Studies on the identity of the parasitoids Aphidius colemani and Aphidius transcaspicus (Hymenoptera: Braconidae)

The taxonomic status of the aphid parasitoid Aphidius colemani Viereck has been questioned, especially in regard to Aphidius transcaspicus Telenga (Hymenoptera: Braconidae). The genetic association between A. colemani and A. transcaspicus was studied by cross mating individuals of A. colemani and A. transcaspicus (A.c.& × A.t.% and A.c.% × A.t.&) and applying appropriate molecular methods. The cross mating resulted in offspring (female and males) that were fertile. Therefore, the cross mating assays performed in an artificial environment showed that these two populations are potentially compatible. The mean number of mummies that developed and the sex ratio of the offspring of each cross were similar. Most of the male and female offspring from each cross were assigned to A. transcaspicus. Furthermore, the genetic divergence between the ribosomal internal transcribed spacers (ITS2) of the A. colemani and A. transcaspicus studied was 16%. These results indicate that A. colemani might be a complex of species with different morphological and biological characters attacking different host aphids.


INTRODUCTION
In the Aphidiinae, a subfamily of Braconidae, 400 species are described in 60 genera and subgenera (Starý, 1988).Members of this subfamily are endoparasitoids of aphids and include genera in which many species are important biological control agents.The genus Aphidius Nees is the largest genus of aphid parasitoids with about 70 species recorded throughout the world (Pungerl, 1986).
A. colemani and A. transcaspicus are very similar morphologically and difficult to separate.Starý (1975) considered A. colemani as synonymous with A. transcaspicus.Rabasse et al. (1985) compared populations of A. colemani from southern France and Brazil reared in Melanaphis donacis (Passerini) and Aphis nerii (Boyer), respectively.Their detailed investigation indicates that these populations differed in the number of antennal segments; the females of the population from southern France had 16-17 segments (rarely 15) and those from Brazil 15-16 segments (rarely 14).These populations also differed in their esterase patterns detected by electrophoresis.The R F of the population from southern France was 0.80 and that of the Brazil population 0.63 and 0.70.Pennacchio (1990) describes A. colemani reared from A. nerii and Hyalopterus spp.The A. colemani females have 15-16 antennal segments and their labial palps are 2 segmented (rarely 3 segmented).Takada (1998) states that Aphidius magdae Meschloff and Rosen (possibly = A. transcaspicus) is a species of Palearctic origin and occurs in Japan and Israel.The antennae of females of A. magdae have 16 (rarely 15 or 17) segments and their labial palps are 3 segmented.This species mainly specializes in parasitizing Hyalopterus spp., but has also been reared from Schizaphis rotundiventris (Signoret) and Toxoptera aurantii (Boyer de Fonscolombe).Kavallieratos & Lykouressis (1999) examined specimens of A. transcaspicus that emerged from Hyalopterus spp.and compared them with specimens of A. colemani reared from several aphid species in Greece.They report that A. transcaspicus females can easily be distinguished from those of A. colemani because the antennae of the former have 16-17 segments (rarely 15) and the latter 15 segments.The labial palps of A. transcaspicus are 3 segmented (rarely 2 segmented) rather than 2 segmented as in A. colemani.In addition, those authors state that A. transcaspicus differs morphologically from A. colemani in having a lighter F1, lighter coloured posterior terga, more slender pterostigma, and a less triangular and shorter metacarpus.
In fact, in terms of systematics Aphidius is a difficult genus (Pungerl, 1983(Pungerl, , 1986;;Mescheloff & Rosen, 1990).Some species within this genus are separated on the basis of their host aphids, despite being very difficult to distinguish morphologically, whilst laboratory experiments have shown that some of the morphological characters used for adult parasitoid identification can display considerable environmentally-induced variation (Pungerl, 1986).This approach was followed in the case of A. colemani and A. transcaspicus but has caused difficulties and given inconsistent results.
Molecular studies are becoming increasingly important in resolving taxonomic relationships in insects.Different genes are used to evaluate the genetic divergence of closely related species or populations (Caterino et al., 2000).The nuclear ribosomal repeat cistrons are widely used for phylogenetic studies of protistan, plant, and animal species.Nuclear RNA gene sequences have already proven valuable for studying higher taxonomic levels of insects (Pelendakis & Solignac, 1993;Malafronte et al., 2007;Nelson et al., 2008).A region of these repeats more suitable for genus and species comparisons is the second Internal Trancribed Spacer region (ITS2), i.e., the one between the 5.8S and 28S RNA genes.The ITS2, a noncoding rapidly evolving region, has highly repetitive sequences that can differ among closely related populations, which have proven useful for comparing closely related insect species, subspecies, or populations (Collins & Paskewitz, 1996;Fenton et al., 1998;Silva et al., 1999;Rokas et al., 2002;Rozenberg et al., 2006).
Studying genetic associations between closely related organisms is a topic that has recently attracted a large amount of attention.This is associated with the evolutionary concept concerning the genetic structure of populations and the process of speciation.This is especially important when using insect parasitoids in the biological control of aphid pests.This was recognized as a main ele-ment hindering the control of Hyalopterus spp.by A. transcaspicus in California (Lozier et al., 2008b).
The aim of the current study is to investigate the genetic association between A. colemani and A. transcaspicus.In particular two questions were addressed.Firstly, whether cross mating between individuals of A. colemani and A. transcaspicus is possible since cross mating indicates compatibility and thus, a close genetic relationship between two populations.Secondly, the genetic divergence between A. colemani and A. transcaspicus was determined using molecular methods.The combined use of these procedures provides a further contribution to the investigation of this taxonomic problem.

