1. Introduction
Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), was discovered infesting urban citrus in San Diego County California, USA, in 2008 [1]. The invasion of this pest in California is significant because D. citri transmits a bacterial pathogen, Candidatus Liberibacter asiaticus (CLas) [2], which causes a lethal citrus disease, huanglongbing [3]. The first tree with CLas was found in Los Angeles County, California, in 2012 and infestations are largely restricted to urban areas in southern California [4]. Backyard citrus trees in southern California are ubiquitous and can harbor D. citri, which facilitates the transmission and acquisition of CLas [5]. Should CLas spread from urban citrus into commercial production zones, especially the San Joaquin Valley, the major citrus producing region in the state, it would potentially jeopardize ~262,000 fruit-bearing acres [6], which supports a total of citrus-related revenue worth an estimated USD 7.1 billion per year [7].
A biological control program against D. citri in California was initiated in 2010 [8,9]. This program focused on two nymphal parasitoids, Diaphorencyrtus aligarhensis Shafee, Alam, and Argarwal (Hymenoptera: Encyrtidae) and Tamarixia radiata Waterston (Hymenoptera: Eulophidae), both of which were imported from Punjab, Pakistan, an area with a good climatic match to southern California [8]. Imported parasitoid species were subjected to host range and host specificity testing and approved for release [10,11]. In urban areas, biological control of D. citri is viewed more favorably by homeowners than large-scale treatments of backyard trees with insecticides [12], while the reduction of vector densities is critical for reducing the rate of spread of CLas by D. citri [13,14]. Consequently, the goal of the biological control program with D. aligarhensis and T. radiata was to use parasitoid complementarity to reduce densities of D. citri with the concomitant goal of slowing rates at which CLas could spread from urban areas into commercial citrus production zones.
Complementarity biological control of D. citri by T. radiata and D. aligarhensis aimed to reestablish in California the two species parasitoid guild from part of the presumptive native range of the target pest (from Punjab, Pakistan) [8] and to exploit parasitoid species preferences for different host stages. At the time this classical biological control program was initiated, it was reported that D. aligarhensis, an arrhenotokous endoparasitoid, favorably parasitized second and third instar D. citri nymphs [15], while T. radiata, an arrhenotokous ectoparasitoid, favorably parasitized fourth and fifth instar D. citri [16]. Subsequently, laboratory studies with both species of parasitoid sourced from Punjab, Pakistan, indicated that D. aligarhensis and T. radiata exhibited a shared life stage preference for fourth instar nymphs, a finding not previously documented [17]. Both parasitoid species can kill D. citri via feeding on nymphs [18,19,20].
In California, T. radiata was first released against D. citri in December 2011 [8]. Since this time, >23 million T. radiata have been released at >19,000 sites [21], with evidence of establishment at >90% of release sites in southern California, including recoveries in areas >10 km from release sites indicating extensive natural dispersal [22]. Impact studies have documented reductions >75% in D. citri densities in some areas, due in part to parasitism by T. radiata, which was recorded at >60% during periods of peak parasitoid activity [23]. The release of D. aligarhensis in California began in December 2014 [11]. Subsequently, >700,000 D. aligarhensis have been released at >300 sites in southern California since 2014 [21]. In an attempt to reduce any negative interspecific interactions that could affect the establishment of incipient populations [24], initial releases of D. aligarhensis were made in areas infested with D. citri that had not received releases of T. radiata.
This decision to release D. aligarhensis in areas with no or little competition from T. radiata was further enforced by reports that this parasitoid failed to establish in Florida, despite multiple releases of two thelytokous populations sourced from Taiwan (released 2000–2002) and China (released 2007–2009) [25]. Tamarixia radiata was first released in Florida in 1999 [26] and had reportedly established by 2001 [27] but interspecific interference could have been a factor in the lack of establishment for D. aligarhensis [25,28]. Other factors affecting outcomes of interspecific competition between D. aligarhensis and T. radiata and subsequent establishment likelihoods could be preferences for performance differences across heterogeneous climates and varieties of citrus cultivars, which may be more favorable for one species but not the other [29]. Range partitioning by different species of biocontrol agents attacking a shared citrus pest has been formerly recorded in California [30]. At the time this study was conducted, it was unknown whether D. aligarhensis had established in California, if establishment rates and efficacy of D. aligarhensis would vary across diverse climatic regions in southern California or different citrus cultivars, or how interactions with T. radiata would affect the collective impact of both parasitoid species on suppression D. citri populations.
