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Effects of host species and host age on biological parameters of Anagrus virlai (Hymenoptera: Mymaridae), an egg parasitoid of Dalbulus maidis (Hemiptera: Cicadellidae) and Peregrinus maidis (Hemiptera: Delphacidae)
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Biological Control journal homepage: www.elsevier.com/locate/ybcon
Effects of host species and host age on biological parameters of Anagrus virlai (Hymenoptera: Mymaridae), an egg parasitoid of Dalbulus maidis (Hemiptera: Cicadellidae) and Peregrinus maidis (Hemiptera: Delphacidae) ⁎
Jorge G. Hilla, Erica Luft Albarracina, , Maria V. Coll Araoza, Eduardo G. Virlaa,b,1 a b
PROIMI- Biotecnología, CONICET, Av. Belgrano y Pje. Caseros (T4001MVB), San Miguel de Tucumán, Argentina Instituto de Entomología, Fundación Miguel Lillo. Miguel Lillo 251, San Miguel de Tucumán, Tucumán, Argentina
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Corn leafhopper Corn planthopper Development Morphometry Parasitism Thelytokous strain
Dalbulus maidis (DeLong) (Hemiptera: Cicadellidae) and Peregrinus maidis (Ashmead) (Hemiptera: Delphacidae) are important pests for maize production in America. Anagrus virlai Triapitsyn (Hymenoptera: Mymaridae) is a potential biological control agent for both hopper species. We provide information on the effects of size, age and host species on the time of development, parasitism and emergence, and morphometry of A. virlai. Additionally, we evaluate if A. virlai females can discriminate between the eggs of both host species using a Y-tube olfactometer. The wasps were reared in breeding chambers under controlled conditions, and one, three and five daysold eggs of D. maidis and P. maidis were used for bioassays. Anagrus virlai developed in eggs from the three age classes of both hopper species; however, the number of parasitized eggs and emergence of wasps was significantly higher on eggs of D. maidis than on those of P. maidis. Wasps that emerged from D. maidis eggs presented larger size compared to those reared on P. maidis eggs. Host species not only influenced the parasitism, emergence, and size of the wasps, but also development time. In olfactometric tests, A. virlai chose maize leaves containing D. maidis eggs, instead of those containing P. maidis eggs. Our results show that A. virlai, formerly known as generalist parasitoid, would only use the planthopper P. maidis as an occasional host.
⁎
Corresponding author. E-mail address: [email protected] (E. Luft Albarracin). 1 ex aequo. https://doi.org/10.1016/j.biocontrol.2018.12.002 Received 5 October 2018; Received in revised form 29 November 2018; Accepted 4 December 2018 1049-9644/ © 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Hill, J.G., Biological Control, https://doi.org/10.1016/j.biocontrol.2018.12.002
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1. Introduction
development time, parasitism and emergence, and size of wasps. Additionally, we assess the influence of host age on these biological parameters. Finally, we evaluate if A. virlai females can detect specific olfactory cues associated with the offered host species using a Y-tube olfactometer.
Maize (Zea mays L.) (Poaceae) is the most prominentcrop worldwide, surpassing even rice and wheat (FAOSTAT, 2017). During the last decades, the growing demand for foods at global scale lay out the need to enhance crop yield replacing old farming practices by high inputs of pesticides and by the use of genetically modified (GM) germplasms (Castañera et al., 2016). However, resistance phenomena in maize pests are being reported denoting a latent risk for future management strategies (Gassmann et al., 2014). Much of the yield losses in annual crops are due to the action of insect pests (Pimentel, 2009). Particularly, hoppers (Hemiptera: Auchenorrhyncha) are conspicuous threats for maize crops in the world; causing damages that vary from necrosis to severe physiological alterations produced by their feeding and/or oviposition habits, and its ability to transmit pathogens (Luft Albarracin et al., 2008; Singh and Seetharama, 2008). The corn leafhopper, Dalbulus maidis (DeLong) (Cicadellidae), is a vector of three important maize pathogens in the Americas: “maize rayado fino virus” (Tymovirales: Marafivirus), “corn stunt spiroplasma” (Mollicutes: Spiroplasmataceae: Spiroplasma kunkelii Whitcomb), and “maize bushy stunt phytoplasma” (Mollicutes: Acholeplasmataceae: Candidatus Phytoplasma ateris) (Laguna and Giménez Pecci, 2012). For its part, the corn planthopper Peregrinus maidis (Ashmead) (Delphacidae) is vector, among others, of “Mal de Rio Cuarto Virus”, an important threat for corn production in Argentina (Redinbaugh and Pratt, 2009) and considered as a quarantine-disease in other countries (Oliveira et al., 2013). A large number of natural enemies of hoppers are recognized, although egg parasitoids are the best known. Species of Cicadellidae and Delphacidae families are the main hosts of Anagrus Haliday wasps (Hymenoptera: Mymaridae) (Krugner et al., 2008; Zhu et al., 2013; Segoli, 2016). Anagrus virlai Triapitsyn (Hymenoptera: Mymaridae), misidentified previously as Anagrus breviphragma Soyka or Anagrus incarnatus Haliday (Triapitsyn, 2015a; Triapitsyn et al., 2018), is the most important egg parasitoid of the corn leafhopper because of its high levels of parasitism along its distribution range (Virla et al., 2009; Luft Albarracin et al., 2017). It is considered a generalist parasitoid, that has been reported in eggs of two Delphacidae and nine Cicadellidae species (Triapitsyn, 2015b; Triapitsyn et al., 2018). Except for the corn leafhopper, the capacity of development and its incidence on the populations of other hoppers have not yet been determined. Field observations made by our group along years in diverse corn crops in subtropical areas of South America, reveal that the levels of parasitism of A. virlai recorded on D. maidis are much higher than those obtained for P. maidis, for whom the attack seems to be occasional (E. Luft Albarracin and E. Virla, data not published). According to Clausen (1940), Mymaridae wasps are unable to parasitize eggs with distinguishable embryos inside, being this assumption later discussed in other contributions (Irvin and Hoddle, 2005; Jacob et al., 2006; Mutitu et al., 2013). The only study on the biological aspects of A. virlai on D. maidis (Virla, 2001) reveals that this wasp parasitizes 1–3 days-old eggs and is not able to develop in eggs containing advanced embryos. Nevertheless, recent observations exposing embryonated eggs of the corn leafhopper revealed that a thelytokous parthenogenetic strain of A. virlai can successfully attack and develop on older eggs (J. Hill, personal observation). All previous field and laboratory studies were conducted on A. virlai populations with sexual reproduction and arrhenotokous parthenogenesis, and the biological parameters of a thelytokous parthenogenetic strain are completely unknown. Considering that these two pest species can coexist simultaneously in corn crops in South America, and due to the lack of information available on preference and host use by A. virlai, the aim of this contribution was to evaluate the biological parameters of a thelytokous parthenogenetic strain of A. virlai developed on two known hosts, D. maidis and P. maidis. We ask whether the host species affects the
