were removed, and before any nymphs emerged, the total number of D. maidis eggs was counted. Each maize leaf with D. maidis eggs was placed in a Petri dish with wet absorbent paper at the bottom to prevent drying out of the maize leaf. The Petri dishes were sealed with transparent plastic and placed in the controlled conditions described above. Petri dishes were examined every other day for 40 days. For each species of parasitoid (A. virlai and P. subflava), the number of D. maidis eggs exposed, the number of parasitoids that emerged, and the percentage of emergence were quantified. In total, for A. virlai, the procedure was performed 30 times (30 leaves), and for P. subflava, it was performed 38 times (38 leaves). The percentage of parasitism was not obtained in the laboratory experiments because nymphs of D. maidis emerged before coloration in the parasitized eggs.

Statistical Analysis

The number of D. maidis eggs, the number of adult parasitoids that emerged, the percentage of parasitism, and the percentage of emergence among treatments were compared with the generalized linear model (GLM; family = Poisson). For all pairwise comparisons, the estimated marginal means were calculated. The abundance between the two emerged species and between females and males was compared with the Mann–Whitney U test. Statistical analysis was performed with SPSS version 22.0.

Results

Field Experiments

First experiment (August and September): bait plants kept 5 days in the field.

The oviposited maize bait plants used for attraction of parasitoids had different numbers of D. maidis eggs, with more eggs available in September than in August (Wald chi square test: X² = 42.88, df = 3, P = 0.0001). In August, the mean number of eggs with microfilaments per maize leaf was 14.50 (SE = 2.43), similar significantly (t = 1.83, P = 0.07) to the mean number of eggs without microfilaments, which was 9.29 (SE = 1.46). On the other hand, in September, the mean number of eggs with microfilaments per maize leaf was 57.60 (SE = 10.61), which is significantly similar (t = 0.07, P = 0.94) to the mean number of eggs without microfilaments, which was 58.56 (SE = 8.41).

A. virlai and P. sublava were the 2 species of adult parasitoids that emerged from the sentinel maize plants. The total abundance of parasitoids differed significantly between August and September (Wald chi-square test: X² = 22.59, df = 3, P = 0.0001), with a greater number of individuals in September than in August. However, no difference was found in the total number of parasitoids that emerged from eggs with vs. without microfilaments in August and in September (Fig. 2A). A similar number of A. virlai emerged from eggs without microfilaments and from eggs with microfilaments in August (Mann–Whitney U; P = 0.53) and in September (Mann–Whitney U; P = 0.15). The same result was found for P. subflava; no significant difference was found in the number of adults emerged from eggs without and with microfilaments in August (Mann–Whitney U; P = 0.35) and in September (Mann–Whitney U; P = 0.92).

The total percentage of parasitism differed significantly by month (Wald chi-square test: X² = 21.98, df = 3, P = 0.0001), with a greater percentage of parasitism in September than in August. No difference was found in the percentage of parasitism in eggs with and without microfilaments in August and in September (Fig. 2B).

The total percentage of the emergence of parasitoids differed significantly by month (Wald chi-square test: X² = 10.85, df = 3, P = 0.013), with a greater percentage of emergence in September than in August. Also, no difference was found in the percentage of emergence from eggs with and without microfilaments in August and September (Fig. 2C).

Second experiment (November): bait plants kept 1 day in the field.

The oviposited maize bait plants used for parasitoid attraction had similar numbers of D. maidis eggs (Wald chi-square test: X² = 0.42, df = 1, P = 0. 51). The average number of eggs with microfilaments per maize leaf was 52.00 (SE = 5.46) and the average number of eggs without microfilaments was 47.18 (SE = 4.88).