2. Materials and Methods
2.1. Parasitoid Release and Monitoring
2.1.1. Study Sites
Fifteen residential sites, five coastal (United States Department of Agriculture [USDA] Hardiness Zone 10b), five intermediate (USDA Hardiness Zone 10a), and five desert (USDA Hardiness Zones 9a/9b) sites [31] for the release and monitoring of D. aligarhensis were established across five southern California counties: San Diego, Orange, Riverside, Los Angeles, and San Bernardino (Table 1; [31]). Temperature and humidity data for each of the 15 study sites were obtained from the closest meteorological station (Table 1; [32]). Each site had one lemon (Citrus limon L.) and one orange (C. sinensis L.) tree for a total of 30 study trees older than 5 years. No insecticides were applied to study trees for the duration of this project (Table 1). The release and monitoring of D. aligarhensis activity across study sites took place between 10 August 2015 and 29 March 2017, and 1 January 2016 and 31 December 2017, respectively (Table 1). No releases of T. radiata were made at these 15 sites during this time and each D. aligarhensis study site was located ~1–10 km from the nearest T. radiata parasitoid release locations ([21]; Table 1 and Table S1). The sites were not examined for the presence of T. radiata prior to the release of D. aligarhensis in 2015.
2.1.2. Parasitoid Sources and Culture
Individuals of D. aligarhensis used for releases were collected from colonies maintained at the University of California Riverside Insectary and Quarantine Facility (UCR-IQF) and the California Department of Food and Agriculture Mt. Rubidoux Station Facility. At UCR-IQF, parasitoids were reared on second through fourth-instar D. citri nymphs cultured on potted 1- to 2-yr old “Volkamer” lemon plants (C. volkameriana V.Ten. & Pasq. [11]. All plants were grown from seed in temperature- and humidity-controlled greenhouses at UCR Ag. Operations, at 27 ± 1 °C, 50 ± 20% RH, under natural light, before they were moved to rearing cages at UCR-IQF [11]. At UCR-IQF, plants were pruned regularly to produce new leaves required for oviposition by D. citri females and subsequent development of nymphs [11,18]. Five D. aligarhensis isocage breeding lines representing different collection localities and dates from foreign exploration trips to Punjab, Pakistan, were maintained to preserve the genetic diversity of mass-produced parasitoids [33,34,35]. Colonies of D. aligarhensis were maintained in one of the five Bugdorms (model 2120F [MegaView Science Co., Ltd., Taichung, Taiwan]) labeled by locality and date of collection. Rearing cages were individually kept inside a larger Bugdorm (model 2400F). This double-cage containment was implemented to reduce the likelihood of accidental escape. All colonies were maintained at 27 °C, 40% RH, and a long-day photoperiod of 14 h [11]. Parasitoids were given water to drink and honey as an edible carbohydrate source. Newly emerged female and male parasitoids were collected daily by aspiration into individual 200 μL centrifuge tubes (model 89004-308, VWR, Radnor, PA, USA). Parasitoids were maintained at 12 ± 2 °C with droplets of 50% honey-water solution for 1–7 d prior to field release.
At the CDFA Mt. Rubidoux station rearing facility, D. aligarhensis colonies were maintained on mature curry leaf plants (Bergera koenigi L., formerly Murraya koenigi) infested with mixed stages of D. citri [21]. Parasitoid colonies were maintained in organdy-screened cages (BugDorm-2 400F, insect rearing tent; length 75 cm × width 75 cm × height 115 cm, 150 × 150 mesh size) held inside glasshouses at 27 ± 2 °C, 50 ± 6% relative humidity (RH), with a 14:10 (L:D) h photoperiod [21,36]. These colonies were initiated and maintained, in part, with D. aligarhensis from the original five UCR-IQF isocage lines sourced from Pakistan (see above). Adult parasitoids were aspirated daily into 150 mL plastic vials (Thermo Fisher Scientific, Waltham, MA, USA) from rearing cages. Tissue paper moistened with water was placed inside the vial to provide water, while honey, a carbohydrate resource, was streaked on vial lids. Parasitoids were maintained at 12 ± 2 °C for 1–7 d prior to their field release [21].
2.1.3. Field Release and Monitoring
Releases of D. aligarhensis were conducted between 10 August 2015 and 29 March 2017 (see Section 2.1.1; Table 1). At each study site, parasitoid releases were contingent upon the presence of D. citri life stages susceptible to D. aligarhensis attack and available flush on study trees susceptible to D. citri infestation. The number of D. aligarhensis liberated on any release date varied, ranging from 14 to 1860 females per release date and site. This variation in numbers of parasitoids released resulted from varying levels of insectary production during the course of this study. Parasitoids contained within plastic vials with honey streaks were released onto D. citri-infested citrus between 8:00 a.m. and 1:00 p.m.