2. Materials and methods 2.1. Insects colony origin and parasitoid breeding Dalbulus maidis colony was established from individuals collected in Los Nogales, Tucumán, Argentina (S 26° 42′, W 65°13′, 588 m a.s.l.), and Peregrinus maidis colony was established from individuals collected at Cabeza de Buey (S 24° 48′, W 65° 02′, 771 m a.s.l., Salta province). The hoppers were fed using potted maize plants (landrace sweet white maize variety “maizón”) within cages built with PVC pipes (50 × 50 × 50 cm) and protected by voile fabric to facilitate catch, prevent escape and avoid accidental parasitism. To start Anagrus virlai colony, D. maidis females were allowed to oviposit for 24 h on corn leaves. Plants with sentinel eggs were later exposed in a cornfield near to the Biological Control Division at PROIMI laboratory and, after 5 days, they were moved to climate-controlled plant growth chambers (Percival AR−36L, Iowa, USA) under controlled conditions (25 ± 1 °C, 50 ± 10% RH, and 12:12 h L:D). When Anagrus pupae were visible (parasitized eggs acquire reddish coloration), leaves were cut and transferred to Petri dishes containing moistened plaster on the bottom. A clear plastic food wrap was used to prevent desiccation and escape of adult parasitoids. Newly emerged females of wild Anagrus were allowed to oviposit for 24 h on leaves with D. maidis eggs, and these leaves were later separated and maintained in breeding chambers (under the previously described conditions). Identification of wasps was made using specific keys (Triapitsyn, 2015b) and voucher specimens were deposited in the entomological collection at the Miguel Lillo Foundation, Tucumán, Argentina (IMLA). Experiments were conducted from September 2016 to February 2018, during spring and summer using only parasitoids reared on D. maidis eggs, due to the low number of emerged wasps obtained from P. maidis eggs (see results section). 2.1.1. Host effect on biological parameters Six to ten hopper females were allowed to oviposit for 24 h on maize plants in the vegetative stage (six leaves) by enclosing them in circular cages (8 cm diameter) that held a maize leaf inside. Cages were built with two wire rings surrounded by polyurethane foam (1 cm wide) covered with voile fabric and were spliced and fastened to the leaf of potted plants through metal clips. As in other species belonging to Cicadellidae and Delphacidae, the eggs were always laid endophytically inside the plant tissues. To evaluate the performance of A. virlai on both host species, the number of parasitized eggs, proportion of emerged parasitoids and development time were registered. A total of 762 one day-old eggs (n = 12 leaves) of D. maidis and 1131 one day-old eggs (n = 15) of P. maidis were used. Eggs called here as one day-old had ≤ 24 h. Only A. virlai females were used because it was a thelytokous parthenogenetic colony. Parasitoids and leaves containing eggs were confined individually for a period of 24 h into glass tubes (25 cm × 2.5 cm diameter) with a honey supplement placed on a small piece of opaline paper. Cotton plugs were placed at the end of the tubes to confine the parasitoid inside the arena. Later, these leaves were isolated into tubular cages (20 cm × 4 cm diameter) built with voile fabric (to avoid accidental contact with other parasitoids) and maintained in controlled environment chamber (25 ± 1 °C, 50 ± 10% RH, and 12:12 h L:D) during the development of the parasitoids. Subsequently, leaves were cut off and transferred to Petri dishes for 15 days. When leaves dried, confirmation of successful parasitism (in not hatched eggs) was determined by subsequent dissections. 2
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averages of developmental times, successful parasitism and number of emerged adults using one day-old eggs of the hosts were compared by Wilcoxon Mann–Whitney non-parametric test. Previously, a ShapiroWilk test was performed to verify the normality of the data set. The eggs volume of hoppers and morphometric parameters were also compared using Wilcoxon Mann–Whitney test. A principal component analysis (PCA) was performed using the measured morphometric variables (see 2.1.2 Section) in two representative dimensions. Interaction among factors, host egg age and host species, were compared with KruskallWallis test. Post hoc, a Dunń’s multiple comparison test to contrast differences between groups was calculated. Frequencies obtained from Y-tube olfactometer bioassays were analysed with an exact binomial test (α = 0.05), validating the data when 95% confidence interval of choice proportions was greater than 0.50 (Quinn and Keough, 2002). Females that did not choose any of the arms were excluded from the statistical analysis. Since the data corresponding to the time wasps took to make a choice did not have a normal distribution, an exact Wilcoxon Mann–Whitney test was carried out. All tests were performed in RStudio (version 1.0.143) using R software version 3.3.1 (R Development Core Team 2016).
2.1.2. Size influence on morphometric variables Dissections of corn leaves containing D. maidis and P. maidis eggs of < 48-h-old were performed using 50x magnification in a Zeiss Stemi 2000c stereoscopic microscope. To facilitate cutting and avoid drying of the eggs, physiological saline solution was used. Assuming that those eggs had a prolate spheroid structure, we used the formula V = 1/6 µ w2 L (w = width; L = length) to calculate volume of eggs (Berrigan, 1991). To compare differences between wasps born from D. maidis and P. maidis eggs, 10 newly emerged females from each host were used to measure the following morphometric parameters: total body length, forewing maximum width and length, fore-tibia length and ovipositor length. 2.2. Parasitoid performance on different age eggs To make data comparable with those taken in one day-old eggs, the number of parasitized eggs, proportion of emerged parasitoids and development time were also measured exposing three and five days-old eggs. A total of 773 three days-old (n = 9 leaves) and 904 five days-old eggs (n = 12) of D. maidis, and 436 three days-old (n = 7) and 474 five days-old eggs (n = 6) of P. maidis were offered to A. virlai females. Eggs corresponding to three days-old had between 60 and 84 h-old, and those of five days-old had between 108 and 132 h-old. D. maidis and P. maidis females were allowed to oviposit for 24 h on corn leaves, and these plants were then isolated and kept under controlled conditions until they were used in the bioassays. A. virlai females were allowed to oviposit for 24 h and the next steps were essentially the same as those detailed in Section 2.1.1.
3. Results 3.1. Performance of A. virlai developed on D. maidis and P. maidis eggs Females did not have a period of preoviposition and were able to oviposit immediately after the emergence. Wasps were offered a mean of 63.5 ± 5.95 (n = 12) D. maidis one day-old eggs and 75.4 ± 6.96 (n = 15) P. maidis one day-old eggs. The mean number of parasitized eggs of D. maidis was significantly higher (Wilcoxon Mann–Whitney test, Z = 4.4091, p < 0.001) than that recorded for P. maidis (29.8 ± 1.92; 2 ± 0.47, respectively; values following means represent standard errors; Fig. 1a). A similar pattern was found in the proportion of emerged wasps (0.80 ± 0.03; 0.07 ± 0.04; Z = 4.5968, p < 0.001; Fig. 1b). The development time of parasitoids on one dayold eggs was not affected by the host species (16.1 ± 6.10 days; 20 ± 400 days; Z = −1.0449, p = 0.29), probably because of a lower proportion of emerged adults in P. maidis eggs than D. maidis eggs and the wide range of days in which those adults emerged (Fig. 1c).
2.3. Olfactory response to host To check the influence of chemical cues in the plant-host-parasitoid system, a Y-tube olfactometer was used following the methodology detailed in Lou et al. (2005) and Chiappini et al. (2012). The olfactometer consisted of a central arm (21 cm × 1.5 cm diameter) and two side arms (15 cm length × 1.5 cm diameter) connected in V-shape with a 45° angle. The airflow (150 ml/min) inside the olfactometer was provided by an aquarium air pump, air leaving the pump passed through an active carbon filter and a humidifier consisting on a Büchner flask filled with deionized water prior to the olfactometer device. The system was illuminated from above with led tubes. All bioassays were carried out between 10:00·h AM to 17:00·h PM in a chamber maintained under constant temperature of 25 ± 2 °C and 60% RH. The odor sources consisted on potted maize plants (sweet white maize variety “maizón”) with two or three fully unfolded leaves containing newly deposited eggs (< 24-h-old) of D. maidis or P. maidis. Maize leaves used as odor sources contained a mean number of 40.33 ± 23.75 D. maidis eggs and 20.66 ± 15.63 P. maidis eggs. The response of 45 newly emerged females was registered. Parasitoids were acclimatized for a period of 30 min prior to the test. A single female was placed in the central arm of the olfactometer using as intermediary a small test glass tube and the time it took to choose the odor source was registered. We considered a choice as valid when the parasitoid crossed more than 25% of the length of a side arm and remained there for 60 s. If a female exceeded 10 min in the central olfactometer arm, it was removed and recorded as no choice. To reduce incidence of the location, learning, light and device contamination, the olfactometer was cleaned with acetone and rotated previously to each release. In addition, after the release of five females, the position of the odor sources was alternated, and after 10 samples, plants with eggs were changed by new ones.
