Lethal effect of Goniozus legneri on Cactoblastis cactorum: A potential biocontrol agent for inundative releases

Abstract

Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), the cactus moth, is native to South America with a widespread distribution in Argentina. The larvae consume the interior of Opuntia spp. (Cactaceae) plants. The moth was used as a biocontrol agent against invasive non-native Opuntia spp. in many countries around the world. The cactus moth arrived unintentionally in Florida, USA, expanded its range and threatened Opuntia-based agriculture and natural ecosystems in southern North America. The insect is also a pest of cultivated O. ficus-indica L. in Argentina. An endemic South American parasitoid, Goniozus legneri Gordth (Hymenoptera: Bethylidae), is used in inundative biological control programmes against lepidopteran pests. The goal of this work was to evaluate G. legneri as a biocontrol agent to be used in inundative releases against C. cactorum. Mortality of C. cactorum by G. legneri was assessed at different spatial scales, as well as the interactions with Apanteles opuntiarum Martínez & Berta (Hymenoptera: Braconidae), a common Argentine natural enemy of C. cactorum. The ability of G. legneri to paralyse, parasitise and kill C. cactorum was confirmed. The paralysis inflicted on C. cactorum larvae reduced larval damage to the plants by 85%. Using two parasitoid species increased the mortality of C. cactorum larvae, but it was highly dependent on the order of their arrival. The combined mortality caused by both parasitoids was higher than a single one, in particular when G. legneri arrived first (56 ± 1%), suggesting asymmetric competition due to the preference of G. legneri attacking previously parasitised larvae. Goniozus legneri has potential as an inundative biocontrol agent of C. cactorum, but its interaction with the classical biocontrol agent A. opuntiarum needs to be considered.

Introduction

The cactus moth, Cactoblastis cactorum (Berg) (Lepidoptera: Pyralidae), is native to South America and has a wide distribution in Argentina (Briano et al., 2012; Varone et al., 2014). The larvae feed gregariously on Opuntia spp. (Cactaceae) plants, consuming the interior tissues. Burrowing activity usually causes secondary bacterial activity which hastens the destruction of cladodes (a modified flattened stem) and can kill the plant (Starmer et al., 1988). Opuntia ficus-indica L. is native to Mexico (Kiesling, 1998), but has been introduced in many parts of the world as a crop and ornamental (Ervin, 2012). The fruit and cladodes are used for human and cattle consumption; the cladodes also have multiple uses (Pimienta-Barrios and Muñoz-Urias, 1995; Soberón et al., 2001; Rodrigues et al., 2023). In the 1980s, C. cactorum arrived unintentionally in Florida, USA (Dickel, 1991), and expanded its range representing a threat to Opuntia-based agriculture and natural ecosystems in the southern USA and Mexico (Solis et al., 2004; Hight and Carpenter, 2009). In Argentina, this pest is present in around 40% of the orchards with cultivated O. ficus-indica (Varone et al., 2014), and produces a detrimental impact on its fruit production (Fuentes Corona et al., 2021).

Sustainable and low-environmental impact management tools are being developed to control C. cactorum, including classical biological control in invaded areas (Varone et al., 2015; Srivastava et al., 2019). Field surveys conducted in Argentina to search for natural enemies identified the koinobiont larval endoparasitoid Apanteles opuntiarum Martínez & Berta (Hymenoptera: Braconidae) (Mengoni Goñalons et al., 2014). Field experiments determined that it has the potential as a biological control agent, accounting for 80–90% of the total parasitism of C. cactorum (Varone et al., 2019) in almost half of the natural populations of C. cactorum (Varone et al., 2015). Highly specific to the genus Cactoblastis in Argentina (Varone et al., 2015), A. opuntiarum was exported to the USA where underwent host specificity testing on North American potential hosts (Srivastava et al., 2019). The parasitoid is considered host specific, not a risk to non-target species, and a request was submitted seeking approval to release it as a biological control agent in the North America against C. cactorum (Personal Communication, N. Benda, Florida Department of Agriculture and Consumer Services, Bureau of Methods Development and Biological Control, Gainesville, FL, USA).