Post-release monitoring was conducted at all study sites every two weeks from 1 January 2016 through 31 December 2017 (see Section 2.1.1; Table 1). Experimental trees were divided into quadrants (north, east, west, and south) to control for differences in psyllid densities related to cardinal direction [23]. The numbers of larval and adult D. citri, and the number of nymphs parasitized by either D. aligarhensis or T. radiata, were recorded per quadrant per tree sampled [23,37,38]. Citrus flush density was assessed on each sampling date by randomly selecting eight branches around the study tree, within arm’s reach and ~1.5–2 m above the ground [23,37,39]. The total number of D. citri individuals per colony and parasitism status were estimated by excising two randomly selected flush growths from each cardinal quadrant for a total of eight samples. If fewer than eight flush shoots were present, all were collected. Collected flush samples were placed singly into sealable bags in an insulated cooler with ice in the field and transported back to the laboratory for processing in compliance with CDFA permit 2870. Samples were refrigerated at 4 °C for 1–2 days before being processed. The length (cm) of each collected flush sample was recorded so that numbers of larval and adult psyllids could be estimated by the length of the sampled flush [23,39]. All psyllid mummies with parasitoid exit holes were counted, identified by parasitoid species, and removed: T. radiata emerges from the anterior thoracic region [20], whereas D. aligarhensis emerges from the posterior abdominal region [15,19] of mummified D. citri.
Dead nymphs lacking exit holes were removed from flush and inspected under a dissecting microscope to determine causes of mortality and the frequency of successful parasitism. Fourth and fifth instar nymphs were inspected on ventral sides for the presence of larval T. radiata [17,40,41]. Second through fourth instar nymphs with no obvious presence of T. radiata eggs or larvae were dissected for the presence of larval D. aligarhensis [19]. Under a dissecting microscope, each nymph was placed in a drop of saline solution on a depression slide and dissected using stainless steel insect pins (size 000) attached to wooden satay stick handles. Eggs, larvae, and pupae found within D. citri nymphs were classified as D. aligarhensis, added to the appropriate parasitoid tallies, and preserved in labeled vials in 95% ethanol for possible future genetic analyses. The total rates of parasitism were calculated across the two parasitoid species for each tree by study site and sampling date. Species-specific parasitism rates were calculated after excluding host instars that were unsuitable for the development of immature parasitoids.
2.2. Parasitoid Establishment
Establishment was monitored at 39 residential sites at which no releases of T. radiata were made (15 previously received D. aligarhensis and 24 served as controls) in February–March 2018 and September–October 2018 (Table 1 and Table S1). No insecticide applications were made at these study sites. The control sites were >1 km apart from the closest D. aligarhensis and T. radiata release sites. The methods described above were used to assess parasitoid establishment through recovery of D. citri mummies with parasitoid exit holes and dissections of D. citri nymphs to detect D. aligarhensis eggs, larvae, or pupae. Surveys of selected sites were timed to coincide with the presence of citrus flush infested with D. citri life stages that D. aligarhensis could attack. If citrus flush and D. citri nymphs were not present at sites, repeat visits were made until optimal conditions for parasitoid activity occurred. If parasitoids were not detected after two consecutive surveys at sites where conditions were suitable for their activity, the site was classified as “not established”.
2.3. Data Analysis
All statistical results were generated using SAS (version 9.4; [42]). The relationship between observed daily temperatures and climate type was estimated with a Gaussian linear mixed-effects model and PROC MIXED procedure in SAS. The observed mean daily temperatures of each study site were aggregated into mean monthly values that were considered repeated measurements. The fixed effects included in the model were year, climate type, and sampling month. All two-way interactions of the fixed models were included in the initial models. The site main effect and all its interactions with climate type, year, and sampling month were considered random. This analytical approach controls for potential temporal effects of climate on observed psyllid populations and parasitism rates related with temperature differences [23].
Psyllid densities were analyzed with hierarchical generalized linear mixed models (GLMMs) with a log link function and Poisson error structure. Parameters for the GLMMs were estimated using the PROC GLIMMIX in SAS [42]. Biweekly sampling periods of study sites were classified into sampling seasons, namely, cool (10–15 °C; sampling periods 1–4, 23, and 24), moderate (15.01–20 °C; sampling periods 5–10 and 19–22), and warm (20.01–25 °C; sampling periods 11–18), which facilitated the interpretation of potential temporal effects (see [23]). The initial full model included the main effects of climate type (coastal, intermediate, desert), host plant (orange, lemon), season (cool, moderate, warm), and their interactions as fixed effects. For the covariate percentage of shoot samples with flush, year was included as a blocking variable. Random effects included site and host tree identity to account for the non-independence of observations taken from within each site and repeated measures, respectively. This analytical approach accounts for the pseudo-replication inherent in hierarchical experimental designs [23]. The models also included measurements of the percent parasitism from the previous sampling period as a covariate to control for possible time-lagged impacts of parasitism on recorded psyllid densities [23].