3.2. Morphometric analysis of the wasps The mean volume of D. maidis eggs (0.017 ± 0.0006 mm3, n = 10) was significantly higher than that recorded for P. maidis eggs (0.012 ± 0.0003 mm3, n = 11) (Wilcoxon Mann–Whitney test, Z = 3.8806, p < 0.001). All measured morphometric parameters (except fore-tibia length) were statistically different among hosts, and wasps that emerged from D. maidis eggs were larger than those emerged from P. maidis eggs. In multivariate analysis, the first two axes of the PCA explained 87.6% of the data variance, and PC1 (70.3%) markedly grouped the females emerged from D. maidis with respect to those emerged from P. maidis eggs (Fig. 2). 3.3. Effect of host egg age on the performance of A. virlai The number of attacked eggs and the emergence of parasitoids were not affected by host age. However, when the interaction between different egg ages and host species was compared, results were significant (Table 1). Anagrus virlai was able to develop in all age classes offered (one- to five-day-old eggs) regardless of host offered. For D. maidis, mean number of parasitized and mean rate of emergence of adults in all age classes (31.5 ± 1.49; 0.78 ± 0.04, respectively) were significantly higher than that recorded for P. maidis (4.36 ± 1.09; 0.11 ± 0.06, respectively). When parasitoids used D. maidis as host, all offered leaves with eggs were parasitized, while a large number of the
2.4. Statistical analysis Each leaf with eggs of D. maidis and P. maidis was considered an independent replicate for the analysis. Differences between rank3
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considering all age classes was 15.95 ± 0.07 days using D. maidis as host, and 19.34 ± 1.68 days on P. maidis. In all cases, except for oneday old eggs, A. virlai developed significantly faster in D. maidis that on P. maidis eggs (Table 1). Parasitoids developed significantly faster on three days-old egg of D. maidis than on one or five days-old eggs; while for P. maidis one-day-old eggs derived in the slowest development (Fig. 1c). The biological traits of the studied strain of A. virlai developed on D. maidis and on P. maidis eggs of different ages are summarized in Table 2. 3.4. Chemical discrimination of host volatiles The response of A. virlai females to semiochemicals from the planthost interaction was evaluated. Wasps moved more frequently into arms of the Y-olfactometer containing odors from plants that had been oviposited by D. maidis compared to those containing P. maidis eggs (exact binomial test, p = 0.02; Fig. 3). Comparatively, a low number of non-responses was recorded. In most cases, females made a constant antennal movement on the olfactometer walls and then they quickly walked or flew to the arm with the most attractive stimulus. The average time of choice was 88.1 ± 20.9 s for D. maidis and 22.4 ± 5 s for P. maidis, and this was not influenced by host species (Wilcoxon Mann–Whitney test, p = 0.17). 4. Discussion The studies conducted here provide valuable information concerning the biology of a formerly considered generalist egg parasitoid of Cicadellidae and Delphacidae. Our data show that A. virlai would only use the planthopper P. maidis as an occasional host. The age of the host egg did not considerably affect the biological parameters, while host species had a stronger influence than egg ages on parasitism, rates of emergence and development time. The egg volume and host species conditioned the size of the emerged wasps. We detected that A. virlai was able to exploit one resource better than another and identify among different hosts through olfactory cues. Although cases of thelytokous parthenogenesis in other species of Anagrus genus were previously recorded (Cronin and Strong, 1996; Choudhury and Copland, 2003), this contribution is the first biological study of a thelytokous parthenogenetic strain of Anagrus in the Neotropical Region. From a biological control point of view, parasitoids with asexual reproduction (thelytoky) would exhibit major advantages in costs of mass rearing, faster population growth after release, and the easier establishment of populations compared to sexual reproduction populations (Ardeh et al., 2005). In crop systems, both populations (sexual and asexual) are present suggesting low probabilities of genetic drift and potential extinction. A successful parasitism may be controlled by different factors, which define the parasitoid-host interaction (Vinson, 1976). Host species, host size and host age usually affect the performance of parasitoids on a given host (Vinson and Iwantsch, 1980). Generalist and specialist parasitoids present differential behaviors that are related to the ways they have to exploit the resources (Stilmant et al., 2008). Comparatively, generalists exhibit more efficiency for exploiting different resources, whereas specialists are better adapted to take advantage on a particular resource (Devictor et al., 2010). Hosts of the Mymaridae family include a wide range of orders: Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, Odonata, Orthoptera, Psocoptera, and Thysanoptera (Huber, 1986); whereas hosts of the Anagrus genus include members of several Hemiptera, some Odonata (mainly Zygoptera), and possibly also a few Gyrinidae (Coleoptera) and Psocoptera (Triapitsyn, 2015b). Anagrus virlai was able to parasitize both species of hoppers; however, when wasps used the planthopper P. maidis as host, all the measured parameters were significantly worse than those registered for the leafhopper D. maidis. Tritrophic coevolution hypothesis (Maize – D. maidis – A. virlai system) can provide insight
Fig. 1. Effect of host and egg age on the performance of A. virlai at 25 °C. a) Number of parasitized eggs; b) proportion of emerged adults; c) developmental time, in days. Dalbulus maidis = white boxes; Peregrinus maidis = grey boxes. Horizontal line in each box is the median, boxes define the hinge (25–75% quartiles, continue lines hinge to the maximum or minimum values) and points outside the intervals are represented as dots. Asterisks indicate significant differences in Kruskal–Wallis test among hosts (*p < 0.05; **p < 0.01; ***p < 0.001; n.s: not significant). Different letters indicate significant differences among ages based on a post-hoc Dunn's multiple comparison test (lowercase letters = D. maidis; capital letters = P. maidis).
leaves with P. maidis eggs were no parasitized under the same testing conditions. The average developmental time from egg to adult for A. virlai 4
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Fig. 2. Principal component analysis (PCA) of different morphometric variables measured on Anagrus virlai developed on Dalbulus maidis and Peregrinus maidis eggs. Variables used were total body length, forewing maximum width and length, fore-tibia length and ovipositor length. Plots are based on the two first components, the first and the second axis explaining 88% of total variation. Points and triangles depict a female for each host. Ellipses group 95% confidence interval of the data set (grey color = P. maidis; clear color = D. maidis. N = 10 individuals per host).
on the host preference previously found both in the field and the laboratory. Maize originated from wild annual teosinte Zea mays ssp. parviglumis (Iltis & Doebley) in Central America (Matsuoka et al., 2002), which would coincide with the center of origin established for D. maidis (Nault and DeLong, 1980). Several contributions on D. maidis egg parasitoids on different species of teosintes and maize plants were conducted demonstrating the close relationship between A. virlai and the corn leafhopper (Virla et al., 2013; Moya Raygoza and Triapitsyn, 2017). Peregrinus maidis, would have adapted only in post-Columbian times to maize as a host (Nault, 1983). Irvin and Hoddle (2010) demonstrated that the egg parasitoid Cosmocomoidea ashmeadi Girault (Hymenoptera: Mymaridae), who has a long history of coevolution with its host, the glassy-winged sharpshooter, has a better ability to make use of different ages of host eggs compared to a recently released Cosmocomoidea Howard exotic species. Presence of suboptimal hosts could be beneficial for the long-term maintenance of the system (Heimpel et al., 2003), because parasitoids do not completely suppress to usual host species and can use alternative hosts that are already established in maize agroecosystems. Such biological and ecological traits could be interesting for conservation biological control programs, which can be enhanced through habitat and culture management (Khan et al., 2008; Lou et al., 2014). Furthermore, we observed that many parasitized eggs of P. maidis presented larvae or pharate adults that had not completed their development. Apparently, the nutritional components of P. maidis eggs were not adequate enough to ensure A. virlai development. This last assumption may be reasonable given the low rate of emergence and the differential size found in the emerged wasps from D. maidis and P. maidis eggs. Egg parasitoids can present innate preferences for a given host that will depend mainly on the physical characteristics of eggs (Mansfield and Mills, 2004). The corn leafhopper and corn planthopper eggs vary in volume: D. maidis
Table 2 Influence of egg age and host species on different biological parameters of Anagrus virlai developed on Dalbulus maidis and Peregrinus maidis eggs at temperature 25 °C. Host species and host agesa
Dalbulus maidis 1 3 5 Peregrinus maidis 1 3 5 Mean ± SE are presented.