A. virlai and P. sublava were the 2 species of adult parasitoids that emerged from the sentinel maize plants. The total abundance of parasitoids that emerged differed significantly between the eggs with vs. without microfilaments (Wald chi square test: X² = 7.06, df = 1, P = 0. 008), with a greater number of individuals of both species in eggs without microfilaments (Fig. 3A). Although more A. virlai emerged from eggs without microfilaments than from eggs with microfilaments (Mann–Whitney U; P = 0.005), the number of emerged A. virlai males and females did not differ significantly between eggs without microfilaments (Mann–Whitney U; P = 0.84) and eggs with microfilaments (Mann–Whitney U; P = 0.55). On the other hand, a similar number of P. subflava emerged from eggs with and without microfilaments (Mann–Whitney U; P = 0.09). Furthermore, for P. subflava, no difference occurred in the number of females and males emerging from eggs without microfilaments (Mann–Whitney U; P = 0.24) vs. with microfilaments (Mann–Whitney U; P = 97).

The total percentage of emergence of adult parasitoids differed significantly on the basis of the presence of microfilaments (Wald chi-square test: X² = 6.38, df = 1, P = 0.012), with a greater percentage of emergence from eggs without microfilaments than from eggs with microfilaments (Fig. 3B).

Laboratory Experiments

Anagrus virlai.

The eggs with vs. without microfilaments on the maize bait plants used for the A. virlai parasitoids differed significantly (Wald chi-square test: X² = 16.81, df = 2, P = 0. 0001). The mean numbers of eggs per maize leaf in the 3 treatments examined were as follows: In the 48 h (without microfilaments because the eggs were too young to produce microfilaments) treatment, the mean number of eggs was 26.56 (SE = 5.28). In the 6 days and with microfilaments treatment, the mean number of eggs was 69.70 (SE = 9.11). In the 6 days and without microfilaments treatment, the mean number of eggs was 66.33 (SE = 10.34).

The abundance of emerged A. virlai parasitoids differed significantly among the treatments (Wald chi square test: X² = 6.86, df = 2, P = 0.048), with greater abundance in the treatments without microfilaments (48 h/without microfilaments and 6 days/without microfilaments) (Fig. 4A). For the treatment 48 h/without microfilaments (young eggs), no difference was seen in the female:male ratio emerged from D. maidis eggs (Mann–Whitney U; P = 0.84). The female:male ratio also did not differ significantly in the 6-day groups (6 days/with microfilaments: Mann–Whitney U; P = 0.70; 6 days/without microfilaments: Mann–Whitney U; P = 0.43).

The percentage of emergence did, however, differ significantly between the eggs with and without microfilaments (Wald chi-square test: X² = 8.34, df = 2, P = 0.015), with a greater percentage of emergence from eggs without microfilaments (48 h/without microfilaments and 6 days/without microfilaments) than from eggs with microfilaments (6 days/with microfilaments) (Fig. 4B).

Paracentrobia subflava.

The oviposited maize plants used for the P. subflava parasitoid had similar numbers of D. maidis eggs available for parasitism (Wald chi-square test: X² = 1.88, df = 2, P = 0. 39). The mean number of eggs per maize leaf in the treatment 48 h/without microfilaments was 32.75 (SE = 5.03); in the treatment 6 days/with microfilaments, 33.81 (SE = 3.99); and in the treatment 6 days/without microfilaments, 26.18 (SE = 4.86).

The abundance of P. subflava parasitoids emerged differed significantly between young eggs and mature eggs (Wald chi-square test: X² = 15.64, df = 2, P = 0.0001), with greater abundance in the 48 h/without microfilaments (young eggs) treatment than in the 6 days/without microfilaments and 6 days/with microfilaments (mature eggs) treatments (Fig. 5A). No significant differences were seen in female:male ratio in any of the groups: 48h/without microfilaments (Mann–Whitney U; P = 0.23), 6 days/ with microfilaments (Mann–Whitney U; P = 0.50), and 6 days/without microfilaments (Mann–Whitney U; P = 0.18).