GLMMs with the same model formula and binomial error structure [43] were used to assess the effects of climate type, season, year, and host plants on parasitism by each of the two parasitoid species over time. The model also included psyllid densities from the prior sampling period as a covariate. This analytical approach controls for possible delayed density-dependent mortality from parasitoids. Only the densities of nymphal instars vulnerable to attack by each species of parasitoid (second through fourth stage nymphs for D. aligarhensis parasitism and fourth and fifth stage nymphs for T. radiata parasitism) were utilized in species-specific models. Climate effects were assessed using 3 lag periods of 0, 1, and 2 sampling months prior to that being analyzed. Best fit time lag (i.e., one month prior to the sampling period being analyzed) was selected based on Akaike’s and Bayesian information criteria (AIC and BIC, respectively) [23].
The full regression models included all the variables. Insignificant variables were removed using a critical p-value of 0.15, while the AIC and BIC analyses were performed until no further improvements to fits were achieved [23,44,45]. The Kenward–Roger adjustment (option ddfm = kr in SAS) was used to calculate the denominator degrees of freedom for the fixed effects [23,42]. The Tukey–Kramer HSD test was used to see which group mean pairs were significantly different at α < 0.05.
3. Results
3.1. Effects of Climate Type and Sampling Month on Mean Daily Temperatures
Daily mean temperatures varied with climate zones (F = 4.17; df = 2, 11; p = 0.04) and sampling months (F = 612.12; df = 11, 1418; p < 0.001), while climate × sampling month interaction was also significant (F = 8.35; df = 22, 1418; p < 0.001). Mean temperatures were similar in 2016 and 2017 (p > 0.28 for main and interaction effects of year; Figure 1). Average monthly temperatures ranged from 9.3–24.9 °C for the coastal climate, from 10.6–29.1 °C for the intermediate climate, and from 11.2–32.8 °C for the desert climate throughout the study. Daily temperatures averaged 17.65 ± 0.18 °C (±SE throughout text) at coastal sites, 19.18 ± 0.2 °C at intermediate sites, and 19.89 ± 0.24 °C at desert sites. Major differences in temperatures between climate zones were recorded during warm seasons (climate × month effect; Figure 1). The coastal climate zone had lower temperatures during warm months than the intermediate and desert climates but temperatures were insignificantly different during cool and moderate months (Figure 1). Temperatures for the intermediate climate closely matched the desert climate during warm months (Figure 1).
3.2. Effects of Climate Type, Season, and Parasitism on D. citri Densities
Psyllid densities differed with season but not with climate zone (Table 2A). Psyllid densities were negatively correlated with parasitism rates from the previous sampling period (Table 2A, Figure 2), indicating the existence of time-delayed negative impacts on psyllid densities. Psyllid densities differed between cool and warm months and moderate and warm months, but not between cool and moderate months (Table 2A). Psyllid densities experienced two peaks in February through May and September through November each year irrespective of climate type (Figure 2). Mean psyllid densities were positively correlated with citrus flushing patterns (Table 2). Psyllid population trends were similar on lemons and oranges over time (Table 2). Psyllid densities were 50% higher in 2016 than in 2017 (Table 2A, Figure 2) irrespective of climate zone. None of the interactions between tested variables were significant (p > 0.15).
3.3. D. aligarhensis Establishment and Parasitism Rates
A total of 39,990 females and 30,371 males of D. aligarhensis were released from August 2015 through March 2017 at the 15 sites monitored (Table 1). Parasitism by D. aligarhensis was exceptionally low, averaging 0.62 ± 0.3% from 2015 through 2017 (Figure 2). Parasitism rates of D. aligarhensis averaged 0 ± 0%, 0.89 ± 0.2%, and 0.53 ± 0.1% during the cool, moderate, and warm seasons, respectively (Figure 2). Parasitism rates of D. aligarhensis averaged 0.63 ± 0.4% at coastal sites, 0.91 ± 0.6% at intermediate sites, and 0.39 ± 0.2% at desert climate sites. Because of this scarcity of recovery data for D. aligarhensis, no definite conclusions could be drawn about the importance of climate effects on parasitism rates. We recovered 12 D. aligarhensis (all larvae and eggs) from nine trees that were surveyed in 2016 and 2017. All recoveries were from just six sites and occurred within one month of release. No D. aligarhensis were recovered in 2018 from all 39 study sites, 15 of which previously received D. aligarhensis and 24 of which served as controls (Table 1 and Table S1). Hence, it was concluded that D. aligarhensis had failed to establish and all subsequent references to parasitism refer to T. radiata only.
