performed in a Mastercycler® ep gradient S (Eppendorf North America Inc., New .... squashed onto Whatman® FTA® PlantSaver Cards (Whatman Inc., Florham ...
Introduction
Results
Trichogrammatid wasps are egg parasitoids, mainly of Lepidoptera (Figure 1). Their small size and lack of clear morphological features has made this group difficult to identify to the species level. While males of many species can be identified using morphological characters, no keys exist for females of these species. This can present a major problem, since the field sex ratio of these species is often very female biased. Furthermore, several species are completely thelytokous and consequently consists of females only. Over the last decade, the DNA sequences of the ITS2 spacer have been successfully exploited for the identification of many Trichogrammatid species. Identifications are made with the use of dichotomous keys based on the size of the ITS2 spacer and the banding pattern it produces following digestion with various restriction enzymes (e.g., Stouthamer et al. 1999). While these methods are useful and work well, for the routine identification of large number of specimens known to belong to only a few species, a cheaper and faster option is the design of a multiplex PCR method, capable of identifying all present species in a single PCR reaction. Here we describe the development and application of a species-specific multiplex PCR capable of identifying the five common Trichogrammatid species found on the Hawaiian island of Kauai. In addition we describe the use of FTA® cards for the preservation and shipment of wasp DNA. Over a six week period in the fall of 2007, extensive collections of lepidopteron eggs were made on Kauai, and the emerged parasitoids were identified using this multiplex method. The data show that the Trichogrammatid fauna of Kauai has changed since the last extensive survey (Oatman et al. 1982). In addition to three previously known species , three new exotic species were found.
The preservation and shipping of wasp DNA on the FTA® cards, combined with species-specific multiplex PCR, proved a highly efficient means of identifying field-caught wasps. Over the 6 week period, a total of 1,358 eggs of the three lepidopteron species were collected from 17 locations across the island of Kauai (Figure 2). Of these, 550 were found to be parasitized and the emerging parasitoids from 468 of those were successfully identified using our species-specific multiplex PCR (Figure 5). Parasitism rates varied greatly across the three hosts, with less than 2% of the collected Monarch eggs being parasitized, but 63% and 48% respectively, of croton moth and Peablue eggs being parasitized (Table 2). The few parasitized Monarch eggs produced only T. pretiosum, while eggs of the croton moth were parasitized to a varying degree by three Trichogramma species; in declining order of abundance, T. chilonis, T. papilionis, and T. acheaea (Table 2). Eggs of the Peablue were parasitized by all five species included in our multiplex PCR, plus an additional thelytokous species of Trichogrammatoidea. By far the most common parasitoid of the Peablue was Trichogrammatoidea sp. 1 (Table 2).
Materials & Methods
Molecular diagnostics of Hawaiian Trichogrammatidae (Hymenoptera) using species-specific oligonucleotide primers and multiplex PCR Richard
Stouthamer1,
Paul
Rugman-Jones1,
Robin
Bromley1
Table 2. Association of six Trichogrammatid species with three species of lepidopteron host eggs, and in turn their respective plant hosts, on the island of Kauai.
Sumer2
& Fahriye
1Department
of Entomology, University of California, Riverside, CA 92507, USA 2Erciyes Üniversitesi, Fen-Edebiyat Fakultesi, Biyologi Bölümü, 38039 Kayseri, Turkey
Materials & Methods (cont.)
Collection Over a six week period in fall of 2007, eggs of the monarch butterfly (Danaus plexippus), the croton moth (Acheae janata) and the Peablue (Lampides boeticus) were collected in several locations on Kauai (Figure 1). Eggs were collected from the main host plant of each of the lepidopteran species, the crownflower (Calotropis gigantea), castor bean (Ricinus communis) and Crotalaria sp. respectively. Eggs of the Monarch and the Croton Moth were found by scanning the lower leaf surface for eggs, while eggs of the Peablue were found in the flower heads. Eggs were removed from the plant, by excising a small part of the plant tissue containing the egg, and placed in an “eppendorf” vial. Collected eggs were placed in a cooler and returned to the lab. Here, they were maintained at room temperature and checked daily for wasp or caterpillar emergence. From host eggs where wasps emerged (i.e. parasitized eggs), one to two iso-female lines were initiated using irradiated Ephestia kuehniella eggs as hosts. These cultures were maintained until the specific identity of the parasitoids was determined (see below).
