Data from: Defensive aphid symbiont Hamiltonella defensa effects on Aphelinus glycinis and Aphelinus atriplicis

Endosymbiont interactions with hosts have important effects on fitness, including the fitness of many pest and beneficial species. Among these interactions, facultative endosymbiotic bacteria can protect aphids from parasitoids. Aphis craccivora and Acyrthosiphon pisum can harbor the symbiotic bacteria Hamiltonella defensa and its bacteriophage APSE. Infection by H. defensa defends these aphids against some but not all parasitoid species in the hymenopteran family Braconidae. Here, we report results on the effect of H. defensa on parasitism of these aphids by species in the other major lineage of aphid parasitoids, Aphelinus species in the family Aphelinidae. Parasitism of aphids infected with H. defensa /APSE by two Aphelinus species did not differ from that of uninfected aphids. While Aphelinus atriplicis showed no difference in fitness components between infected and uninfected aphids, Aphelinus glycinis actually produced more adult progeny and larger female progeny on infected than on uninfected aphids. Aphelinus glycinis may increase host quality for itself by changing the titer of nutritional versus protective bacteria in such a way that aphids infected with H. defensa can be made more suitable for parasitoid development than uninfected aphids. Our results and reasoning suggest that these Aphelinus species may be less prone to harm by H. defensa /APSE that affect eggs because they have anhydropic, heavily chorionated eggs, which may not absorb toxins during embryogenesis.

Insect sources and rearing conditions

Aphelinus glycinis was collected in the Peoples Republic of China under a Memorandum of Understanding between their Ministry of Agriculture and the United States Department of Agriculture (USDA). Aphelinus atriplicis was collected by employees of the USDA, Agricultural Research Service (ARS), in the Republic of Georgia with the permission of that government. The parasitoids were imported into the USDA, ARS, Beneficial Insect Introductions Research Unit containment facility in Newark, Delaware, under permits from the USDA, Animal and Plant Health Inspection Service (Permit Numbers P526P-08-02142 and P526P-09-01929). No specific permissions were required to collect Aphis craccivora or Acyrthosiphon pisum because these are cosmopolitan aphids that occur in the field throughout North America. None of the species collected or studied are endangered or protected.

Aphis craccivora is a cosmopolitan pest of legumes and is anholocyclic, i.e. reproduces asexually, throughout most of its range (Blackman and Eastop, 2006). Acyrthosiphon pisum is an almost cosmopolitan pest of legumes and is holocyclic, i.e. alternates by asexual and sexual reproduction in temperature regions (Blackman and Eastop, 2006). Aphis craccivora in these experiments were collected originally in Kentucky, USA, in 2009, and Ac. pisum were collected in Utah, USA, in 2007. The aphids were cultured on fava bean, Vicia faba L., in 12 cm pots in plant growth chambers at 20°C and a 16L:8D h photoperiod. Isofemale lines were initiated from single female aphids on individual V. fava plants. Lines from the original populations tested positive for infection with only H. defensa using Denaturing Gradient Gel Electrophoresis (DGGE) analysis of PCR-amplified fragments of 16S rRNA and confirmed with diagnostic PCR (Russell et al., 2013). APSE type 3 was found in H. defensa in Ac. pisum and APSE type 4 was found in H. defensa in Ap. craccivora. Aphid lines without H. defensa were generated by a selective curing technique in which aphids were fed an artificial diet that was treated with equal parts of the antibiotics gentamycin, defatoxiamine and ampicillin for 3 days, after which the aphids were placed on V. fava plants (Dykstra et al., 2014). Clearing of H. defensa from offspring of aphids from these lines was confirmed by PCR (Russell et al., 2013). Cured (AC1H-) and uncured lines (AC1H+) from Ap. craccivora and cured (AS3H-) and uncured lines (AS3H+) from Ac. craccivora were used for the experiments reported here. They were shipped to Newark, Delaware, USA, in spring 2012, where they were maintained for 40-50 generations prior to these experiments. Both cured and uncured lines were tested using diagnostic PCR for the presence of H. defensa and APSE prior to assays.