% parasitized eggs
% emerged wasps
Development timeb
n (total number of offered eggs)
49.3 ± 3.11 43.7 ± 6.81 46.9 ± 6.11
80.1 ± 2.52 72.9 ± 4.81 81.1 ± 3.73
16.1 ± 6.10 15.7 ± 6.26 16.1 ± 8.92
12 (7 6 2) 9 (7 7 3) 12 (9 0 4)
2.65 ± 0.52 7.86 ± 3.45 18.7 ± 8.46
6.89 ± 3.96 7.85 ± 4.68 19.9 ± 10.7
20 ± 400 21.3 ± 75 16.8 ± 30.3
15 (1131) 7 (4 3 6) 6 (4 7 4)
a
Host ages are expressed in days. Corresponding to 226, 287 and 307 wasps born from D. maidis eggs; and 3, 4 and 17 emerged wasps from P. maidis eggs. Development time is expressed in days. b
eggs had a 30% more volume than those measured for P. maidis. Larger eggs potentially provide more food and nutrients to support progeny development (Brotodjojo and Walter, 2006). Our results are similar to those obtained from a previous study with Trichogramma euproctidis (Girault) (Hymenoptera: Trichogrammatidae) in three different lepidopteran hosts, where the size of the adult parasitoid was influenced by the size and quality of the host (Martel et al., 2011). Likewise, in the Trichogrammatidae Trichogrammatoidea lutea Girault, the preferred host for oviposition among three lepidopteran host species was the one
Table 1 Results of a Kruskal–Wallis test in multiple comparisons among ages and interaction between age and host (Dalbulus maidis and Peregrinus maidis) for: number of parasitism, rates of emergence and development time of Anagrus virlai. Vector species
Parasitized eggs: 1, 3 and 5 days Adult emergence: 1, 3 and 5 days Development time: 1, 3 and 5 days
Dalbulus maidis
Peregrinus maidis
D. maidis vs P. maidis
Interaction between age and host
X2
df
P-value
X2
df
P-value
X2
df
P-value
X2
df
P-value
2.841 1.909 29.346
2 2 2
0.242 0.385 < 0.001
4.559 3.297 8.157
2 2 2
0.102 0.192 < 0.001
43.092 44.316 15.095
1 1 1
< 0.001 < 0.001 < 0.001
45.229 45.702 48.522
5 5 5
< 0.001 < 0.001 < 0.001
5
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Fig. 3. Response of Anagrus virlai females to volatile blends released from maize plants containing Dalbulus maidis or Peregrinus maidis eggs. Asterisks indicate significant differences in exact binomial test (*p < 0.05). N = 45 individuals.
confirm these assumptions and provide information about the chemical cues used during host selection by the studied egg parasitoid. This work contributes to a better understanding of an egg parasitoid and its relation to two hosts involved in corn diseases transmission, preventing the hatching of eggs of these vectors thus disrupting the epidemiological cycle (Ballal, 2013).
with the largest eggs (Mawela et al., 2013). A major volume of eggs, in turn, could be fundamental for the searching behaviour, identification and later acceptance of host (Moreau et al., 2009). Hence, we were unable to successfully maintain lab populations of A. virlai using solely P. maidis as host, due to the scarce the low number of parazited eggs, emergence of females, and incomplete development of most parasitoids. Vinson (1998) mentioned that the oophagous (sensu stricto) parasitoids are able to reject the older host eggs. However, Virla et al. (2005) suggested that the mymarids can belong to two groups based on their ability to exploit host eggs of different ages: those that can attack and develop only on newly laid eggs (without embryos) and those that attack eggs in all their developmental stages. In Cicadellidae and Delphacidae eggs, the appearance of anatomical structures distinguishable through egg chorion (in ≥90 h-old eggs) such as head cap and eyespot are visible characteristic of the stage of development of the embryo (AlWahaibi and Morse, 2009). In our laboratory tests, eyespots were observed 5 days after oviposition. Contrary to our expectations, parasitism and emergence did not differed when A. virlai used 1 to 5-days eggs of both hosts indicating that this thelytokous strain could have a certain plasticity in the selection of host age. Chantarasa-ard et al. (1984) found that A. incarnatus parasitized all immature stages of development (1–9 days old) of the brown planthopper Nilaparvata lugens (Stål), without considerable differences in parasitism and emergence. Anagrus delicatus (Dozier), an egg parasitoid of the planthopper Prokelisia marginata (Van Duzee), attacked eggs of all ages, but its levels of parasitism were higher in 5–6 days old eggs (Cronin and Strong, 1990). The fairyfly Cosmocomoidea morrilli (Howard), parasitized all egg ages of the glassy-winged sharpshooter Homalodisca vitripennis (Germar), reducing its performance in older (> five-day old) egg masses (Krugner, 2014). Numerous studies evaluating the host preference were conducted in the Trichogrammatidae family. Several species can detect host age, preferring young eggs and reducing clutch size as host age increases (Godin and Boivin, 2000). Contrary to this prediction, the studied population of A. virlai oviposited both in newly laid and old eggs of its habitual host (D. maidis), without significant differences in the number of attacked eggs. This amplitude in resource-use could be advantageous for intra and inter-specific competition (Irvin and Hoddle, 2005), although A. virlai ecological response when carrying out future massive releases in cornfields, should be evaluated subsequently. Hosts must be located using different strategies in response to longrange and short-range cues (Fatouros et al., 2008). Odor cues produced by herbivore-plant interaction and contact host kairomones play a key role in location behavior and host discrimination when parasitoids search a host to deposit their eggs (Meiners et al., 2000; Colazza et al., 2007). In our olfactometry assay, A. virlai tended to choose maize leaves oviposited with D. maidis eggs more frequently than those containing P. maidis eggs. Our results highlight that this species could be able to identify among patches of hosts with differential characteristics and quality, and in this way, focus its efforts in the more suitable hosts (Colazza et al., 2014). Some authors have reported that plant-induced volatiles themselves are not the major attractants for Anagrus parasitoids, but rather there is synergy among those and kairomones from eggs within plant tissues (Honda and Walker, 1996; Chiappini et al., 2012). We believe that the host preference could be influenced by qualitatively or quantitatively different volatile profiles that vary according to the hosts attacking the plant. Future studies are necessary to
5. Author statement J.G.H. carried out the experiments. E.L. A. and E.G.V. supervised the project. M.V.C.A. revised the language. All authors helped shape the research, analysis and manuscript. Acknowledgements This work was supported by PICT N° 1147 (FONCYT, Fondo para la Investigación Científica y Tecnológica, Argentina). Jorge G. Hill thanks CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina) for a scholarship. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biocontrol.2018.12.002. References Al-Wahaibi, A.K., Morse, J.G., 2009. Egg morphology and stages of embryonic development of the glassy-winged sharpshooter (Hemiptera: Cicadellidae). Ann. Entomol. Soc. Am. 102 (2), 241–248. Ardeh, M.J., Jong, P.W., Van Lenteren, J.C., 2005. Intra-and interspecific host discrimination in arrhenotokous and thelytokous Eretmocerus spp. Biol. Control 33 (1), 74–80. Ballal, C.R., 2013. Other egg parasitoids: research for utilisation. In: Sithanantham, S., Ballal, C., Jalali, S., Bakthavatsalam, N. (Eds.), Biological control of insect pests using egg parasitoids. Springer, New Delhi, pp. 223–270. Berrigan, D., 1991. The allometry of egg size and number in insects. Oikos 60, 313–321. Brotodjojo, R.R., Walter, G.H., 2006. Oviposition and reproductive performance of a generalist parasitoid (Trichogramma pretiosum) exposed to host species that differ in their physical characteristics. Biol. Control 39 (3), 300–312. Castañera, P., Farinós, G.P., Ortego, F., Andow, D.A., 2016. Sixteen years of Bt maize in the EU hotspot: why has resistance not evolved? PloS one 11 (5), e0154200. Chantarasa-ard, S., Hirashima, Y., Hirao, J., 1984. Host range and host suitability of Anagrus incarnatus Haliday (Hymenoptera: Mymaridae): an egg parasitoid of delphacid planthoppers. Appl. Entomol. Zool. 19 (4), 491–497. Chiappini, E., Salerno, G., Berzolla, A., Iacovone, A., Cristina Reguzzi, M., Conti, E., 2012. Role of volatile semiochemicals in host location by the egg parasitoid Anagrus breviphragma. Entomol. Exp. Appl. 144 (3), 311–316. Choudhury, D.A.M., Copland, M.J.W., 2003. A new record in thelytoky in the egg parasitoid Anagrus atomus (Linnaeus) (Hymenoptera: Mymaridae). Pak. J. Biol. Sci. 6, 500–504. Clausen, C.P., 1940. Entomophagous Insects. McGrawHill, New York. Colazza, S., Aquila, G., De Pasquale, C., Peri, E., Millar, J.G., 2007. The egg parasitoid Trissolcus basalis uses n-nonadecane, a cuticular hydrocarbon from its stink bug host Nezara viridula, to discriminate between female and male hosts. J. Chem. Ecol. 33 (7), 140–1420. Colazza, S., Cusumano, A., Giudice, D.L., Peri, E., 2014. Chemo-orientation responses in hymenopteran parasitoids induced by substrate-borne semiochemicals. BioControl 59 (1), 1–17. Cronin, J.T., Strong, D.R., 1990. Biology of Anagrus delicatus (Hymenoptera: Mymaridae), an egg parasitoid of Prokelisia marginata (Homoptera: Delphacidae). Ann. Entomol. Soc. Am. 83 (4), 846–854. Cronin, J.T., Strong, D.R., 1996. Genetics of oviposition success of a thelytokous fairyfly parasitoid, Anagrus delicatus. Heredity 76 (1), 43. Devictor, V., Clavel, J., Julliard, R., Lavergne, S., Mouillot, D., Thuiller, W., Venail, P., Villéger, S., Mouquet, N., 2010. Defining and measuring ecological specialization. J. Appl. Ecol. 47 (1), 15–25.