Cactoblastis cactorum is a naturally occurring Opuntia pest in South America. In spite of the fact that A. opuntiarum is found attacking half of the C. cactorum populations in Argentina, larval damage to some Opuntia plantations can be serious (Lobos, 2006; Folgarait et al., 2018). A second parasitoid is under evaluation as an additional control measure against C. cactorum in Argentina; Goniozus legneri Gordh (Hymenoptera: Bethylidae), an endemic idiobiont ectoparasitoid (Legner and Silveira-Guido, 1983). Goniozus legneri is largely used in inundative biological control programmes in Argentina against lepidopteran pests on apple, pear, stone fruits and in nut orchards (Garrido et al., 2018a; Garrido et al., 2019). Some habits such as paralysing larvae and host-feeding, are considered positive attributes for a biocontrol agent (Skinner et al., 1990; Legner and Gordh, 1992; Balasubramanian, 2017). Goniozus legneri was introduced into California in 1979 to control the almond pest (Legner and Silveira-Guido, 1983; Legner and Gordh, 1992). The parasitoid is currently available in the USA for purchase and release from commercial insectaries (Wilson et al., 2020).

In general, koinobiont parasitoids (such as A. opuntiarum) do not paralyse or kill their hosts immediately after oviposition, and because parasitised hosts continue to develop for some time, it is possible for another parasitoid to attack the previously parasitised host (Wang et al., 2008; Magdaraog et al., 2012). Interspecific competition among parasitoids can occur when their larvae develop in or on the same host (Wang et al., 2008; Magdaraog et al., 2012; Yang et al., 2013; Costi et al., 2022). This type of competition can affect the establishment of a biocontrol agent and decrease the efficacy of parasitoids released for biological control (Denoth et al., 2002; Wang et al., 2008). Therefore, the effects of multiparasitism have received attention in many biological control programmes that involved the release of multiple parasitoid agents (Pedata et al., 2002; Rossbach et al., 2008; Ulyshen et al., 2010). Adding multiple species of biocontrol agents can significantly increase pest mortality and lead to a greater extent of control (DeBach, 1966; Stiling and Cornelissen, 2005; Aguirre et al., 2021). The justification to add G. legneri with A. opuntiarum into a biological control programme against C. cactorum will be influenced by the type of interaction between the parasitoid species and their overall impact on the target host population.

The goals of this study were to assess the lethal effect of G. legneri on larvae of C. cactorum, as well as to evaluate its interaction with A. opuntiarum, to determine the potential of G. legneri as a biological control agent for inundative releases against C. cactorum. To explore the interactions between parasitoids, we performed a series of laboratory and cage studies, coupled with black-box survival and functional response models, to investigate the effect of sequential exposure of host C. cactorum to different parasitoid species. Use by the two parasitoids of the same host larva will be determined, and the potential for competition between the two parasitoid species will be evaluated.

Materials and methods

Insect colonies and life cycles

Laboratory colonies of C. cactorum were maintained to provide host larvae for testing parasitism by G. legneri and A. opuntiarum. Insect rearing was conducted under 25°C, 70% RH, and a photoperiod of 16:8 (L:D) at the Fundación para el Estudio de Especies Invasivas (FuEDEI), Hurlingham, Buenos Aires, Argentina. Cactoblastis cactorum larvae were originally collected in a large O. ficus-indica plantation located near Santiago del Estero city, Santiago del Estero province, Argentina (S 27° 62′ 24.2″, W 62° 34′ 39.1″). Larvae were transported to the laboratory to initiate a laboratory colony of the cactus moth. Females lay eggs as a linear structure called an ‘eggstick’ (Dodd, 1940) that hatch simultaneously; larvae gregariously chew a hole to enter the plant and tunnel through the cladode while feeding. Larvae complete their development by building galleries as they feed on soft interior tissues and discard their feaces outside the plant. The final (sixth) instars leave the cladode, spin a cocoon, and pupate inside the cocoon mostly within plant litter near the base of the host plant. The complete life cycle takes from 4 to 6 months, depending on the host plant species, giving rise to two or three generations per year (Zimmermann et al., 2004; Varone et al., 2019).

Females of A. opuntiarum came from wild larvae of C. cactorum collected in the same O. ficus-indica plantation during the last host larval generation before winter, previously determined to have high parasitism rates (Varone et al., 2019). Larvae were lab-reared in groups of 50 until pupation when parasitised larvae were identifiable. Parasitised C. cactorum were individually stored in 20 ml plastic cups and checked daily until emergence of the adult parasitoids. Upon wasp emergence, females were confined with double the amount of males (4–8 females with 8–16 males) for 24 h to allow mating in a 3 L plastic canning jar with a modified 80-mesh lid. Added to the mating jar were a slice of O. ficus-indica (6 × 10 cm), a tablespoon of C. cactorum larval frass to stimulate female oviposition (Varone et al., 2020), and a damp strip of paper towel (1 × 3 cm) saturated with water and honey to provide moisture and food.

