Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Liviidae), was discovered in California USA in 2008. D. citri vectors ‘Candidatus Liberibacter asiaticus', a putative causative agent of huanglongbing (HLB), a lethal disease of citrus (Hoffman et al. 2013; Wang & Trivedi 2013). HLB was detected in California in Mar 2012 (Leavitt 2012). To mitigate the threat posed by D. citri-HLB to California's citrus industry, a biological control program using Tamarixia radiata (Waterston) (Hymenoptera: Eulophidae) sourced from Pakistan was initiated (Hoddle 2012). Diaphorencyrtus aligarhensis (Shafee, Alam, and Agarwal) (Hymenoptera: Encyrtidae), a second parasitoid of D. citri also collected from Pakistan, is currently in quarantine at the University of California, Riverside (UCR). The purpose of this study was to confirm that Chartocerus sp. and P. crassiculme, both suspected hyperparasitoids, are not primary parasitoids of D. citri.

Materials and Methods

Parasitized D. citri host material returned from Punjab Pakistan to quarantine at UCR (15–22 Apr 2013) yielded previously collected T. radiata and D. aligarhensis, along with several species of known (Marietta leopardina Motschulsky [Hymenoptera: Aphelinidae], Aprostocetus (Aprostocetus) sp. [Hymenoptera: Eulophidae] [Hoddle et al. 2013]) or suspected (Chartocerus sp. [Hymenoptera: Signiphoridae], Pachyneuron crassiculme Waterston [Hymenoptera: Pteromalidae] and Psyllaphycus diaphorinae [Hymenoptera: Encyrtidae]) hyperparasitoids.

To confirm that Chartocerus sp. (Fig. 1A. male, B. female) and P. crassiculme (Fig. 2A. male, B. female) are not primary parasitoids of D. citri, exposure trials using 10 sets of 4-7 Chartocerus sp. and 10 pairs of 1 male and 1 female P. crassiculme that emerged from material collected in Pakistan were rotated through each of 4 treatment types between 26 Apr and 24 May, 2013 in quarantine at UCR. It was not possible to reliably sex live Chartocerus sp., so this species was exposed in groups (assumed to contain at least 1 female each) unless a pair was otherwise observed mating. Exposure treatments consisted of: (A) nymphs parasitized by T. radiata (n = 8 replicates of 5-10 parasitized nymphs for Chartocerus sp. and 9 replicates of 5 parasitized nymphs for P. crassiculme), 5-9 days post-exposure to T. radiata; (B) nymphs parasitized by D. aligarhensis (n = 8 replicates of 5-10 parasitized nymphs for Chartocerus sp. and 10 replicates of 5 for P. crassiculme), 10-14 days post-exposure to D. aligarhensis; (C) unparasitized third to fourth instar D. citri nymphs (n = 9 replicates of 5-10 unparasitized nymphs for Chartocerus sp. and 10 replicates of 5 nymphs for P. crassiculme); and (D) each of the 3 previously listed host types (A, B, and C) presented simulta- neously in a choice cage (n = 9 replicates of 5-10 of each host type for Chartocerus sp. and 9 replicates of 5 of each host type for P. crassiculme).

Fig. 1.

Chartocerus sp. male (A) and female (B). This figure is shown in color in the supplementary document in Florida Entomologist 97(2) (2014) online at  http://purl.fcla.edu/fcla/entomologist/browse.

Fig. 2.

Pachyneuron crassiculme male (A) and female (B). This figure is shown in color in the supplementary document in Florida Entomologist 97(2) (2014) online at  http://purl.fcla.edu/fcla/entomologist/browse

Each replicate was comprised of host material for each treatment type exposed to a group of potential hyperparasitoids for 24 h each. Hosts were exposed sequentially in a different order for each replicate to prevent bias due to presentation order. Emergence rates of T. radiata (n = 5 parasitized nymphs on each of 10 cuttings) and D. aligarhensis (n = 5 parasitized nymphs on each of 10 cuttings) determined baseline mortality for primary parasitoids in the absence of hyperparasitoids. Unparasitized D. citri nymphs (n = 5 fourth instar nymphs on each of 10 plants) provided data on nymph mortality in the absence of hyperparasitoids. Mummies of T. radiata and D. aligarhensis used in exposure experiments were sourced from colonies maintained in quarantine at UCR.

Diaphorina citri nymphs parasitized by either T. radiata or D. aligarhensis for no-choice treatments were presented on small Citrus volkameriana cuttings. Citrus volkameriana seedlings grown in 114 mL Cone-tainersTM (SC7 Stubby, 3.8 cm diameter, Stewe and Sons Inc., Oregon) and infested with D. citri nymphs were used to expose unparasitized D. citri nymphs to Chartocerus sp. and P. crassiculme. Clear plastic vials (Thornton Plastic Co. 148 mL capacity, Salt Lake City, Utah) with three 12 mm diam ventilation holes covered with ultra-fine organza were inverted and placed over the top of the plant and fitted into the corresponding vial lid, which had a hole cut in the center to allow it to be fitted around the cone (Irvin et al. 2009).

