Molecular and morphological identification of the alfalfa weevil larval parasitoids Bathyplectes anura and Bathyplectes curculionis to estimate the rate of parasitism

Alfalfa (Medicago sativa L.) is the most valuable cultivated forage crop in the world (Orloff 1997). In Spain, it is a traditional component of crop rotations covering 250,000 ha, accounting for ~ 20% of the alfalfa land area in Europe (Delgado and Lloveras 2020). Alfalfa is also an important reservoir for pest insects that infest alfalfa as well as surrounding crops (Pons and Nuñez 2020; Madeira et al. 2022).

The alfalfa weevil (Hypera postica Gyllenhal; Coleoptera: Curculionidae) is native to Eurasia (Hoffmann 1963) but has spread globally and is now one of the most destructive alfalfa pests (Goosey 2012; Hoff et al. 2002; Pons and Nuñez 2020; Saeidi and Moharramipour 2017; Soroka et al. 2019). In Spain, the weevil larvae cause serious damage to the first cutting (from March to the end of April). There is little information in the scientific literature about the ecology and pest status of this species in Europe. Natural enemies can reduce alfalfa weevil populations (Soroka et al. 2020). Alfalfa weevil larvae are parasitized by solitary endoparasitoid wasps of the genus Bathyplectes (Hymenoptera: Ichneumonidae) which is native to the Old World (Kingsley et al. 1993; Kuhar et al. 1999; Radcliffe and Flanders 1998). B. anura and B. curculionis were introduced to North America with remarkable success as a weevil control strategy (Radcliffe and Flanders 1998; Rand 2013). Although eight Bathyplectes species have been recorded in Spain (Ribes 2012), only B. anura Thomson and B. curculionis Thomson are associated with alfalfa. The identification of adult wasps is challenging because there are only slight morphological differences between species (Pons and Nuñez 2020), especially in the male (Soroka et al. 2020). The puparia are easier to distinguish because B. anura forms a hard, dark-brown puparium with a narrow, raised, white horizontal band, whereas the B. curculionis puparium is light brown with a flat, diffuse, white horizontal band (Day 1970; Dysart and Day 1976). For definitive parasitoid identification, each H. postica larva must therefore be reared until pupation, which requires optimal environmental conditions and feeding, and the avoidance of other natural factors that cause mortality. Using this approach, the rates of parasitism recorded in Spain are generally low but highly variable (Levi-Mourao et al. 2021; Pons & Nuñez 2020).

In contrast to the delayed results from rearing experiments, DNA analysis allows parasitism to be followed in real time, and does not require the use of captive insects in controlled-environment chambers (Liang et al. 2015, 2018; Wolf et al. 2018; Agustí et al. 2020; Molina et al. 2021). The high sensitivity and fidelity of molecular methods also facilitate detailed studies of trophic interactions that are otherwise inaccessible (Traugott et al. 2013). Such methods require the development of specific molecular probes to detect target organisms. In arthropods, the evolution of mitochondrial genes has been studied in detail, and divergent sequences in related populations provide a source of species-specific PCR primers (Black et al. 1989; Simon et al. 1994). Several studies have used cytochrome C oxidase subunit I (COI) mitochondrial DNA fragments as targets to increase the specificity of detection (Agustí et al. 2003a, 2005). Here we report for the first time the development of COI primers to detect and identify the main parasitoids of alfalfa weevil (B. curculionis and B. anura) in order to estimate the rate of parasitism in H. postica larvae compared to the classical rearing method.

We have developed a new molecular method based on the amplification of the mitochondrial COI gene for the quick detection of B. anura and B. curculionis in H. postica larvae. The direct analysis of parasitoid DNA is an alternative to the time-consuming morphological analysis of puparia, which requires the rearing of insects in containment. The mitochondrial COI gene has proven useful for the reliable identification of other morphologically similar species (Nanini et al. 2019; Solà et al. 2018; Traugott and Symondson 2008). We therefore designed specific primers that discriminate between the B. curculionis and B. anura COI genes, a strategy that has been successful for other parasitoids and predators (Agustí et al. 2003b, 2005). The new strategy can be used to investigate interactions between H. postica and its larval parasitoids in more detail.