Individuals of G. legneri were obtained from the Instituto Nacional de Tecnologia Agropecuaria mass-rearing institute located in Alto Valle, Río Negro, Argentina. Mass-rearing of this parasitoid is carried out with fifth instar larvae of Galleria melonella (L.) (Lepidoptera: Pyralidae) as hosts because oviposition is higher in this species and produces a more efficient generational increase (Garrido et al., 2018b). Parasitoids were received at FuEDEI as pupae in 20 ml plastic cups. When female parasitoids emerged, they were immediately used in the experiments. Copulation was ensured because males of G. legneri emerge before females and enter the female cocoons for mating. After mating, the female seeks out a host larva and injects a poison through its stinger, causing paralysis that facilitates oviposition (Gordh et al., 1983).

Study 1: Lethal effect of G. legneri on C. cactorum

Three experiments were conducted, with increasing experimental spatial scale, to assess the lethal effect of G. legneri on C. cactorum larvae (fig. 1a–c). The first experiment was performed to test the ability of G. legneri to attack (kill and/or parasitise) C. cactorum in the presence or absence of a small refuge for the host larvae, and with different larval instars exposed to the parasitoid. Although C. cactorum larvae have internal feeding habits in the cladodes, they occasionally exit the cladode, either to move to a new one or to pupate, exposing themselves to parasitoids. Cactoblastis cactorum larvae were confined in a 300 ml vented container with or without a piece of cactus and with a single mated G. legneri (fig. 1a) female. Treatments with cacti present in the container consisted of either three to five second instar larvae (L2) or three to five fourth instar larvae (L4) together with a 3 × 3 cm cube-shaped piece of O. ficus-indica that served as a refuge for the host larvae. Larvae were placed together with the cactus cubes 24 h before the release of the female parasitoid, allowing larvae to feed and penetrate the cactus. For the treatment without cactus cubes (no refuge), only L4 host larvae were placed in the 300 ml test arena because they could survive starvation during the parasitoid exposure time. Female parasitoids were confined with larvae for 24 h and removed. Between 10 and 12 replicates of each treatment were conducted. An additional set of five replicates with three to five L4 host larvae plus cactus cubes, without parasitoids, served as controls. Cactoblastis cactorum larvae were fed with fresh O. ficus-indica cladodes as needed until they either completed development and pupated, or died. Containers were checked three times a week to record parasitised, dead, or pupated larvae. Parasitised larvae were isolated in 20 ml plastic cups until the emergence of G. legneri adults. The number and sex ratio of adult wasps per container were recorded.

Figure 1. Experimental designs to assess the lethal effect of Goniozus legneri on Cactoblastis cactorum larvae with increasing experimental spatial scale (a to c), and the interaction between G. legneri and Apanteles opuntiarum (d).

A second experiment tested the mortality of C. cactorum with increasing numbers of G. legneri females and larger pieces of O. ficus-indica (fig. 1b). Because C. cactorum larvae reduced cactus mass by feeding inside the cladode, the piece of cactus was weighed before and after the trial to estimate weight loss due to feeding. Groups of 20 L4 C. cactorum larvae were placed in an 8 l plastic vented container with half a cladode of O. ficus-indica of similar size, and larvae were allowed to penetrate the plant for 24 h. After 24 h, 5, 10, 15, or 20 G. legneri mated females were added to the containers with a solution of honey and water as a food source. After the death of all the females, containers were checked three times a week to record parasitised, dead, and pupated C. cactorum larvae until all larval hosts completed development or died. Groups of 20 larvae feeding on similar sized cladodes, unexposed to parasitoids, were placed in 8 l containers and served as controls for larval survival. Ten replicates of each treatment (and control) were conducted.