The percentage of emergence, however, did differ significantly between eggs with and without microfilaments (Wald chi-square test: X² = 12.63, df = 2, P = 0.002), with a greater percentage of emergence from eggs without microfilaments; 48 h without microfilaments and 6 days without microfilaments, vs. 6 days/with microfilaments (Fig. 5B).

Discussion

The egg of an insect is vulnerable to predators and parasitoids (Hilker et al. 2023). Eggs are attacked by idiobiont hymenopteran parasitoids that develop within the nongrowing egg stage (Jervis et al. 2008), and little is known about how insect eggs respond to parasitism by hymenopterans. In the present study, field and laboratory experiments were conducted to investigate the function of microfilaments produced by D. maidis eggs parasitized by the wasp species A. virlai and P. subflava, which are biological control agents of the corn leafhopper pest.

Field Experiments

A similar level of parasitism by A. virlai and P. subflava was observed in maize plant-bearing D. maidis eggs with and without microfilaments in field conditions when healthy eggs were exposed to parasitism for 5 days. Maize sentinel plants bearing D. maidis eggs with and without microfilaments showed no difference in the abundance of adult parasitoids that emerged, the percentage of parasitism, and the percentage of emergence, after 5 days’ exposure to parasitism in the field. In other words, when egg microfilaments were removed from sentinel plants 72 h after the oviposition period, before being transported to the field, and 2 days after being placed in the field, the parasitism rate remained similar to that of sentinel maize plants bearing microfilaments of D. maidis eggs. Heady and Nault (1984) reported that microfilaments are reformed 24 h after removal. Results found in the present study suggest that the rapid reforming of microfilaments contributed to the lack of a difference in the level of parasitism between D. maidis eggs with and without microfilaments in field conditions. Likely, the rapid reforming of microfilaments by D. maidis eggs is a mechanism to avoid parasitism over a long period of exposure time.

Differences in parasitism were observed between months (August and September) during the maize-growing season. More eggs were oviposited by D. maidis on the sentinel maize plants in September than in August, and this difference in the number of available eggs probably contributed to an increase in the abundance of adult parasitoids that emerged and the percentage of parasitism by A. virlai and P. subflava in September. Moya-Raygoza (2020) found in the maize crop a strong, positive, density-dependent association between the number of D. maidis eggs and the abundance of adult egg parasitoids during the maize-growing season. In addition, a density-dependent association was found between leafhoppers and Anagrus egg parasitoids in other agroecosystems. For instance, a density-dependent association exists between Anagrus daanei Triapitsyn and the leafhopper host Erythroneura spp. in vineyard habitat in California, USA (Segoli and Rosenheim 2013).

In November, different levels of parasitism were observed in D. maidis eggs with and without microfilaments when eggs were exposed to parasitism for a short period of 1 day in the maize field. This difference was seen in eggs parasitized by both A. virlai and P. subflava. Potted maize plants bearing D. maidis eggs without microfilaments showed a higher abundance of adult parasitoids and a higher percentage of emergence than plants bearing D. maidis eggs with microfilaments. This result indicates that A. virlai and P. subflava parasitized the eggs of D. maidis to a greater degree after microfilaments were removed and before microfilaments were reformed during the first 24 h.

Laboratory Experiments

A greater percentage of emergence was shown by A. virlai and P. subflava in plants bearing D. maidis eggs without microfilaments than in those with microfilaments when mature eggs (6 days old) were exposed for a short period of time (1 day). These results suggest that the presence of microfilaments, at least over short periods of time, decreases the percentage of emergence of A. virlai and P. subflava in mature D. maidis eggs. It is likely the case that microfilaments interfere with egg parasitism at all timepoints, but that this effect is obscured in longer-term experiments because of the phenomenon of filament regrowth after 24 h. A higher rate of adult emergence is important in biological control because adult parasitoids are responsible for controlling the host population (Pizzol et al. 2012).