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Biological Control journal homepage: www.elsevier.com/locate/ybcon
Effects of host species and host age on biological parameters of Anagrus virlai (Hymenoptera: Mymaridae), an egg parasitoid of Dalbulus maidis (Hemiptera: Cicadellidae) and Peregrinus maidis (Hemiptera: Delphacidae) ⁎
Jorge G. Hilla, Erica Luft Albarracina, , Maria V. Coll Araoza, Eduardo G. Virlaa,b,1 a b
PROIMI- Biotecnología, CONICET, Av. Belgrano y Pje. Caseros (T4001MVB), San Miguel de Tucumán, Argentina Instituto de Entomología, Fundación Miguel Lillo. Miguel Lillo 251, San Miguel de Tucumán, Tucumán, Argentina
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Corn leafhopper Corn planthopper Development Morphometry Parasitism Thelytokous strain
Dalbulus maidis (DeLong) (Hemiptera: Cicadellidae) and Peregrinus maidis (Ashmead) (Hemiptera: Delphacidae) are important pests for maize production in America. Anagrus virlai Triapitsyn (Hymenoptera: Mymaridae) is a potential biological control agent for both hopper species. We provide information on the effects of size, age and host species on the time of development, parasitism and emergence, and morphometry of A. virlai. Additionally, we evaluate if A. virlai females can discriminate between the eggs of both host species using a Y-tube olfactometer. The wasps were reared in breeding chambers under controlled conditions, and one, three and five daysold eggs of D. maidis and P. maidis were used for bioassays. Anagrus virlai developed in eggs from the three age classes of both hopper species; however, the number of parasitized eggs and emergence of wasps was significantly higher on eggs of D. maidis than on those of P. maidis. Wasps that emerged from D. maidis eggs presented larger size compared to those reared on P. maidis eggs. Host species not only influenced the parasitism, emergence, and size of the wasps, but also development time. In olfactometric tests, A. virlai chose maize leaves containing D. maidis eggs, instead of those containing P. maidis eggs. Our results show that A. virlai, formerly known as generalist parasitoid, would only use the planthopper P. maidis as an occasional host.
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Corresponding author. E-mail address: [email protected] (E. Luft Albarracin). 1 ex aequo. https://doi.org/10.1016/j.biocontrol.2018.12.002 Received 5 October 2018; Received in revised form 29 November 2018; Accepted 4 December 2018 1049-9644/ © 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Hill, J.G., Biological Control, https://doi.org/10.1016/j.biocontrol.2018.12.002
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1. Introduction
development time, parasitism and emergence, and size of wasps. Additionally, we assess the influence of host age on these biological parameters. Finally, we evaluate if A. virlai females can detect specific olfactory cues associated with the offered host species using a Y-tube olfactometer.
Maize (Zea mays L.) (Poaceae) is the most prominentcrop worldwide, surpassing even rice and wheat (FAOSTAT, 2017). During the last decades, the growing demand for foods at global scale lay out the need to enhance crop yield replacing old farming practices by high inputs of pesticides and by the use of genetically modified (GM) germplasms (Castañera et al., 2016). However, resistance phenomena in maize pests are being reported denoting a latent risk for future management strategies (Gassmann et al., 2014). Much of the yield losses in annual crops are due to the action of insect pests (Pimentel, 2009). Particularly, hoppers (Hemiptera: Auchenorrhyncha) are conspicuous threats for maize crops in the world; causing damages that vary from necrosis to severe physiological alterations produced by their feeding and/or oviposition habits, and its ability to transmit pathogens (Luft Albarracin et al., 2008; Singh and Seetharama, 2008). The corn leafhopper, Dalbulus maidis (DeLong) (Cicadellidae), is a vector of three important maize pathogens in the Americas: “maize rayado fino virus” (Tymovirales: Marafivirus), “corn stunt spiroplasma” (Mollicutes: Spiroplasmataceae: Spiroplasma kunkelii Whitcomb), and “maize bushy stunt phytoplasma” (Mollicutes: Acholeplasmataceae: Candidatus Phytoplasma ateris) (Laguna and Giménez Pecci, 2012). For its part, the corn planthopper Peregrinus maidis (Ashmead) (Delphacidae) is vector, among others, of “Mal de Rio Cuarto Virus”, an important threat for corn production in Argentina (Redinbaugh and Pratt, 2009) and considered as a quarantine-disease in other countries (Oliveira et al., 2013). A large number of natural enemies of hoppers are recognized, although egg parasitoids are the best known. Species of Cicadellidae and Delphacidae families are the main hosts of Anagrus Haliday wasps (Hymenoptera: Mymaridae) (Krugner et al., 2008; Zhu et al., 2013; Segoli, 2016). Anagrus virlai Triapitsyn (Hymenoptera: Mymaridae), misidentified previously as Anagrus breviphragma Soyka or Anagrus incarnatus Haliday (Triapitsyn, 2015a; Triapitsyn et al., 2018), is the most important egg parasitoid of the corn leafhopper because of its high levels of parasitism along its distribution range (Virla et al., 2009; Luft Albarracin et al., 2017). It is considered a generalist parasitoid, that has been reported in eggs of two Delphacidae and nine Cicadellidae species (Triapitsyn, 2015b; Triapitsyn et al., 2018). Except for the corn leafhopper, the capacity of development and its incidence on the populations of other hoppers have not yet been determined. Field observations made by our group along years in diverse corn crops in subtropical areas of South America, reveal that the levels of parasitism of A. virlai recorded on D. maidis are much higher than those obtained for P. maidis, for whom the attack seems to be occasional (E. Luft Albarracin and E. Virla, data not published). According to Clausen (1940), Mymaridae wasps are unable to parasitize eggs with distinguishable embryos inside, being this assumption later discussed in other contributions (Irvin and Hoddle, 2005; Jacob et al., 2006; Mutitu et al., 2013). The only study on the biological aspects of A. virlai on D. maidis (Virla, 2001) reveals that this wasp parasitizes 1–3 days-old eggs and is not able to develop in eggs containing advanced embryos. Nevertheless, recent observations exposing embryonated eggs of the corn leafhopper revealed that a thelytokous parthenogenetic strain of A. virlai can successfully attack and develop on older eggs (J. Hill, personal observation). All previous field and laboratory studies were conducted on A. virlai populations with sexual reproduction and arrhenotokous parthenogenesis, and the biological parameters of a thelytokous parthenogenetic strain are completely unknown. Considering that these two pest species can coexist simultaneously in corn crops in South America, and due to the lack of information available on preference and host use by A. virlai, the aim of this contribution was to evaluate the biological parameters of a thelytokous parthenogenetic strain of A. virlai developed on two known hosts, D. maidis and P. maidis. We ask whether the host species affects the