Multiplex-PCR Design Species-specific oligonucleotide primers (SSO) were designed manually using the following criteria: •They should complement a universal forward primer •At least one nucleotide at the 3’ end must be unique to a single species •Complementarity between primers should be minimal •They should work at similar annealing temperatures •They should produce a PCR product of a unique size in the multiplex PCR
Figure 2. Collection locations (solid diamonds) of lepidoteran host eggs.
Materials & Methods (cont.) Preservation and shipping of wasp DNA On completion of oviposition (i.e., the initiation of iso-female lines), the field-caught female wasps were squashed onto Whatman® FTA® PlantSaver Cards (Whatman Inc., Florham Park, NJ) using sterile pestles (Figure 3). FTA® Cards contain chemicals that lyse cells, denature proteins and protect nucleic acids from nucleases, oxidative and UV damage. FTA® Cards also rapidly inactivate organisms, including blood-borne pathogens, and prevent the growth of bacteria and other microorganisms (for more information see panel below). The FTA® Cards were placed in a Zip-loc bag along with a desiccant pack and shipped overnight (via FedEx) to The University of California, Riverside.
No. collected
No. parasitized1
No. of broods identified2
PR
P
C
A
T
T2
Danaus plexippus / Calotropis gigantea
309
5
5
5
-
-
-
-
-
Acheae janata / Ricinus communis
272
172
161
-
64
99
2
-
-
Lampides boeticus / Crotalaria sp.
777
373
302
11
16
16
19
237
1
Total
1358
550
468
17
81
115
21
237
1
PR = T. pretiosum, P = T. papilionis, C = T. chilonis, A = T. acheaea, T1 = Trichogrammatoidea sp. 1, T2 = Trichogrammatoidea sp. 2 1Parasitized
eggs did not always produce a brood of wasps. 2The numbers of each species identified may total greater than the number of broods identified since some host eggs produced mixed broods.
Several primers were designed and the specificity of each was tested, individually and in multiplex PCR, against each of the 5 target species. These preliminary tests resulted in a set of species-specific reverse primers, which together with the common forward primer, gave the best combination of the desired characteristics (Table 1; Figure 4).
Discussion FTA® Cards Squashing Trichogrammatids onto FTA® cards provided a quick, simple, reliable and cost effective means of preserving wasp DNA and moving that DNA overseas. Essentially, these cards maintain the integrity of DNA at room temperature, and on a flat piece of filter paper, making them much easier to ship than material preserved in ethanol. One disadvantage of this method is that while traditional DNA extraction methods may produce enough template DNA (even from a single Trichogramma) for twenty or more PCRs, each card disc (i.e. each extraction) can only be used 3 or 4 times (unpublished data).
Table 1. Species-specific oligonucleotide primers used in the multiplex PCR
Figure 1. Trichogramma sp. ovipositing into its lepidopteran egg host.
Host egg / host plant
Species
Primer name
Sequence
Trichogramma acheae
ac 2-rev
5’-AGGCGACGTCTCTGTAGGGCC-3’
T. chilonis
ch 2-rev
5’-TGCTGCTGTTGTTGATACAACC-3’
T. papilionis
pa 1-rev
5’-AAAGTCTCGTTCAGCAAAAAGC-3’
T. pretiosum
pr 2-rev
5’-TCGCGCGCGTTTTACACACGTG-3’
Trichogrammatoidea sp.1
to 2-rev
5’-GACGTTCGTCGTCGAGCTACC-3’
Common forward
ITS2-forward
5’-TGTGAACTGCAGGACACATG-3’‡
‡Campbell
et al. 1993
Identification of specimens from FTA® cards Individual wasps were “collected” from the FTA® card as a 1.2 mm diameter “punched” disc, which was then transferred to a 0.2 mL PCR tube. Individual discs were incubated in 200 µL FTA® Purification Reagent for 5 min at room temperature and then washed with 200 µL of TE0.1 for a further 5 min, again at room temperature. The discs were then allowed to air dry for at least 30 min before being used directly as template for multiplex. Thus, for each specimen, DNA isolation/purification and multiplex PCR were performed in a single PCR tube. Following PCR, FTA® discs were retrieved from the PCR tubes, washed with 200 µL of TE0.1 for 5 min at room temperature, air dried, and stored at 4oC for use in future PCR. The Reaction DNA from individual wasps was amplified in 25 µL multiplex reactions containing 1xThermoPol Reaction Buffer (New England BioLabs, Ipswich, MA), 20 µM each dNTP, 0.6 µM common forward primer, 0.2 µM each reverse primer, 1 U Taq polymerase (NEB), and a single FTA® template DNA disc. Amplification was performed in a Mastercycler® ep gradient S (Eppendorf North America Inc., New York, NY) programmed for: an initial denaturing step of 3 min at 95oC; followed by 37 cycles of 45 sec at 92oC, 30 sec at 58.5oC, and 45 sec at 72oC; and, a final extension of 10 min at 72oC. PCR products were visualized by electrophoresis on a 2% agarose gel stained with ethidium bromide (Figure 5).