3.4. Effects on T. radiata Parasitism Rates and Recovery Success
Parasitism of D. citri nymphs by T. radiata was found at all 15 D. aligarhensis release sites. Overall, T. radiata accounted for ~97% of total parasitism observed (Figure 2). Time of year, but not climate, affected T. radiata parasitism rates (Table 2B). Parasitism rates were significantly correlated with the previous month’s density of large D. citri nymphs (instars four and five are the preferred stages for parasitism) (Table 2B, Figure 3). The mean proportions of nymphs parasitized by T. radiata differed between cool and warm months and moderate and warm months, but not between cool and moderate months (Table 2B, Figure 3). Parasitism rates were highest in February through May and October through December each year irrespective of climate zone (Figure 2 and Figure 3). Parasitism by T. radiata was relatively consistent across coastal (21.4 ± 2.4%), intermediate (21.9 ± 2.4%), and desert (17.6 ± 2.8%) climate sites (Table 2B). Parasitism rates were positively correlated with citrus flushing patterns over time (Table 2B). Average parasitism rates of T. radiata were not affected by citrus hosts or year (Table 2B). None of the interactions between the tested variables were significant (p > 0.32). In 2018, T. radiata was recovered at 34 of the 39 sites surveyed (i.e., 87% of the total sites surveyed) (Table 1 and Table S1). This included 21 control sites that were located up to 5 km away from the closest T. radiata release site, indicating that parasitoids had dispersed naturally from release sites. In addition, ~87% of D. aligarhensis release sites were colonized by T. radiata.
4. Discussion
The results presented strongly suggest that despite releasing ~40,000 female D. aligarhensis, this parasitoid failed to establish at the 15 release sites used in this study and D. aligarhensis was not subsequently recovered from surveyed study sites after releases ceased. A total of >700,000 female and male D. aligarhensis released over 2014–2018 across >300 sites dispersed over all three climatic zones also appear to have failed to establish and mass rearing and releases of this species ceased in 2019 (DJW Morgan CDFA, unpublished data). When recoveries of D. aligarhensis were made at release sites, parasitoid activity was only found in the month following release, parasitism rates were low (<1%), and this parasitoid likely contributed very little to observed reductions in D. citri densities at study sites.
The reasons for the low levels of parasitism by D. aligarhensis and failure to establish are not conclusively known, but may be due, in part, to competition from T. radiata that had naturally invaded and established at release sites. Previous studies using D. aligarhensis and T. radiata from the same source areas (i.e., Punjab, Pakistan) used in this study indicated that competition between D. aligarhensis and T. radiata for D. citri nymphs is asymmetric and T. radiata dominates to the detriment of D. aligarhensis [17,40,41]. Additionally, female D. aligarhensis parasitize fewer hosts per unit time than T. radiata [18] and when multiparasitism occurs, D. aligarhensis larvae are inferior intrinsic competitors and are killed by T. radiata larvae [19]. The superior competitiveness of T. radiata may have prevented the establishment of D. aligarhensis in Florida [25,28], and possibly also in California.
In Punjab, Pakistan, where D. aligarhensis was sourced, this parasitoid exhibited a very strong association with Diaphorina aegyptiaca Puton, infesting leaves of Assyrian plum, Cordia myxa L. (SZ Khan, CD Hoddle, and MS Hoddle, pers. obs.). Diaphorina aegyptiaca on C. myxa was also reported as a host for D. aligarhensis in India where D. aligarhensis is also native [46]. Cordia myxa is a fruit-bearing tree that may be grown in close proximity to citrus orchards or interspersed through orchards. Infestations of D. aegyptiaca could act as reservoirs from which D. aligarhensis disperse and attack less preferred or less suitable hosts such as D. citri infesting neighboring citrus trees. From foreign exploration efforts in Pakistan, nearly half as many D. aligarhensis (1023) as T. radiata (2021) were reared at UCR-IQF from D. citri collected [47]. A lack of a preferred alternative host species, such as D. aegyptiaca, could also explain, in part, the failure of D. aligarhensis to establish in California and its prevalence in Pakistani citrus orchards where it coexists with T. radiata. Further, in comparison to T. radiata, D. aligarhensis is more likely to be attacked by hyperparasitoids of which at least 17 species are known from Southeast Asia and Pakistan [47,48,49,50,51], and at least 8 of these genera are present in California [50]. Although not documented, an additional impediment to establishment of D. aligarhensis may have been the acquisition of hyperparasitoid species already resident in California.
All study sites had steady year-round Argentine ant (Linepithema humile Mayr) activity (data not shown). Argentine ants infest >90% of all residential citrus trees growing in southern California where they protect more than 55% of D. citri colonies from natural enemies, in exchange for a sugar-rich honeydew [52]. This mutualistic relationship can impair parasitoid oviposition performance and reduce efficacy of T. radiata by >80% [23,52,53]. Field observations revealed that the majority of interactions between Argentine ants and T. radiata on citrus branches infested with ants resulted in mortality of this parasitoid or its deterrence by tending ants [54]. Nearly all oviposition attempts by T. radiata were frequently interrupted by Argentine ants, allowing psyllids to escape parasitism. In the context of this study, L. humile likely impeded D. aligarhensis’s efficacy, even though putative negative impacts were not quantified.