2. Materials and methods 2.1. Insects colony origin and parasitoid breeding Dalbulus maidis colony was established from individuals collected in Los Nogales, Tucumán, Argentina (S 26° 42′, W 65°13′, 588 m a.s.l.), and Peregrinus maidis colony was established from individuals collected at Cabeza de Buey (S 24° 48′, W 65° 02′, 771 m a.s.l., Salta province). The hoppers were fed using potted maize plants (landrace sweet white maize variety “maizón”) within cages built with PVC pipes (50 × 50 × 50 cm) and protected by voile fabric to facilitate catch, prevent escape and avoid accidental parasitism. To start Anagrus virlai colony, D. maidis females were allowed to oviposit for 24 h on corn leaves. Plants with sentinel eggs were later exposed in a cornfield near to the Biological Control Division at PROIMI laboratory and, after 5 days, they were moved to climate-controlled plant growth chambers (Percival AR−36L, Iowa, USA) under controlled conditions (25 ± 1 °C, 50 ± 10% RH, and 12:12 h L:D). When Anagrus pupae were visible (parasitized eggs acquire reddish coloration), leaves were cut and transferred to Petri dishes containing moistened plaster on the bottom. A clear plastic food wrap was used to prevent desiccation and escape of adult parasitoids. Newly emerged females of wild Anagrus were allowed to oviposit for 24 h on leaves with D. maidis eggs, and these leaves were later separated and maintained in breeding chambers (under the previously described conditions). Identification of wasps was made using specific keys (Triapitsyn, 2015b) and voucher specimens were deposited in the entomological collection at the Miguel Lillo Foundation, Tucumán, Argentina (IMLA). Experiments were conducted from September 2016 to February 2018, during spring and summer using only parasitoids reared on D. maidis eggs, due to the low number of emerged wasps obtained from P. maidis eggs (see results section). 2.1.1. Host effect on biological parameters Six to ten hopper females were allowed to oviposit for 24 h on maize plants in the vegetative stage (six leaves) by enclosing them in circular cages (8 cm diameter) that held a maize leaf inside. Cages were built with two wire rings surrounded by polyurethane foam (1 cm wide) covered with voile fabric and were spliced and fastened to the leaf of potted plants through metal clips. As in other species belonging to Cicadellidae and Delphacidae, the eggs were always laid endophytically inside the plant tissues. To evaluate the performance of A. virlai on both host species, the number of parasitized eggs, proportion of emerged parasitoids and development time were registered. A total of 762 one day-old eggs (n = 12 leaves) of D. maidis and 1131 one day-old eggs (n = 15) of P. maidis were used. Eggs called here as one day-old had ≤ 24 h. Only A. virlai females were used because it was a thelytokous parthenogenetic colony. Parasitoids and leaves containing eggs were confined individually for a period of 24 h into glass tubes (25 cm × 2.5 cm diameter) with a honey supplement placed on a small piece of opaline paper. Cotton plugs were placed at the end of the tubes to confine the parasitoid inside the arena. Later, these leaves were isolated into tubular cages (20 cm × 4 cm diameter) built with voile fabric (to avoid accidental contact with other parasitoids) and maintained in controlled environment chamber (25 ± 1 °C, 50 ± 10% RH, and 12:12 h L:D) during the development of the parasitoids. Subsequently, leaves were cut off and transferred to Petri dishes for 15 days. When leaves dried, confirmation of successful parasitism (in not hatched eggs) was determined by subsequent dissections. 2
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averages of developmental times, successful parasitism and number of emerged adults using one day-old eggs of the hosts were compared by Wilcoxon Mann–Whitney non-parametric test. Previously, a ShapiroWilk test was performed to verify the normality of the data set. The eggs volume of hoppers and morphometric parameters were also compared using Wilcoxon Mann–Whitney test. A principal component analysis (PCA) was performed using the measured morphometric variables (see 2.1.2 Section) in two representative dimensions. Interaction among factors, host egg age and host species, were compared with KruskallWallis test. Post hoc, a Dunń’s multiple comparison test to contrast differences between groups was calculated. Frequencies obtained from Y-tube olfactometer bioassays were analysed with an exact binomial test (α = 0.05), validating the data when 95% confidence interval of choice proportions was greater than 0.50 (Quinn and Keough, 2002). Females that did not choose any of the arms were excluded from the statistical analysis. Since the data corresponding to the time wasps took to make a choice did not have a normal distribution, an exact Wilcoxon Mann–Whitney test was carried out. All tests were performed in RStudio (version 1.0.143) using R software version 3.3.1 (R Development Core Team 2016).
2.1.2. Size influence on morphometric variables Dissections of corn leaves containing D. maidis and P. maidis eggs of < 48-h-old were performed using 50x magnification in a Zeiss Stemi 2000c stereoscopic microscope. To facilitate cutting and avoid drying of the eggs, physiological saline solution was used. Assuming that those eggs had a prolate spheroid structure, we used the formula V = 1/6 µ w2 L (w = width; L = length) to calculate volume of eggs (Berrigan, 1991). To compare differences between wasps born from D. maidis and P. maidis eggs, 10 newly emerged females from each host were used to measure the following morphometric parameters: total body length, forewing maximum width and length, fore-tibia length and ovipositor length. 2.2. Parasitoid performance on different age eggs To make data comparable with those taken in one day-old eggs, the number of parasitized eggs, proportion of emerged parasitoids and development time were also measured exposing three and five days-old eggs. A total of 773 three days-old (n = 9 leaves) and 904 five days-old eggs (n = 12) of D. maidis, and 436 three days-old (n = 7) and 474 five days-old eggs (n = 6) of P. maidis were offered to A. virlai females. Eggs corresponding to three days-old had between 60 and 84 h-old, and those of five days-old had between 108 and 132 h-old. D. maidis and P. maidis females were allowed to oviposit for 24 h on corn leaves, and these plants were then isolated and kept under controlled conditions until they were used in the bioassays. A. virlai females were allowed to oviposit for 24 h and the next steps were essentially the same as those detailed in Section 2.1.1.
3. Results 3.1. Performance of A. virlai developed on D. maidis and P. maidis eggs Females did not have a period of preoviposition and were able to oviposit immediately after the emergence. Wasps were offered a mean of 63.5 ± 5.95 (n = 12) D. maidis one day-old eggs and 75.4 ± 6.96 (n = 15) P. maidis one day-old eggs. The mean number of parasitized eggs of D. maidis was significantly higher (Wilcoxon Mann–Whitney test, Z = 4.4091, p < 0.001) than that recorded for P. maidis (29.8 ± 1.92; 2 ± 0.47, respectively; values following means represent standard errors; Fig. 1a). A similar pattern was found in the proportion of emerged wasps (0.80 ± 0.03; 0.07 ± 0.04; Z = 4.5968, p < 0.001; Fig. 1b). The development time of parasitoids on one dayold eggs was not affected by the host species (16.1 ± 6.10 days; 20 ± 400 days; Z = −1.0449, p = 0.29), probably because of a lower proportion of emerged adults in P. maidis eggs than D. maidis eggs and the wide range of days in which those adults emerged (Fig. 1c).