The third experiment was conducted in cages to evaluate the lethal effect of G. legneri on C. cactorum when the larvae were feeding inside live prickly pear plants (fig. 1c). Two-year-old O. ficus-indica potted plants (with two cladodes each) were infested with 40 L4 larvae of C. cactorum. Prior to the release of the parasitoids, and so that they could penetrate into the plant, larvae were placed in an open 20 ml plastic cup, and attached to the plant in a manner that the larvae were in contact with the surface of a cladode. Potted plants were placed inside a 120 × 50 × 50 cm rectangular mesh cage. After larvae feed for 48 h, 40 G. legneri mated females were released in the centre of the cage. A solution of honey and water was also provided as food for the females. Five replicates were conducted and five plants with C. cactorum larvae, without parasitoids, were used as controls. Since larvae leave the plant to pupate, a piece of cloth was placed at the base of each plant, which served as a pupation substrate. After the parasitoids' death, cages were checked three times a week to record the number of pupating C. cactorum, meaning that those larvae survived exposure to the parasitoids. Parasitised or dead larvae remaining inside the plants were not recorded since this would have required the destruction of the entire living plant system. After all larvae pupated (or died), damage to the plants produced by larval feeding was estimated as the percentage of visible surface damage to plant tissue (categories of 0, 25, 50, and 100% surface damage). The damage estimate, along with the number of successfully pupated larvae, were compared between treatments with and without G. legneri to identify the impact of the parasitoid on the test host larvae.

Study 2: Interaction between G. legneri and A. opuntiarum

The mortality produced by G. legneri and A. opuntiarum, acting together or separately, was tested with increasing amounts of C. cactorum host larval densities. In addition, the influence of refugia on parasitoid interaction was included in the host density tests; with a piece of cactus that served as a refuge for the larvae or without a piece of cactus (no refugia) (fig. 1d). Figure 2 highlights the trophic relationships between species that were investigated in this study.

Figure 2. Venn diagram of trophic relationships in this study. The yellow circles are the species, and the blue lines indicate the trophic relationships between species. The light pink filled circle indicates the entire group of hosts/prey of Cactoblastis cactorum (Cc) exposed to natural enemies. The medium-light pink circle indicates the subgroup attacked only by Apanteles opuntiarum (Ao, shown as Cc -> Ao), while the medium-dark pink circle indicates the subgroup attacked only by Goniozus legneri (Gl, shown as Cc -> Gl). The dark pink area indicates the Cc larvae parasitised by Ao and attacked by Gl (Cc -> Ao -> Gl).

The interaction study consisted of four treatments: host larvae exposed to only one species of the two parasitoids, host larvae exposed to one parasitoid species for 24 h and then the sequential exposure to the other parasitoid species (G. legneri followed by A. opuntiarum, and vice versa). In all four treatments, five larval densities (one repetition each) of 20, 25, 30, 35, and 40 L3 C. cactorum were confined with a single female parasitoid for 24 h in a 500 ml vented arena. In the treatments with sequential exposure of the two species of parasitoids, the female was also with the larvae for 24 h. After the first 24 h with one parasitoid species, the parasitoid was removed and the other species was immediately introduced for another 24 h. A piece of cactus was added in the refugia-present design when host larvae were placed in the arena, allowing them to enter and use it as food and refuge (fig. 1d). The same design was repeated (four treatments of parasitoids exposed to five larval densities) but only a slim piece of cactus was available as a food source, insufficient in thickness to be used as a refuge. At the end of all parasitoid 24 h exposures, host larvae were transferred to 3 l vented containers with a more significant piece of O. ficus-indica cladode for feeding and development. Larvae were checked thrice weekly to record parasitised, dead, or pupated larvae.

Data analysis

The lethality of G. legneri on C. cactorum was analyzed in the first and third experiments of Study 1 which compared larval mortality among treatments. For the first experiment, the number of G. legneri adults and the sex ratio of the F1 that emerged from the parasitised C. cactorum larvae were compared. All comparisons were made using a Kruskal–Wallis test because of the non-normal distribution of the data, and groups were identified post hoc using Duncan's test. For the second experiment of Study 1, a linear regression was conducted between larval survival, or relative loss of cladode weight, and the increasing number of female G. legneri exposed. All the analyses were conducted in Infostat version 2020e (Di Rienzo et al., 2008).

The interaction between G. legneri and A. opuntiarum of Study 2 was evaluated by measuring the effectiveness in the order of the release of the species causing host mortality through the development of a series of multiplicative models of cumulative mortality coupled with functional responses. The best-explaining model in terms of more explanatory power and less complexity was selected by means of the Schwartz criterion, or Bayesian Information Index, called BIC hereon (Schwarz, 1978).