Factors promoting the field persistence of D. aligarhensis on D. citri need determination. For example, self-introduced populations of D. aligarhensis into various citrus-growing regions, including Réunion Island [55], Saudi Arabia [56,57], Taiwan [58], Colombia [59], and the Philippines [60], generally provided low levels of D. citri control, especially in areas where T. radiata was already present (e.g., Réunion Island and Taiwan) or later introduced. For example, in the Philippines, where D. aligarhensis was widespread, releases of T. radiata dominated the D. citri parasitoid guild within 12 months of release at study sites [61]. A similar outcome was observed in Saudi Arabia where D. aligarhensis was recorded inflicting mortality of 60–70% on D. citri in limes [57]. Releases of T. radiata at sites with established D. aligarhensis populations resulted in the suppression of D. aligarhensis and T. radiata emerged as the dominant parasitoid species [56].
An important, albeit unintentional, byproduct of this work assessing the establishment and impact of D. aligarhensis on D. citri was the opportunity to simultaneously investigate the impacts of T. radiata on this pest in urban areas. At sites where D. aligarhensis was released, T. radiata accounted for >95% of total parasitism rates. For the 2016–2017 survey, T. radiata parasitism rates averaged 21%, similar to levels reported in Pakistan [39], southern California [23,37], Texas [62], and Florida [28,63], and lower than rates reported from Réunion Island [55] and Puerto Rico [64]. Secondly, with respect to T. radiata analyses of parasitism rates, delayed density-dependent regulation of D. citri populations with a lag of ~12 months was detected. Following the peaks of D. citri activity in the spring and fall of 2016, mean T. radiata parasitism increased to a peak of over 46%, before D. citri levels declined in 2017 to levels that were ~50% of previous recorded densities. Finally, in the 2018 surveys, T. radiata was recovered at 34 of 39 sites (i.e., 87%) monitored, indicating a strong propensity for natural dispersal and establishment. The highest psyllid densities and frequencies of parasitized D. citri nymphs coincided with citrus flushing periods. Consequently, parasitism of D. citri by T. radiata peaked in March through June and October through November each year when daily temperature averages consistently fell within an optimal temperature range of 20–28 °C for development [65]. The results of work reported here are supported by previous studies that also indicated the significant role that natural enemies such as T. radiata have played in suppressing D. citri densities in urban citrus [23,37].
In conclusion, classical biological control introductions into the USA of thelytokous (Florida) and arrhenotokous (California) populations of D. aligarhensis failed to establish. The reasons for this are uncertain, however, competition from T. radiata is suspected as a significant contributing factor. With respect to establishment success in classical biological control, an elucidation of factors adversely affecting the successful establishment of natural enemies is important. The identification and understanding of barriers (e.g., interspecific competition, hyperparasitism, intraguild predation, or poor climate matching) that impede agent establishment can inform decision-making during critical steps of program development [66,67,68]. In this regard, interspecific competition may be important and could deserve consideration when classical biological programs for D. citri are being developed. Consequently, classical biological control programs targeting D. citri should probably only consider the use of T. radiata. This suggestion is supported by results from meta-analyses that indicate considerable suppression of insect pests is often attained by a single species of natural enemy [69] and that increased levels of control are usually not achieved by releasing multiple species of natural enemies [70]. Documentation of successes and failures of natural enemy releases in the context of classical biological control, both in terms of establishment and suppression of target pest populations, is recommended so that factors contributing to observed outcomes can be better understood and used to inform the development of future projects [68].
Conceptualization, I.M., M.A.V., D.J.W.M., C.D.H., R.E.M. and M.S.H.; methodology, I.M., M.A.V., D.J.W.M., C.D.H., R.E.M. and M.S.H.; formal analysis, I.M.; investigation, I.M., M.A.V., D.J.W.M., C.D.H., R.E.M. and M.S.H.; data curation, I.M., M.A.V., D.J.W.M., C.D.H. and R.E.M.; project administration, I.M., M.A.V., D.J.W.M., C.D.H., R.E.M. and M.S.H.; writing—original draft, I.M. and M.S.H.; writing—review & editing, I.M., M.A.V., D.J.W.M., C.D.H., R.E.M. and M.S.H.; funding acquisition, M.S.H. All authors have read and agreed to the published version of the manuscript.
This work was supported, in part, from funds provided by the California Citrus Research Board (award number 5500-191) and the United States Department of Agriculture Multi-Agency Coordination (USDA-MAC) Agreement Number #15-8130-0336-CA.