2.3. Olfactory response to host To check the influence of chemical cues in the plant-host-parasitoid system, a Y-tube olfactometer was used following the methodology detailed in Lou et al. (2005) and Chiappini et al. (2012). The olfactometer consisted of a central arm (21 cm × 1.5 cm diameter) and two side arms (15 cm length × 1.5 cm diameter) connected in V-shape with a 45° angle. The airflow (150 ml/min) inside the olfactometer was provided by an aquarium air pump, air leaving the pump passed through an active carbon filter and a humidifier consisting on a Büchner flask filled with deionized water prior to the olfactometer device. The system was illuminated from above with led tubes. All bioassays were carried out between 10:00·h AM to 17:00·h PM in a chamber maintained under constant temperature of 25 ± 2 °C and 60% RH. The odor sources consisted on potted maize plants (sweet white maize variety “maizón”) with two or three fully unfolded leaves containing newly deposited eggs (< 24-h-old) of D. maidis or P. maidis. Maize leaves used as odor sources contained a mean number of 40.33 ± 23.75 D. maidis eggs and 20.66 ± 15.63 P. maidis eggs. The response of 45 newly emerged females was registered. Parasitoids were acclimatized for a period of 30 min prior to the test. A single female was placed in the central arm of the olfactometer using as intermediary a small test glass tube and the time it took to choose the odor source was registered. We considered a choice as valid when the parasitoid crossed more than 25% of the length of a side arm and remained there for 60 s. If a female exceeded 10 min in the central olfactometer arm, it was removed and recorded as no choice. To reduce incidence of the location, learning, light and device contamination, the olfactometer was cleaned with acetone and rotated previously to each release. In addition, after the release of five females, the position of the odor sources was alternated, and after 10 samples, plants with eggs were changed by new ones.
3.2. Morphometric analysis of the wasps The mean volume of D. maidis eggs (0.017 ± 0.0006 mm3, n = 10) was significantly higher than that recorded for P. maidis eggs (0.012 ± 0.0003 mm3, n = 11) (Wilcoxon Mann–Whitney test, Z = 3.8806, p < 0.001). All measured morphometric parameters (except fore-tibia length) were statistically different among hosts, and wasps that emerged from D. maidis eggs were larger than those emerged from P. maidis eggs. In multivariate analysis, the first two axes of the PCA explained 87.6% of the data variance, and PC1 (70.3%) markedly grouped the females emerged from D. maidis with respect to those emerged from P. maidis eggs (Fig. 2). 3.3. Effect of host egg age on the performance of A. virlai The number of attacked eggs and the emergence of parasitoids were not affected by host age. However, when the interaction between different egg ages and host species was compared, results were significant (Table 1). Anagrus virlai was able to develop in all age classes offered (one- to five-day-old eggs) regardless of host offered. For D. maidis, mean number of parasitized and mean rate of emergence of adults in all age classes (31.5 ± 1.49; 0.78 ± 0.04, respectively) were significantly higher than that recorded for P. maidis (4.36 ± 1.09; 0.11 ± 0.06, respectively). When parasitoids used D. maidis as host, all offered leaves with eggs were parasitized, while a large number of the
2.4. Statistical analysis Each leaf with eggs of D. maidis and P. maidis was considered an independent replicate for the analysis. Differences between rank3
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considering all age classes was 15.95 ± 0.07 days using D. maidis as host, and 19.34 ± 1.68 days on P. maidis. In all cases, except for oneday old eggs, A. virlai developed significantly faster in D. maidis that on P. maidis eggs (Table 1). Parasitoids developed significantly faster on three days-old egg of D. maidis than on one or five days-old eggs; while for P. maidis one-day-old eggs derived in the slowest development (Fig. 1c). The biological traits of the studied strain of A. virlai developed on D. maidis and on P. maidis eggs of different ages are summarized in Table 2. 3.4. Chemical discrimination of host volatiles The response of A. virlai females to semiochemicals from the planthost interaction was evaluated. Wasps moved more frequently into arms of the Y-olfactometer containing odors from plants that had been oviposited by D. maidis compared to those containing P. maidis eggs (exact binomial test, p = 0.02; Fig. 3). Comparatively, a low number of non-responses was recorded. In most cases, females made a constant antennal movement on the olfactometer walls and then they quickly walked or flew to the arm with the most attractive stimulus. The average time of choice was 88.1 ± 20.9 s for D. maidis and 22.4 ± 5 s for P. maidis, and this was not influenced by host species (Wilcoxon Mann–Whitney test, p = 0.17). 4. Discussion The studies conducted here provide valuable information concerning the biology of a formerly considered generalist egg parasitoid of Cicadellidae and Delphacidae. Our data show that A. virlai would only use the planthopper P. maidis as an occasional host. The age of the host egg did not considerably affect the biological parameters, while host species had a stronger influence than egg ages on parasitism, rates of emergence and development time. The egg volume and host species conditioned the size of the emerged wasps. We detected that A. virlai was able to exploit one resource better than another and identify among different hosts through olfactory cues. Although cases of thelytokous parthenogenesis in other species of Anagrus genus were previously recorded (Cronin and Strong, 1996; Choudhury and Copland, 2003), this contribution is the first biological study of a thelytokous parthenogenetic strain of Anagrus in the Neotropical Region. From a biological control point of view, parasitoids with asexual reproduction (thelytoky) would exhibit major advantages in costs of mass rearing, faster population growth after release, and the easier establishment of populations compared to sexual reproduction populations (Ardeh et al., 2005). In crop systems, both populations (sexual and asexual) are present suggesting low probabilities of genetic drift and potential extinction. A successful parasitism may be controlled by different factors, which define the parasitoid-host interaction (Vinson, 1976). Host species, host size and host age usually affect the performance of parasitoids on a given host (Vinson and Iwantsch, 1980). Generalist and specialist parasitoids present differential behaviors that are related to the ways they have to exploit the resources (Stilmant et al., 2008). Comparatively, generalists exhibit more efficiency for exploiting different resources, whereas specialists are better adapted to take advantage on a particular resource (Devictor et al., 2010). Hosts of the Mymaridae family include a wide range of orders: Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, Odonata, Orthoptera, Psocoptera, and Thysanoptera (Huber, 1986); whereas hosts of the Anagrus genus include members of several Hemiptera, some Odonata (mainly Zygoptera), and possibly also a few Gyrinidae (Coleoptera) and Psocoptera (Triapitsyn, 2015b). Anagrus virlai was able to parasitize both species of hoppers; however, when wasps used the planthopper P. maidis as host, all the measured parameters were significantly worse than those registered for the leafhopper D. maidis. Tritrophic coevolution hypothesis (Maize – D. maidis – A. virlai system) can provide insight
Fig. 1. Effect of host and egg age on the performance of A. virlai at 25 °C. a) Number of parasitized eggs; b) proportion of emerged adults; c) developmental time, in days. Dalbulus maidis = white boxes; Peregrinus maidis = grey boxes. Horizontal line in each box is the median, boxes define the hinge (25–75% quartiles, continue lines hinge to the maximum or minimum values) and points outside the intervals are represented as dots. Asterisks indicate significant differences in Kruskal–Wallis test among hosts (*p < 0.05; **p < 0.01; ***p < 0.001; n.s: not significant). Different letters indicate significant differences among ages based on a post-hoc Dunn's multiple comparison test (lowercase letters = D. maidis; capital letters = P. maidis).
leaves with P. maidis eggs were no parasitized under the same testing conditions. The average developmental time from egg to adult for A. virlai 4
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Fig. 2. Principal component analysis (PCA) of different morphometric variables measured on Anagrus virlai developed on Dalbulus maidis and Peregrinus maidis eggs. Variables used were total body length, forewing maximum width and length, fore-tibia length and ovipositor length. Plots are based on the two first components, the first and the second axis explaining 88% of total variation. Points and triangles depict a female for each host. Ellipses group 95% confidence interval of the data set (grey color = P. maidis; clear color = D. maidis. N = 10 individuals per host).