Model formulation

The starting point of the model development was a null model in which the host mortality is constant:

(1)$$N_d\sim B( {N_o, \;p_d} ) $$

where N_o is the number of offered hosts and the number of dead hosts (N_d) is a random variable which follows a binomial distribution (B), with a probability of death equal to p_d. To add the effect of parasitoids, we modified the parameter p_d. The mortality caused by a given parasitoid i is:

(2)$$p_r( q ) = ( {q_i > 0} ) p_{di}$$

where p_r is the resulting mortality caused by parasitoid i, q_i is the number of parasitoids of species i in the experimental arena (zero or one), and p_di is the mortality caused by parasitoid i. If parasitoid i is absent, q_i is zero, and then, the resulting mortality is zero, otherwise, p_r is p_di.

As presented here, equation 2 assumes that the parasitoids follow a type-I functional response, with a constant attack rate resulting in a constant proportion of hosts attacked regardless of the number of hosts offered (Holling, 1959). More realistically, if the proportion of attacked hosts decreases with the number of hosts (N) because the handling time of the host is required, it reaches a saturation curve. Replacing equation 2 with a type-2 functional response model (Holling, 1959):

(3)$$p_r( {q_i, \;N} ) = q_ip_p/ ( {1 + p_phNq_i} ) $$

where q_i is the number of parasitoids in the experiment, p_p is the attack rate, and h is the handling time. Because at very low N, p_r is almost the same as p_r from equation 2, in the following equations all the p_r(q_i) or p_r(q_i, N) will be called p_r for simplicity.

If the ‘natural’ mortality of equation 1 is combined with mortality caused by parasitoids from equations 2 and 3, the following model is:

(4)$$\eqalign{& p_{di}( {q_i} ) = 1 - ( {1-p_o} ) ( {1- p_r} ) \cr & {\rm with} \cr & N_d\sim B( {N_o, \;p_{di}( {q_i} ) } ) } $$

where p _o is the natural mortality of the hosts. Consequently, the proportion of the dead hosts is given by those that remain after removing hosts that survived the other causes of mortality and the action of parasitoids.

The next model considers that both parasitoid species cause different mortality levels, thus the second term from equation 2 is incorporated into a new equation:

(5)$$\eqalign{& p_{d12}( {q_1, \;q_2} ) = 1 - ( {1-p_o} ) ( {1-p_{r1}( {1-p_{r2}} ) } ) \cr & N_d\sim B( {N_o, \;p_{d12}( {q_1, \;q_2} ) } ) } $$

where p_r1 and p_r2 are the proportion of hosts attacked by parasitoids 1 and 2, respectively. Given there are two parasitoids q₁ and q₂ acting sequentially the expected proportion of dead hosts is given by the remaining hosts that survived the natural cause of mortality and both parasitoids.

The following model considered that the mortality might also be affected by the availability of refuges (R) (pieces of cacti available in the experimental arena) and that the presence of refugees decreases the hosts' finding by the parasitoid at a certain ratio r. According to the function r(R) = r (R = 0), if there are no refuges R is zero, otherwise, R is one. In the case of R = 0, the mortality increases by r. Equation 4, is then modified as follows:

(6)$$p_{d12}( {q_1, \;q_2} ) = ( {1-r ( R ) } ) ( 1 - ( {1-p_o} ) ( {1-p_r( {q_1} ) ( {1-p_r( {q_2} ) } ) } ) $$

Finally, it is considered that the mortality might increase more than proportionally by the effect of the second parasitoid in the sequence:

(7)$$p_{d12}( {q_1, \;q_2} ) = ( {1-r} ) ( {1 - ( {1-p_o} ) ( {1-p_r( {q_1} ) ( {1-p_r( {q_2} ) } ) } ) p_{e12}} ) $$

where p_e12, is the extra mortality caused by the parasitoid 1, given that the host was first exposed to the parasitoid 2. Alternatively, the mortality might increase less than proportionally after the second parasitoid is added, for example, if there is a preference to attack hosts previously attacked as a form of interference competition. Thus the equation modifies as follows:

(8)$$p_{d12}( {q_1, \;q_2} ) = ( {1-r} ) ( {1 - ( {1-p_o} ) ( {1-p_r( {q_1} ) ( {1-p_r( {q_2} ) p_{e12}} ) } ) } ) $$

where p_e12 is the mortality caused by the second parasitoid, given that the attacked hosts were previously attacked by the first parasitoid.