Aphelinus atriplicis was collected as mummified Diuraphis noxia on wheat near Tbilisi, Republic of Georgia, in 2000; A. glycinis was collected as mummified Aphis glycines Matsumura (Hemiptera: Aphididae) on soybean near Xiuyan, Liaoning Province, Peoples Republic of China, in 2007. Aphelinus glycinis was described from the culture used in this study (Hopper et al., 2012); A. atriplicis was described by Kurdjumov (1913), and the culture used in this study was included in a molecular phylogeny of species in the Aphelinus varipes complex (Heraty et al., 2007). Cultures were established in the quarantine facility at the USDA-ARS, Beneficial Insect Introductions Research Unit, Newark, Delaware, USA. The cultures were divided into 4-6 subcultures, and each subculture was maintained with an adult population size greater than 200 and sex ratio of 1:1 males:females. Aphelinus atriplicis was reared on D. noxia on barley, Hordeum vulgare L. (Poaceae); Aphelinus glycinis was reared on Aphis glycines on soybean, Glycine max (L.) (Fabaceae). The culture of D. noxia was started in 1998 with aphids from southeastern Wyoming, and the culture of A. glycines was started in 2008 with aphids from Newark, Delaware. Neither D. noxia nor A. glycines are known to harbor H. defensa, and diagnostic PCR assays confirmed the absence of H. defensa in our colonies of these species. Although A. glycines harbors another bacterium, Arsenophonus, this secondary symbiont apparently does not protect against parasitoids (Wulff et al., 2013). All cultures were reared on appropriate host plant species in plant growth chambers at 20°C, 50-70% relative humidity, 16:8 h (L:D) photoperiod. Vouchers are maintained at -20°C in molecular-grade ethanol at the Beneficial Insect Introduction Research Unit, Newark, Delaware, USA.

Parasitism and parasitoid fitness on aphids infected with Hamiltonella defensa versus uninfected aphids

To measure the effect of infection with H. defensa harboring APSE on parasitism of Ap. craccivora and Ac. craccivora, as well as on parasitoid fitness components, we exposed individual, mated females of Aphelinus atriplicis or Aphelinus glycinis that were less than 5 days old to either aphids infected with H. defensa or uninfected aphids on V. faba variety Windsor. Females were drawn at random from the replicated sub-populations used for rearing. Seeds were planted in 12 cm diameter x 12 cm tall pots with the same soil mix and fertilizer used for insect rearing and plants and insects were kept under the same conditions as described for insect rearing prior to use in experiments. We put each female parasitoid in a cage (polystyrene, 10 cm diameter by 22 cm tall, with eight 2.5 cm holes in the sides and a 12 cm hole in the top covered with fine-mesh screening) enclosing the foliage of 2-3 young fava bean plants in a pot with about 100 aphids, including 1-4 instar nymphs and adults. The aphids were transferred as colonies on excised leaves from our rearing cultures, and the excised leaves were placed on the experimental plants to allow the aphids to settle and feed two days before the parasitoids were added. Female parasitoids were removed after 7 days. Aphelinus females rarely superparasitize unless they are host limited (Bai and Mackauer, 1990; Hagen and VanDenBosch, 1968). Their lifetime fecundities are 100-300, and given that we exposed them to 100 aphids at the beginning of the experiments, and the aphids would produce more than 2000 progeny during the 7 day exposure, there were far more aphids than they could parasitize during this period so the likelihood of superparasitism is quite low.

The density of aphids, amount of plant material, and cage size meant that parasitoids were not limited by search rate.

Aphids parasitized by these Aphelinus species mummify about 7 days after being parasitized and adult parasitoids emerge 10 days after mummification. To ensure that aphids parasitized throughout the exposure period had time to mummify but would not yet have emerged as adults, we collected mummified aphids 7 days after the end of the exposure of aphids to parasitoids and held them for adult parasitoid emergence. After the adults emerged, we recorded the number of mummified aphids, the number of mummies from which adults emerged, and the number and sex of adult parasitoids. We dried the adult progeny of each female at 50°C for one hour and weighed the sexes separately on a microbalance. Because we scored parasitism after the larval parasitoids killed and mummified their hosts, which occurs during the parasitoid third instar, this measure of parasitism is a combination of acceptance of aphids for oviposition and suitability of aphids for parasitoid survival to third instar.

Design structure

The experiments on parasitism of each aphid species by each wasp species were designed as randomized complete-blocks blocks: location within rearing chamber was the blocking factor and each cage with a female parasitoid, aphids, and plants was an experimental unit. There were 48 experimental units each for Aphelinus glycinis on Ap. craccivora and Aphelinus atriplicis on Ap. craccivora and on Ac. pisum (24 each for Hamiltonella-infected versus uninfected aphids).