on the host preference previously found both in the field and the laboratory. Maize originated from wild annual teosinte Zea mays ssp. parviglumis (Iltis & Doebley) in Central America (Matsuoka et al., 2002), which would coincide with the center of origin established for D. maidis (Nault and DeLong, 1980). Several contributions on D. maidis egg parasitoids on different species of teosintes and maize plants were conducted demonstrating the close relationship between A. virlai and the corn leafhopper (Virla et al., 2013; Moya Raygoza and Triapitsyn, 2017). Peregrinus maidis, would have adapted only in post-Columbian times to maize as a host (Nault, 1983). Irvin and Hoddle (2010) demonstrated that the egg parasitoid Cosmocomoidea ashmeadi Girault (Hymenoptera: Mymaridae), who has a long history of coevolution with its host, the glassy-winged sharpshooter, has a better ability to make use of different ages of host eggs compared to a recently released Cosmocomoidea Howard exotic species. Presence of suboptimal hosts could be beneficial for the long-term maintenance of the system (Heimpel et al., 2003), because parasitoids do not completely suppress to usual host species and can use alternative hosts that are already established in maize agroecosystems. Such biological and ecological traits could be interesting for conservation biological control programs, which can be enhanced through habitat and culture management (Khan et al., 2008; Lou et al., 2014). Furthermore, we observed that many parasitized eggs of P. maidis presented larvae or pharate adults that had not completed their development. Apparently, the nutritional components of P. maidis eggs were not adequate enough to ensure A. virlai development. This last assumption may be reasonable given the low rate of emergence and the differential size found in the emerged wasps from D. maidis and P. maidis eggs. Egg parasitoids can present innate preferences for a given host that will depend mainly on the physical characteristics of eggs (Mansfield and Mills, 2004). The corn leafhopper and corn planthopper eggs vary in volume: D. maidis
Table 2 Influence of egg age and host species on different biological parameters of Anagrus virlai developed on Dalbulus maidis and Peregrinus maidis eggs at temperature 25 °C. Host species and host agesa
Dalbulus maidis 1 3 5 Peregrinus maidis 1 3 5 Mean ± SE are presented.
% parasitized eggs
% emerged wasps
Development timeb
n (total number of offered eggs)
49.3 ± 3.11 43.7 ± 6.81 46.9 ± 6.11
80.1 ± 2.52 72.9 ± 4.81 81.1 ± 3.73
16.1 ± 6.10 15.7 ± 6.26 16.1 ± 8.92
12 (7 6 2) 9 (7 7 3) 12 (9 0 4)
2.65 ± 0.52 7.86 ± 3.45 18.7 ± 8.46
6.89 ± 3.96 7.85 ± 4.68 19.9 ± 10.7
20 ± 400 21.3 ± 75 16.8 ± 30.3
15 (1131) 7 (4 3 6) 6 (4 7 4)
a
Host ages are expressed in days. Corresponding to 226, 287 and 307 wasps born from D. maidis eggs; and 3, 4 and 17 emerged wasps from P. maidis eggs. Development time is expressed in days. b
eggs had a 30% more volume than those measured for P. maidis. Larger eggs potentially provide more food and nutrients to support progeny development (Brotodjojo and Walter, 2006). Our results are similar to those obtained from a previous study with Trichogramma euproctidis (Girault) (Hymenoptera: Trichogrammatidae) in three different lepidopteran hosts, where the size of the adult parasitoid was influenced by the size and quality of the host (Martel et al., 2011). Likewise, in the Trichogrammatidae Trichogrammatoidea lutea Girault, the preferred host for oviposition among three lepidopteran host species was the one
Table 1 Results of a Kruskal–Wallis test in multiple comparisons among ages and interaction between age and host (Dalbulus maidis and Peregrinus maidis) for: number of parasitism, rates of emergence and development time of Anagrus virlai. Vector species
Parasitized eggs: 1, 3 and 5 days Adult emergence: 1, 3 and 5 days Development time: 1, 3 and 5 days
Dalbulus maidis
Peregrinus maidis
D. maidis vs P. maidis
Interaction between age and host
X2
df
P-value
X2
df
P-value
X2
df
P-value
X2
df
P-value
2.841 1.909 29.346
2 2 2
0.242 0.385 < 0.001
4.559 3.297 8.157
2 2 2
0.102 0.192 < 0.001
43.092 44.316 15.095
1 1 1
< 0.001 < 0.001 < 0.001
45.229 45.702 48.522
5 5 5
< 0.001 < 0.001 < 0.001
5
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Fig. 3. Response of Anagrus virlai females to volatile blends released from maize plants containing Dalbulus maidis or Peregrinus maidis eggs. Asterisks indicate significant differences in exact binomial test (*p < 0.05). N = 45 individuals.
confirm these assumptions and provide information about the chemical cues used during host selection by the studied egg parasitoid. This work contributes to a better understanding of an egg parasitoid and its relation to two hosts involved in corn diseases transmission, preventing the hatching of eggs of these vectors thus disrupting the epidemiological cycle (Ballal, 2013).
with the largest eggs (Mawela et al., 2013). A major volume of eggs, in turn, could be fundamental for the searching behaviour, identification and later acceptance of host (Moreau et al., 2009). Hence, we were unable to successfully maintain lab populations of A. virlai using solely P. maidis as host, due to the scarce the low number of parazited eggs, emergence of females, and incomplete development of most parasitoids. Vinson (1998) mentioned that the oophagous (sensu stricto) parasitoids are able to reject the older host eggs. However, Virla et al. (2005) suggested that the mymarids can belong to two groups based on their ability to exploit host eggs of different ages: those that can attack and develop only on newly laid eggs (without embryos) and those that attack eggs in all their developmental stages. In Cicadellidae and Delphacidae eggs, the appearance of anatomical structures distinguishable through egg chorion (in ≥90 h-old eggs) such as head cap and eyespot are visible characteristic of the stage of development of the embryo (AlWahaibi and Morse, 2009). In our laboratory tests, eyespots were observed 5 days after oviposition. Contrary to our expectations, parasitism and emergence did not differed when A. virlai used 1 to 5-days eggs of both hosts indicating that this thelytokous strain could have a certain plasticity in the selection of host age. Chantarasa-ard et al. (1984) found that A. incarnatus parasitized all immature stages of development (1–9 days old) of the brown planthopper Nilaparvata lugens (Stål), without considerable differences in parasitism and emergence. Anagrus delicatus (Dozier), an egg parasitoid of the planthopper Prokelisia marginata (Van Duzee), attacked eggs of all ages, but its levels of parasitism were higher in 5–6 days old eggs (Cronin and Strong, 1990). The fairyfly Cosmocomoidea morrilli (Howard), parasitized all egg ages of the glassy-winged sharpshooter Homalodisca vitripennis (Germar), reducing its performance in older (> five-day old) egg masses (Krugner, 2014). Numerous studies evaluating the host preference were conducted in the Trichogrammatidae family. Several species can detect host age, preferring young eggs and reducing clutch size as host age increases (Godin and Boivin, 2000). Contrary to this prediction, the studied population of A. virlai oviposited both in newly laid and old eggs of its habitual host (D. maidis), without significant differences in the number of attacked eggs. This amplitude in resource-use could be advantageous for intra and inter-specific competition (Irvin and Hoddle, 2005), although A. virlai ecological response when carrying out future massive releases in cornfields, should be evaluated subsequently. Hosts must be located using different strategies in response to longrange and short-range cues (Fatouros et al., 2008). Odor cues produced by herbivore-plant interaction and contact host kairomones play a key role in location behavior and host discrimination when parasitoids search a host to deposit their eggs (Meiners et al., 2000; Colazza et al., 2007). In our olfactometry assay, A. virlai tended to choose maize leaves oviposited with D. maidis eggs more frequently than those containing P. maidis eggs. Our results highlight that this species could be able to identify among patches of hosts with differential characteristics and quality, and in this way, focus its efforts in the more suitable hosts (Colazza et al., 2014). Some authors have reported that plant-induced volatiles themselves are not the major attractants for Anagrus parasitoids, but rather there is synergy among those and kairomones from eggs within plant tissues (Honda and Walker, 1996; Chiappini et al., 2012). We believe that the host preference could be influenced by qualitatively or quantitatively different volatile profiles that vary according to the hosts attacking the plant. Future studies are necessary to
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