Numerical methods (model selection and parameter calculation)

The data analysis was performed in two steps; first, a model selection procedure was performed to select the combination that produced the best explanatory power in terms of the log-likelihood of the model, and less complexity, in terms of the number of parameters. Second, the parameters of the selected model and their statistical distribution were calculated using Monte Carlo methods. A list of candidate models was created using different combinations of the equations described above (table 1). A Markov Chain Monte Carlo procedure using the Metropolis-Hastings algorithm was performed (MCMC) for all the candidate models, according to Gelman et al. (2003). A total of 20,000 iterations were discarded, as a burn-in for optimisation of the parameters, and 50,000 iterations for the parameters and information indexes calculation, named traces.

Table 1. List of models proposed to evaluate the interaction between the parasitoids Goniozus legneri and Apanteles opuntiarum on the mortality of Cactoblastis cactorum (see model selection procedure)

Pr is the mortality caused by a parasitoid, BIC is the Schwarz criterion, and gcd is the generalized coefficient of determination. In bold is the selected model after the procedure.

The BIC information criterion was calculated using these traces that contained the values of the parameters and the log-likelihood function at each iteration (Schwarz, 1978). The model with the lowest BIC value was chosen, and the trace of the iterations was used to calculate the parameters. Additionally, to describe how well the model described the data, we used a generalised r² calculated according to Cox and Snell (2018), because the variable was binomially distributed and therefore the classical r² was not applicable.

The model was written in the Python programming language version 3.10 (Van Rossum and Drake, 2009), with the libraries NumPy version 1.21.5 (Harris et al., 2020), and scipy version 1.7.3 (Virtanen et al., 2020).

Results

Study 1: lethal effect of G. legneri on C. cactorum

Goniozus legneri showed an overall ability to mainly kill, but also parasitise larvae of C. cactorum. This was the first record of the interaction between these two species. As a general trend, females of G. legneri stung the host larvae to first paralyse the host and then oviposited. However, paralysis was much more common than parasitisation. Host feeding was also observed, but not quantified, and likely contributed to host mortality. In the first experiment, all the larvae of C. cactorum were killed by G. legneri, independent of the larval instar exposed and the presence of a refuge (fig. 3, H = 13.04, p < 0.01). The proportion of parasitised larvae did not differ between treatments with different larval stages or the presence of refuge for the larvae (fig. 3, H = 1.11, p = 0.56), and ranged between 0.27 and 0.37. Neither the amount of G. legneri F1 adults nor the sex ratio obtained was different between treatments (H = 0.22, p = 0.23 and H = 0.95, p = 0.64, respectively). When L2 C. cactorum hosts were exposed, 0.56 ± 1.33 adults G. legneri developed, with a 0.87 ± 0.17 proportion of females. In the case of L4 hosts with refuge, 2.0 ± 1.93 adults developed, with 0.93 ± 0.12 proportion of females, while without a refuge, 1.42 ± 3.06 adults emerged and were all females.

Figure 3. Proportion of dead and parasitised larvae of different instars of Cactoblastis cactorum exposed to a single female of Goniozus legneri, with or without refuge for the host larvae. Data were analyzed with a Kruskal–Wallis test. Different letters indicate statistically significant differences.

In the second experiment, the increasing amount of G. legneri females generated increasing mortality of C. cactorum larvae. When 15 or more female parasitoids were exposed per 20 C. cactorum larvae, all larvae died in all replicates (fig. 4, F _{1, 23} = 25.26, p < 0.01). Additionally, the weight loss of the cladodes decreased with increasing amounts of female parasitoids in the experimental arena (fig. 5, F _{1, 23} = 23.01, p < 0.01) because the larvae of C. cactorum died as a consequence of parasitism or paralysis and stopped eating the plant.

Figure 4. Cactoblastis cactorum larvae killed by different numbers of Goniozus legneri female parasitoids.

Figure 5. Weight loss (in grams) of Opuntia ficus-indica cladodes due to feeding by 20 Cactoblastis cactorum larvae exposed to increasing numbers of Goniozus legneri female parasitoids.

In the third experiment, the capacity to kill and paralyse of G. legneri was also confirmed when C. cactorum larvae were completely inside the cactus cladode in a live-standing plant. In the presence of G. legneri, the proportion of dead C. cactorum larvae (0.96 ± 0.05) was significantly higher than in the absence of parasitoids (0.54 ± 0.12) (H = 6.82, p < 0.01). The percentage of visible damage generated to the cladodes by the larval feeding without the presence of the parasitoids was 90.0 ± 22.36%, while with the release of G. legneri, only 6.0 ± 13.41% of the cladode surface showed visible feeding damage.