The classical technique revealed a larger number of emerging adults for B. anura compared to B. curculionis and thus a greater rate of parasitism, suggesting that B. anura is more prevalent than B. curculionis in the study area, as previously reported (Pons and Nuñez 2020). In regions of the New World where the two species are colocalized, B. anura is generally dominant over B. curculionis and can even displace it completely due to greater reproductive capacity and aggression, and more successful host finding (Harcourt 1990). Our new molecular method detected up to seven times as many parasitism events as the classical technique, indicating greater sensitivity and thus more reliability when estimating the rate of parasitism, as suggested for other species (Agustí et al. 2005; Gariepy et al. 2008; Gomez-Polo et al. 2014). Furthermore, the molecular method suggested for the first time that the rate of parasitism caused by B. anura and B. curculionis does not differ by so wide a margin as suggested by the classical method (Pons and Nuñez 2020; Levi-Mourao et al. 2021). The mean rate of parasitism was similar for both species, suggesting that the two species coexist in the alfalfa crops of north-east Spain. Moreover, the classical and molecular methods both showed that B. anura was the prevalent species in March, when H. postica begins to attack alfalfa crops, whereas B.curculionis was slightly more prevalent during April. This indicates a succession from one species to the other, as recently reported (Levi-Mourao et al. 2021) and may explain the absence of multiparasitism by both species.

The major and most effective parasitoid of H. postica in some regions of North America is thought to be B. curculionis (Berberet and Bisges 1998; Radcliffe and Flanders 1998; Soroka et al. 2019). However, its effectiveness is often comprised by the encapsulation of the parasitoid egg by hemocytes in the host hemocoel (Berberet et al. 2003; Salt and van den Bosch 1967; Shoubu et al. 2005). H. postica L1 larvae have little defense against parasitism, but 30–50% of the L2–L4 instars survive as a result of encapsulation (Berberet et al. 1987; van Den Bosch and Dietrick 1959). This may explain why we detected a larger number of B. curculionis parasitism events by PCR compared to conventional rearing. PCR-based methods can overestimate the rate of parasitism because they detect parasitoids that are already neutralized by the host immune system, whereas the classical method allows the direct measurement of parasitoid survival (Traugott et al. 2006). On the other hand, classical rearing techniques are influenced by the mortality of parasitoids under laboratory conditions, which can result in partial data loss (Tilmon et al. 2000). Furthermore, the puparia of each species have different environmental requirements to complete their life cycle, which can also influence the results. For example, B. curculionis can extend its diapause up to 10–12 months in an unfavorable environment (Radcliffe and Flanders 1998), but high emergence rates were achieved by placing the puparia in a refrigerator for 4–6 months before transfer to an environment set at 21 °C with a 12-h photoperiod (Jacob and Evans 2000; 2004). The low effective rate of parasitism we observed in the case of B. curculionis may be due to the maintenance of the puparia in rearing cages under laboratory conditions. In contrast to B. curculionis, B. anura eggs are almost never encapsulated by H. postica (Maund and Hsiao 1991; Puttler 1967). This may explain the correlation between the two methods during 2019, when there was no additional mortality caused by Z. phytonomi.

Our results also showed that B. anura females prefers L3 H. postica larvae because no DNA was found in L2 larvae, agreeing with previous findings (Bartell and Pass 1980; Dowell and Horn 1977). In contrast, B. curculionis targeted L2 larvae, concurring with reports showing that this species favors early-instar H. postica larvae and that this is strictly related to high parasitoid larval survival (Duodu and Davis 1974; Barney et al. 1978). The differences in larval instar preference probably reflects the length of the ovipositor, which is longer for B. curculionis and facilitates the utilization of early instars still hidden in unfolded leaves and buds (Dowell and Horn 1977). Our results also suggest that B. anura has a shorter larval development phase than B. curculionis.