A higher number of A. virlai and P. subflava adults emerged from D. maidis young eggs (48 h after a D. maidis oviposition period; without microfilaments) than D. maidis mature eggs (with or without microfilaments), when eggs were exposed for 1 day to each of these 2 parasitoid species. Therefore, the emergence of adult parasitoids could be affected by the age of the host egg. Similar results have been reported for other hymenopterans that attack the eggs of Lepidoptera and Coleoptera pests, where the presence of microfilaments is not documented. For example, eggs of the lepidopteran pest Lobesia botrana Denis and Schiffermüller that were 3–4 days old were parasitized less than eggs that were 1–2 days old, by Trichogramma cacoeciae Marchal (Hymenoptera: Trichogrammatidae) (Pizzol et al. 2012). Similarly, older eggs are less preferred by the egg parasitoid Trichogramma zahiri Polaszek when attacking the eggs of Dicladispa armigera (Olivier) (Coleoptera: Crysomelidae) (Bari et al. 2016). In addition, the egg parasitoid Telenomus remus Nixon (Hymenoptera: Scelionidae) showed more parasitism of 1- to 2-day-old eggs than 3-day-old eggs of Spodoptera frugiperda (J.E. Smith) (Peñaflor et al. 2012). The results in the present study also concord with the results for Mymaridae wasp egg parasitoids. For instance, the eggs of Homalodisca coagulata (Say) (Hemiptera: Cicadellidae) at 2, 3, and 4 days of age were more susceptible to parasitism by Gonatocerus spp. than eggs at 6–10 days of age (Irvin and Hoddle 2005).

Sex ratio is important for biological control using wasps since only females oviposit into pests. In the present study, the same number of females and males emerged for A. virla and P. subflava in the 3 treatments (48 h/without microfilaments, 6 days/with microfilaments, and 6 days/without microfilaments) conducted under laboratory conditions. This result supports the idea that the age of the parasitized egg and the presence or absence of microfilaments do not affect the sex ratio of the emerged parasitoids. A similar sex ratio has been reported in other studies when egg parasitoids developed in egg hosts of different ages (Godin and Boivin 2000, Zhou et al. 2014). The present study is the first report that the absence of microfilaments in mature eggs (6 days/without microfilaments) of D. maidis did not affect the sex ratio of emerged parasitoids. Previously, Heady and Nault (1984) reported that when microfilaments are removed from D. maidis eggs, egg hatching is not affected.

In conclusion, the present results show that, in field conditions, microfilaments can be reformed after removal, reducing the danger of being parasitized by the egg parasitoids A. virlai and P. subflava. However, greater parasitism was observed in field conditions in D. maidis eggs without microfilaments when exposed to parasitism for a short period of time (24 h), before the microfilaments were reformed. Laboratory experiments employing 24 h exposure to A. virlai and P. subflava separately confirmed a higher adult emergence in the treatments in which microfilaments were absent (48 h/without microfilaments and 6 days/without microfilaments) than in the treatment in which microfilaments were present (6/with microfilaments). Utilizing both young and mature D. maidis eggs without microfilaments could ensure better adult emergence of A. virlai and P. subflava in maize crops. A high rate of adult parasitoid emergence is important in biological control because adults are responsible for controlling the host population of the insect pest. Considering the implications of the present study, a method for removing or inhibiting filament formation could be beneficial to biological control efforts based on egg parasitoids like A. virlai and P. subflava.

Acknowledgments

Thanks to Dra. Claudia S. Copeland (Carpe Diem Biomedical Writing and Editing) for editing the English in the final manuscript.

Author Contributions

Gustavo Moya-Raygoza (Conceptualization [Lead], Data curation [Lead], Formal analysis [Lead], Funding acquisition [Lead], Investigation [Lead], Methodology [Lead], Project administration [Lead], Resources [Lead], Software [Lead], Supervision [Lead], Validation [Lead], Visualization [Lead], Writing—original draft [Lead], Writing—review & editing [Lead])

References