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.
We would like to thank Alejandro Muniz, Grace Radabaugh, and Robert Dempster (California Department of Food and Agriculture), and Martin Castillo, Michael Lewis, Ruth Amrich, and Nagham Melham (University of California Riverside) for their contribution to the mass rearing of released parasitoids, field work, and processing of data. We are grateful to homeowners in southern California who provided unlimited access to their properties to conduct survey work over a three-year period.
The authors declare no conflict of interest.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Enlarge this image.Figure 1. Mean (±SE) temperatures of sampling periods at study sites belonging to either the coastal (CIMIS stations: 62, 75, 174), intermediate (CIMIS stations: 44, 78, 153) or desert (CIMIS stations: 207, 239, 251) climate zones in southern California from January 2016 through December 2017 study period. Sampling months were grouped into three seasons: cool (10–15 °C), moderate (15.01–20 °C), or warm (>20.01 °C).
Enlarge this image.Figure 2. The relationship between Diaphorina citri densities and total parasitism rates across field sites from January 2016 through December 2017. Mean (±SE) total D. citri population densities (pooled across all psyllid life stages) and mean (±SE) total parasitism rates (pooled across Tamarixia radiata and Diaphorencyrtus aligarhensis parasitism rates) from the previous sampling interval (pooled across climate types and host plants) are shown.
Enlarge this image.Figure 3. Mean (±SE) total and species-specific parasitism rates of Diaphorina citri by Tamarixia radiata and Diaphorencyrtus aligarhensis at study sites in southern California from January 2016 through December 2017 (pooled across study years, climate types, and host plants).
Site characteristics and parasitoid release frequencies and numbers for selected D. aligarhensis release sites in southern California, USA.
| Climate Type | County | Site Name | Latitude |
Longitude |
Elev. (m) | Surveyed Tree(s) | CIMIS |
Date of First |
Most Recent |
No. D. aligarhensis |
No. D. aligarhensis |
|---|---|---|---|---|---|---|---|---|---|---|---|
| Coastal | Los Angeles | Alameda | N33°59′ | W118°14′ | 51 | Lemon, Orange | 174 | 31 August 15 | 7 November 16 | 39 (31) | 6388 (4711) |
| Orange | Anaheim | N33°51′ | W117°44′ | 316 | Lemon, Orange | 75 | 17 August 15 | 1 March 17 | 49 (32) | 7006 (5602) | |
| Fullerton | N33°53′ | W117°53′ | 76 | Lemon, Orange | 75 | 17 August 15 | 15 March 17 | 37 (31) | 6419 (4072) | ||
| Irvine | N33°43′ | W117°47′ | 33 | Lemon, Orange | 75 | 8 September 15 | 15 March 17 | 46 (31) | 5615 (4904) | ||
| San Diego | Fallbrook | N33°23′ | W117°10′ | 197 | Lemon, Orange | 62 | 21 September 15 | 8 February 17 | 42 (35) | 4389 (3406) | |
| Intermediate | Los Angeles | Claremont | N34°05′ | W117°42′ | 341 | Lemon, Orange | 78 | 31 August 15 | 27 March 17 | 26 (24) | 2616 (2256) |
| Pomona | N34°03′ | W117°44′ | 255 | Lemon, Orange | 78 | 31 August 15 | 27 March 17 | 39 (31) | 5595 (3512) | ||
| Riverside | Citrus Hill | N33°51′ | W117°19′ | 542 | Lemon, Orange | 44 | 21 September 15 | 8 February 17 | 30 (28) | 3102 (2858) | |
| Woodcrest | N33°54′ | W117°20′ | 479 | Lemon, Orange | 44 | 10 August 15 | 8 February 17 | 30 (23) | 4449 (2929) | ||
| San Diego | Ramona | N33°01′ | W116°49′ | 443 | Lemon, Orange | 153 | 24 August 15 | 22 March 17 | 49 (28) | 6524 (4961) | |
| Desert | Riverside | Hemet | N33°44′ | W116°49′ | 645 | Lemon, Orange | 239 | 8 September 15 | 20 March 17 | 20 (18) | 2603 (1952) |
| San Bernardino | Redlands | N34°02′ | W117°13′ | 403 | Lemon, Orange | 251 | 5 October 15 | 20 March 17 | 29 (21) | 3058 (2029) | |
| Warner * | N34°04′ | W117°10′ | 440 | Lemon, Orange | 251 | 5 October 15 | 29 March 17 | 17 (15) | 1981 (1151) | ||
| Crafton | N34°03′ | W117°07′ | 600 | Lemon, Orange | 251 | 5 October 15 | 29 March 17 | 34 (27) | 3684 (2671) | ||
| San Diego | Valley Ctr. * | N33°15′ | W117°03′ | 348 | Lemon, Orange | 207 | 20 July 15 | 8 February 17 | 36 (29) | 5542 (3191) |
* Indicates study sites where T. radiata activity was not detected in the 2018 surveys. No next-year recoveries were made for D. aligarhensis in 2018.