Given the differences in parasitoid occurrence, development, and host instar preferences, alfalfa crop management during the first cutting could be optimized to enhance the survival and development of B. anura and B. curculionis. Bathyplectes anura can survive and complete its development during alfalfa weevil infestation because it appears earlier in the field, favors late-instar host larvae and develops more quickly (Levi-Mourao et al. 2021). In contrast, the survival of B. curculionis can be seriously compromised by the timing of first cutting because it appears later in the field (mainly during the second half of April, when most H. postica larvae have nearly completed development) and favors young larvae which are increasingly scarce by this time point (Levi-Mourao et al. 2021; Levi et al. in preparation). The commercial cutting of alfalfa at the end of April eliminates almost the entire H. postica population, so bringing this forward to reduce yield losses could severely limit the availability of hosts for B. curculionis. A delay in this practice can help to the survival of this parasitoid species. Beside this, a recent study in the Ebro Basin on the efficacy of a winter alfalfa cutting (Levi-Mourao et al. 2022) to reduce the egg population and to prevent the development of larvae of H. postica at the first alfalfa commercial cutting, shows that the larval density was significantly reduced, whereas the rate of parasitism increased, especially B. anura, the prevalent species at the beginning of the spring. Furthermore, the reduction of alfalfa weevil larvae below the economic thresholds, enhanced by the winter cutting, would allow delaying the date of the first commercial cutting and, in turn, B. curculionis survival.

Parasitism rates in 2020 were lower than in 2019 due to the presence of Z. phytonomi. Epizooties of this fungus occur in years with high rainfall (Barney and Armbrust 1981; Kuhar et al. 1999). This was the case in 2020 but not in 2019, which featured a dry winter. Although, Z. phytonomi kills H. postica larvae and is considered an important biological control agent (Harcourt and Guppy, 1991; Giles and Obricky, 1997), it also kills parasitoid larvae (Giles et al. 1994; Kuhar et al. 1999). Our results show that epizootic infections of this fungus disrupt the alfalfa weevil–parasitoid system under Spanish crop conditions. The lower B. anura parasitism rate in 2020 suggests that this species was probably the most affected by the fungus. B. curculionis appears later in the study area and therefore has an advantage over B. anura because the environmental conditions no longer favor the spread of the disease, increasing the likelihood of host survival until pupation and thus the survival of the parasitoid.

Parasitism rates in 2019 varied at the field level, with maximum values of 37% for B. anura and 17% for B. curculionis. This agrees with other studies in the same area, where variable rates were reported with a maximum of 30% (Pons and Nuñez 2020). These parasitoids were most effective when introduced into North America to control the alfalfa weevil. The rate of parasitism with B. curculionis tended to be high, at times exceeding 90% in the mild San Francisco Bay and Pleasanton areas, but this approach was much less effective in the hotter San Joaquin Valley (Radcliffe and Flanders 1998). Rearing studies conducted in south-western Canada revealed B. curculionis parasitism rates of up to 17% (Soroka et al. 2020). This suggests that environmental conditions play a key role in the success of parasitism, with hotter temperatures inhibiting parasitoid performance, and can explain our lower rates recorded in our study area. In other regions of North America, where B. anura tends to be the prevalent species (as is the case in Spain), the rate of parasitism was similar to our findings (Harcourt 1990; Berberet and Bisges 1998).

In conclusion, our new molecular strategy provides information about the ecology of B. anura and B. curculionis, reveals the prevalence of both species, and contributes to the development of biological control strategies in Europe. Our results show that specific primers can be used to detect and identify both endoparasitoid wasps in alfalfa weevil specimens, and provided an alternative way to estimate the rate of parasitism in the field. One drawback of the new method is its tendency to overestimate the rate of parasitism by counting unsuccessful events. Accordingly, we recommend that DNA analysis should be combined with conventional rearing to determine the effective rate of parasitism and also to accommodate interactions with other species that are not specifically targeted by the molecular assay. In spite of the potential of Bathyplectes sp. as a biological control agent for H. postica, it seems that the alfalfa crop management system currently performed in Spain may be unfavorable to their control capacity. However, the incorporation of a winter cutting and the delay of the first spring alfalfa cutting, which increase the rates of parasitism of B. anura and can help to the survival of B. curculionis, respectively, are tools that can be included in integrated pest management strategies in Spain and, potentially, in other Mediterranean countries.