Choice treatments were set up in 15 cm × 15.3 cm × 15.3 cm (h × w × d) clear plastic boxes (S&W Plastics, Riverside, California) with a 30 cm sleeve sewn from no-see-um netting (Skeeta Mosquito & Other Insect Protection Products, Bradenton, Florida). Unparasitized D. citri nymphs in Conetainers and T. radiata- and D. aligarhensis-parasitized nymphs on C. volkameriana cuttings in water were placed in the cage without ventilated vials on top to allow free access to all 3 host types simultaneously. After 24 h, each host type was enclosed with an inverted ventilated vial to contain all insects that emerged from each host type. All experiments were conducted in quarantine at UCR's Insectary and Quarantine facility, at 27 °C, 50% RH, and 14:10 h L:D. Replicates were observed daily after initial exposure, and total numbers of each emerged species were recorded per treatment.

Results

No-choice treatments resulted in Chartocerus sp. reproducing successfully only on D. aligarhensis (Table 1). Mean emergence time for Char- tocerus sp. offspring from D. aligarhensis was 18.36 days ± 2.34 (SE). Pachyneuron crassiculme produced progeny on D. aligarhensis and T. radiata in no-choice treatments, though parasitism was much higher on D. aligarhensis (Table 2). Mean emergence times for males and females were 12.83 days ± 2.48 (SE) and 11.33 days ± 2.05 (SE), respectively. Pachyneuron crassiculme had a single male emerge from T. radiata after 11 days. Emergence rates for control treatments of T. radiata, D. aligarhensis, and D. citri were 84%, 88%, and 88%, respectively (Table 3). Chartocerus sp. and P. crassiculme failed to reproduce on unparasitized D. citri nymphs.

Table 1.

Emergence and mortality rates for Chartocerus sp. exposed to unparasitized third and fourth instar D. citri nymphs, and nymphs parasitized by T. radiata, and D. aligarhensis in no-choice and choice treatments.

Immature D. aligarhensis exposed to Chartocerus sp. in no-choice tests experienced 47% parasitism, 17% died from undetermined causes, 3% were unaccounted for, and 33% emerged as adult D. aligarhensis. In 20% of trials (i.e., 2 of 10 replicates) Chartocerus sp. exhibited superparasitism, with 11 adults emerging from 9 D. aligarhensis mummies in 1 replicate, and 6 adults emerging from 3 mummies in the second. In no-choice tests, immature T. radiata exposed to Chartocerus sp. exhibited 0% parasitism, 27% of mummies died from unknown causes, 6% disappeared, and 67% emerged as adult T. radiata.

In no-choice tests where P. crassiculme was exposed to immature D. aligarhensis, 28% of hosts were parasitized by P. crassiculme, 19% died from unknown causes, and 53% emerged as adult D. aligarhensis. On T. radiata, P. crassiculme successfully parasitized only 2% of host material (i.e., one host), 40% died from unknown causes, 7% were unaccounted for, and 51% emerged as adult T. radiata. Unknown mortality may be attributable to superparasitism, host feeding, or a combination of both by P. crassiculme.

There was no successful parasitism of any host in choice tests for either Chartocerus sp. or P. crassiculme. However, elevated mortality rates were observed for T. radiata (26% when exposed to Chartocerus sp.; 28% for P. crassiculme) and D. aligarhensis (29%; 13%). In comparison, control mortality for T. radiata and D. aligarhensis were < 13% in the absence of these hyperparasitoids. When viewed collectively, data from exposure trials demonstrates that Chartocerus sp. and P. crassiculme are obligate hyperparasitoids within the D. citri-Tamarixia-Diaphorencyrtus system. Immediately following the conclusion of trials, all Chartocerus sp. and P. crassiculme material was killed in quarantine and preserved in 95% ethanol. Voucher specimens were deposited in the Entomology Museum at UCR (Table 4).

Assuming Chartocerus sp. and P. crassiculme preferentially parasitize D. aligarhensis as these exposure trial data suggest, the frequency of Chartocerus sp. and P. crassiculme emergence in quarantine from material collected from Punjab Pakistan in April 2013 was significant in compar- ison to D. aligarhensis emergence rates. Chartocerus sp. (237 individuals reared), P. crassiculme (181), and D. aligarhensis (743) represented 20%, 16%, and 64% of material reared, respectively, within this complex. A total of 292 T. radiata were reared from April 2013 collections. Exposure trials suggest that the lower numbers of T. radiata obtained from Pakistan in April 2013 were not likely due to hyperparasitism.

Table 2.

Emergence and mortality rates for Pachyneuron crassiculme exposed to unparasitized third and fourth instar D. citri nymphs, and nymphs parasitized by T. radiata and D. aligarhensis in no-choice and choice treatments.

Table 3.

Emergence rates of unparasitized third and fourth instar D. citri nymphs and nymphs parasitized by T. radiata and D. aligarhensis in control treatments not exposed to hyperparasitoids.

Table 4.

Specimen accession numbers for all species used in exposure trials and deposited in the Entomology Museum at the University Of California Riverside.