Results of generalized linear mixed-effects models examining the main and interaction effects of climate type and host plants on recorded (A) total psyllid densities and (B) parasitism rates of T. radiata at field sites from January 2016 through December 2017. The selected regression model for psyllid abundance also included a covariate of total parasitism rates from the prior sampling period (A). The selected regression model for parasitism rates of T. radiata also included a covariate of densities of late fourth and fifth instar psyllid nymphs from the previous sampling period (B). The final minimal and most adequate regression models for each response variable are presented below. The critical level of significance was set at 0.05 *.
| Parameter | Estimate | SE | df | t | p | 95% Conf. Interval | ||
|---|---|---|---|---|---|---|---|---|
| Lower | Upper | |||||||
| (A) Psyllid densities | ||||||||
| Climate type | Coastal_Intermediate | 0.3094 | 0.5231 | 54.33 | 0.59 | 0.82 | −0.7393 | 1.3581 |
| Coastal_Desert | 0.3662 | 0.5796 | 92.48 | 0.63 | 0.81 | −0.7848 | 1.5171 | |
| Desert_Intermediate | −0.0568 | 0.5742 | 70.74 | -0.11 | 0.99 | −1.2018 | 1.0882 | |
| Year | 2016_2017 | 0.3126 | 0.1389 | 634.9 | 2.25 | 0.048 * | 0.0126 | 0.6126 |
| Season | Cool_Moderate | −0.2295 | 0.5874 | 74.82 | -0.39 | 0.92 | −1.3998 | 0.9407 |
| Cool_Warm | 1.5459 | 0.6485 | 105.7 | 2.38 | 0.02 * | 0.6201 | 2.3817 | |
| Moderate_Warm | 1.7755 | 0.5616 | 136.3 | 3.16 | 0.006 * | 0.6648 | 2.8861 | |
| Crop | Lemon_Orange | 0.5701 | 0.4282 | 673.9 | 1.33 | 0.19 | −0.2708 | 1.4109 |
| Flush abundance | 6.6711 | 0.5043 | 689 | 13.23 | <0.001 * | 5.6808 | 7.6611 | |
| Total parasitism | −0.0326 | 0.0119 | 677.8 | −2.74 | 0.006 * | −0.0549 | −0.0092 | |
| (B) Parasitism of T. radiata | ||||||||
| Climate type | Coastal_Intermediate | 0.0964 | 0.4858 | 35.08 | 0.19 | 0.97 | −0.9728 | 1.1655 |
| Coastal_Desert | 0.5147 | 0.5604 | 18.97 | 0.92 | 0.65 | −0.7187 | 1.7481 | |
| Desert_Intermediate | −0.4183 | 0.5509 | 26.24 | −0.76 | 0.73 | −1.6308 | 0.7941 | |
| Year | 2016_2017 | 0.6787 | 0.4112 | 465 | 1.65 | 0.13 | −1.5669 | 0.2098 |
| Season | Cool_Moderate | 0.7115 | 0.4696 | 55.84 | 1.52 | 0.13 | −1.6344 | 0.2113 |
| Cool_Warm | 4.0058 | 0.6947 | 74.18 | 5.77 | <0.001 * | 2.5750 | 5.4365 | |
| Moderate_Warm | 2.3531 | 0.5845 | 76.5 | 4.03 | <0.001 * | 1.1492 | 3.5568 | |
| Crop | Lemon_Orange | 0.2844 | 0.3456 | 413.5 | 0.79 | 0.44 | −0.4816 | 1.0505 |
| Flush abundance | 1.6528 | 0.5425 | 463.4 | 3.05 | 0.009 * | 0.5355 | 2.7700 | |
| Large nymphs | −0.7012 | 0.1989 | 382.6 | 3.52 | <0.001 * | 0.3102 | 1.0922 | |
Supplementary Materials
The following supporting information can be downloaded at
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; Vankosky, Meghan A 2 ; Morgan, David J W 3 ; Hoddle, Christina D 4 ; Massie, Rachael E 3 ; Hoddle, Mark S 1 1 Department of Entomology, University of California, 900 University Ave, Riverside, CA 92521, USA;
2 Department of Entomology, University of California, 900 University Ave, Riverside, CA 92521, USA;
3 California Department of Food and Agriculture, 4500 Glenwood Drive, Riverside, CA 92501, USA;
4 Department of Entomology, University of California, 900 University Ave, Riverside, CA 92521, USA;