PR
C
P
A
C
P
C
P
P
T
C
T
C
T
*
A
*
P
P
T
P
A
P
T
-VE
+VE
T
T
P
-VE
T
A
T
T
T
+VE
The multiplex method Development of a species-specific multiplex PCR for this group of species was aided by large interspecific variation in the DNA sequence of the ITS2 spacer of the species present on Kauai. This multiplex PCR works well with this assembly of species but it is important to note that it is context specific; i.e. PCR primers that are species-specific on Kauai may not be species-specific if other species are also present. A key only works if the characters for the species that are present are known, new species are often not recognized unless they differ substantially form the ones known to occur. A case in point, is our discovery of a second species of Trichogrammatoidea near Hanalei, of which we found only a single thelytokous female. We were particularly interested in the culture line we established from that female because of its thelytokous mode of reproduction. As such, we tried several times to use our multiplex PCR method to identify specimens from the culture, but each time, the PCR failed. General ITS2 primers (Stouthamer et al. 1999) also failed, but we were able to eventually determine the ITS2 sequence of this species using universal primers (White et al. 1990). Since it was so rare, no species-specific primer was developed for this species. Furthermore, it can be argued that out multiplex PCR already identifies the specimen, because no PCR product is formed. Association of hosts with particular Trichogrammatid species There appears to be a clear association between some hosts and a particular parasitoid species. For instance Trichogramma pretiosum emerged from all five parasitized Monarch eggs, and Trichogrammatoidea sp 1 was only collected from Peablue eggs. The croton moth was parasitized by T. papilionis, T. chilonis and T. acheaea. The highest number of trichogrammatid species was found on the Peablue eggs, with as the most commons species Trichogrammatoidea sp.1, T. acheaea, T. papilionis, T. chilonis, T. pretiosum and a second Trichogrammatoidea species. This last species was only found once near Hanalei and is thelytokous. The Trichogrammatoidea sp. 1 appears to be specialized on the Peablue eggs because it was never found on Croton Moth eggs that were present in the same habitat, often the host plants for the croton moth and the Peablue were found right next to each other. Kauai has been sampled intensively before (Oatman et al. 1982) for trichogrammatid species, but neither Trichogramma acheaea, nor the two Trichogrammatoidea species had been collected there earlier. At the moment we do not know the species identity of the two Trichogrammatoidea species, but no Trichogrammatoidea species has been recorded before from Hawaii as far as we have been able to determine. The discovery of three new trichogrammatid species since the last survey is an indication of the large number of insect species that are inadvertently introduced into Hawaii. None of these species was introduced on purpose during this period for biological control. References Oatman ER, Pinto JD, Platner GR (1982) Trichogramma (Hymenoptera: Trichogrammatidae) of Hawaii. Pacific Insects 24:1-24.
Figure 3. Individual Trichogrammatid wasps were squashed onto FTA® Plant Cards (Whatman Inc., Florham Park, NJ).
http://www.whatman.com/UserFiles/File/Data%20 Sheets/Bioscience/FTA%20DNA%20Protection.pdf
Figure 4. Agarose gel (2%) showing the multiplex PCR products of five Trichogrammatid species from the Hawaiian island of Kauai. Lane 1, 100 bp DNA size standard (Fermentas, Glen Burnie, MD); 2, T. acheaea; 3, T. chilonis; 4, T. papilionis; 5, T. pretiosum; 6, Trichogrammatoidea sp. 1; 7, negative control; 8, multiplex-specific DNA size standard; 9, 100 bp DNA size standard.
Figure 5. Specimen identification using the speciesspecific multiplex PCR. A = T. acheaea; C = T. chilonis; P = T. papilionis; Pr = T. pretiosum; T = Trichogrammatoidea sp. 1; and, * = unknown. “Unknowns” were subsequently identified using universal Trichogramma PCR primers (see text).
Stouthamer R, Hu J, Van Kan FJPM, Platner GR, Pinto JD (1999) The utility of internally transcribed spacer 2 DNA sequences of the nuclear ribosomal gene for distinguishing sibling species of Trichogramma. BioControl 43:421–440. White TJ, Burns T, Lee S, Taylor TJ (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, Burlington, MA, pp 315–322.