Study 2: Interaction between G. legneri and A. opuntiarum

The best model in terms of explained variability and complexity using the BIC criterion was the 7th of the 13 models proposed (table 1). According to model 7, the interaction between parasitoids was explained by equation 8, and the equation was expressed as follows based on the estimated parameters:

(9)$$\eqalignno{& p_{dag}( {q_g, \;q_a} ) = ( {1-r} ) ( {1 - ( {1-p_o} ) ( {1-p_r( {q_g} ) ( {1-p_r( {q_a} ) } ) } ) } ) \cr & {\rm with} \cr & sp1 = g( {G. legneri} ) \cr & sp2 = a( {A. opuntiarum} ) \cr & {\rm and}\,p_{ga} = 1 \cr & p_{dga}( {q_a, \;q_g} ) = ( {1-r} ) ( {1 - ( {1-p_o} ) ( {1-p_r( {q_g} ) ( {1-p_r( {q_a} ) p_{ag}} ) } ) } ) \cr & {\rm with} \cr & sp1 = a( {A. opuntiarum} ) \cr & sp2 = g( {G. legneri} )}$$

The first line is for the experiment where G. legneri (sp1) attacked first, and the second is when A. opuntiarum (sp2) attacked first. As there was no effect of A. opuntiarum on G. legneri (the mortality inflicted by G. legneri does not change if A. opuntiarum attacked first), the parameter p_ga was equal to one and was eliminated from the first line of equation 9. As a result of this equation, the mortalities caused by both parasitoids simultaneously, after correcting for the presence of the refuges and the natural mortality, was estimated as:

(10)$$\eqalign{& P_gP_a = ( 1-p_r( {q_g} ) ( {1-p_r( {q_a} ) } ) \cr & P_aP_g = ( {1-p_r( {q_g} ) ( {1-p_r( {q_a} ) p_{ag}} ) } ) ) } $$

where P_gP_a is the mortality caused by both parasitoids when G. legneri attacked first, and P_aP_g is the mortality caused by both when A. opuntiarum attacked first.

Most of the proposed models had a high explanatory power with a gcd higher than 90%, and even some higher than 95%. While the selected model also had a gcd of more than 95%, it contained fewer parameters. The selected model revealed three findings:, the host larvae had a constant baseline natural mortality of near 20% (parameter p_o = without the action of parasitoids); the addition of an Opuntia cladode piece produced a refuge effect that decreased the mortality by nearly 5% (parameter r) (table 1); and within the range of offered hosts, the parasitoids attacked the larvae proportionally to the offered number, as occurs in a type I functional response.

Mortality caused by G. legneri was nearly double that caused by A. opuntiarum (parameters P_pg and P_pa, respectively) and the highest mortality was achieved when both parasitoids were acting together, with G. legneri arriving first (parameter P_aP_g) (table 2). This competitive interaction between both parasitoids was asymmetric, evidenced by the differences in mortalities related to the order in which the parasitoids were released in the experimental arena. The additional mortality caused by using G. legneri in the second place (according to the parameter P_ag) was almost half compared to the expected additional mortality caused if this species was used in the first place (P_ga) (table 2). A mortality of five percentile points higher was determined when A. opuntiarum attacked second, because that parasitoid attacked only the hosts that previously survived G. legneri. Goniozus legneri prefered to attack hosts previously attacked by their competitor A. opuntiarum, killing them in the process.

Table 2. A posteriori mean (μ) and standard deviation (σ) of the estimated parameters of the selected model PMSR + EA, the log-likelihood, and the probability of being killed by both biological control agents

Parameters P_aP_g and P_gP_a are according to equation 10.

Parameter p_ga was not estimated and assumed to be equal to 1.0, thus it has no estimation of error.

Discussion

This work presented the first record of the parasitoid G. legneri successfully parasitising the cactus moth, C. cactorum, and confirmed the lethality of the attack on host larvae. Also supported under laboratory conditions, the potential of using two parasitoid species, G. legneri and A. opuntiarum, together as biological control agents against C. cactorum without diminishing the efficacies of either parasitoid species.

The ability of G. legneri to paralyse, host feed, parasitise and kill C. cactorum was demonstrated even when larvae were feeding inside the cladodes. One of the more promising abilities that produced the lethal effect of G. legneri on C. cactorum larvae was the paralysis inflicted on hosts, followed by death of larvae. Damage to O. ficus-indica plants from larval feeding was decreased by G. legneri attack, likely enhancing plant health and survival. Because C. cactorum has two or three discrete generations per year (Varone et al., 2019), inundative releases of G. legneri during early larval stages could prevent Opuntia plants from severe damage.

Previous parasitisation studies of G. legneri attacking lepidopteran larvae under laboratory conditions reported higher mean numbers of parasitised larvae per female, as in the case of C. pomonella (13 larvae/female) (Laumann et al., 2000), and A. transitella (23 larvae/female) (Gordh et al., 1983). Given the low parasitisation rates found in the present work, the main mechanisms involved in the death of C. cactorum larvae appeared to be paralysis and host feeding. Female bethylid wasps paralyse their hosts by repeated stinging (Steiner, 1986) and produce toxins that permanently paralyze larvae (Skinner et al., 1990). The number of G. legneri stings was quite variable and when paralysis occurred (usually within 30 min), the C. cactorum larvae did not recover. In the present research, host feeding, another mechanism reported for bethylid wasps (Gordh et al., 1983) was observed, but not quantified.

Since G. legneri was first reported in southern Argentina in 2005 (Garrido et al., 2005), a G. legneri mass-rearing protocol was developed using the pyralid host G. mellonella. Numerous successful inundative biocontrol programmes were carried out as part of integrated management approaches to control pests of fruit trees with low negative environmental impact (non-target attacks). Goniozus legneri was also successfully used in inundative biological control programmes against lepidopteran pest species in the US (Gordh et al., 1983; Butler and Schmidt, 1985). In central California, the parasitoid was capable of significant population regulation of the navel orangeworm, A. transitella, due to its ability to increase the percentage of parasitisation with rising host densities (Legner and Silveira-Guido, 1983; Legner and Gordh, 1992). Given the potential of G. legneri, studies were also conducted with the parasitoid to evaluate behavioural responses to the carob moth, Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae), a major pest of pomegranate in Iran (Aleosfoor et al., 2014), and to the jasmine moth, Palpita unionalis Hb. (Lepidoptera: Crambidae), a destructive pest of young olive farms in Egypt and in most of the Mediterranean basin countries (El-Basha and Mandour, 2006). Currently, the commercial biological control industry has developed mass production, shipment and release methods for G. legneri against a variety of pests. In several areas of agriculture, inundative biological control has obtained considerable success and is now a reliable and appreciated element of IPM programmes (van Lenteren, 2012).

We found promising results for considering G. legneri as a biocontrol agent for inundative releases in a biological control programme. The next step will be to conduct field experiments to estimate the extent of control this parasitoid exerts over C. cactorum larvae and the outcome of the interaction with A. opuntiarum. Maximum mortality and improved control of the pest would be achieved with release ratios of 0.75–1 G. legneri females per larva of C. cactorum. Current inundative biological control programmes using G. legneri against fruit pests in northern Patagonia, Argentina, revealed that release ratios of 1:1 were obtainable and optimally controlled the pests (Garrido unpublished data).

In the absence of parasitoid competition, G. legneri produced higher mortality rates than A. opuntiarum. When the two parasitoid species interacted together with the same group of host larvae, C. cactorum larval mortality was increased compared to the use of a single parasitoid species. Although this increment was not additive, since there was asymmetric competition due to the preference of G. legneri to attack previously parasitised A. opuntiarum larvae, introducing both parasitoid species together as biological control agents was recommended because of greater mortality of the hosts. In plantations where A. opuntiarum does not occur naturally, larval mortality would be maximised by releasing G. legneri first, followed by A. opuntiarum. This superior performance of G. legneri was found in other idiobiont ectoparasitoids that paralyse the host by making it less likely for koinobiont endoparasitoids to find the host (Ulyshen et al., 2010; Wang et al., 2010). Moreover, the observed preference of parasitoids to oviposit in already parasitised hosts has previously been documented and can occur given the different competition strategies, competitive power, functional responses, advantages of arriving first, etc. (Bruzzone et al., 2018).

In summary, the results presented here support the idea that despite having found competition between parasitoids, the mortality caused by the two species continues to be higher than the use of only one. However, competition experiments under laboratory conditions need to be coupled with field investigations. The evaluation of host finding ability and adult mobility of the two parasitoids will be critical because it will directly influence the probability of sequential access to the host and confirm the outcome of combined attacks and the competitive interactions. The present work constitutes baseline information for the consideration of G. legneri as an inundative biocontrol agent of C. cactorum, and to further evaluate multiple biocontrol strategies.