Experimental material
A. colemani and A. transcaspicus were reared on M. persicae infesting pepper plants (cv.Vidi).The A. colemani culture was initiated by adults reared from A. gossypii mummies collected on Hibiscus syriacus L. (Malvaceae) and that of A. transcaspicus from Hyalopterus mummies collected on Prunus persica L. (Rosaceae) in Athens.The aphid species was named following Lozier et al. (2008a), with aphids of the genus Hyalopterus infesting peach most likely belonging to H. persikonus.M. persicae colonies were initiated from aphids collected from pepper plants growing on the Campus of the Agricultural University of Athens.Aphid colonies were maintained in wooden framed cages 80 × 80 × 70 cm in a glasshouse at an average temperature of 22.5 ± 2.5°C (mean ± S.D.) and under natural lighting.
Potted pepper plants infested with M. persicae were each covered with a plastic cage.Each cage (11 cm in diameter and 30 cm high) was made of PVC (0.4 mm thick) with 2 openings of 9 × 9 cm each in the sides and the opening at the top covered with fine muslin for ventilation.Newly emerged parasitoids (15-20) (A.colemani or A. transcaspicus) were released into each cage and provided with food in the form of dilute drops of honey placed on the muslin covering the top of each cage.The cages were placed in a growth cabinet at 25 ± 0.5°C, 65 ± 5% R.H. and a 16L : 8D photoperiod.The parasitoid cultures were maintained by transferring, by means of an insect aspirator, two day old parasitoids to another cage.

Cross mating
Mummified aphids were removed from the cultures and transferred individually to small plastic vials (1 cm diameter × 4 cm height).The emerging parasitoids (<1 day old) were sorted by sex.One female (A.colemani or A. transcaspicus) together with one male (A.transcaspicus or A. colemani) were placed in a plastic Petri dish (9 cm in diameter and 1 cm in height, with a 3 cm in diameter opening in its lid covered with fine muslin, on which a droplet of honey was placed).After 24 h, the female parasitoid was transferred to another Petri dish containing about 200 individuals of M. persicae (nymphs of all instars and adults) on an egg plant leaf.The leaf was on a layer of cotton wool, moistened with water, in the base of the dish.The dish was then placed in a growth cabinet at 25 ± 0.5°C, 65 ± 5% R.H. and a 16L : 8D photoperiod.The parasitoid was removed from the dish after 24 h.The leaf in each dish was replaced with a new one every other day and the aphids carefully transferred to the new leaf and reared until mummification.In total, there were thirty replicates (15 with a A. colemani female and 15 with a A. transcaspicus female).The number of parasitoids that emerged from the mummies was recorded for each female.
The number of antennal segments, the number of labial palps of the offspring with 15 antennal segments and the sex of the emerged offspring of each maternal female were recorded.
The fertility of one female offspring from each of the cross mated females was determined.The offspring of each cross mated female were kept in a dish for 24 h after emergence.Then one of the female offspring was transferred to a Petri dish containing approximately 200 nymphs and adults of M. persicae feeding on the leaf of an egg plant and its fertility recorded as described above.The sex of the offspring of each female was recorded.
The number of mummies and number of offspring resulting from the two crosses (A.colemani& × A. transcaspicus% and A. colemani% × A. transcaspicus&) were compared using one way ANOVA of logarithmically transformed data.The emergence rate and sex ratio were compared using one way ANOVA.The percentage of offspring assigned to A. colemani or A. transcaspicus in the two crosses was compared using a two-way ANOVA, with the species of the maternal female and the morphology of the offspring as factors.Similarly, the sex ratio of the offspring of each maternal female was tested for significance using a two-way ANOVA.Data on percentages were arcsine transformed prior to analysis.Means were separated using a Tukey-Kramer HSD test.Analyzes were done using the statistical package JMP (SAS Institute Inc., 2007)

DNA extraction and amplification
Five A. collemani females were reared from A. gossypii mummies collected from five H. syriacus plants and five A. transcaspicus females from H. persiconus mummies collected from five P. persica trees in Athens.
Total genomic DNA was extracted from each individual by mechanical trituration of the tissue (in buffer solution), followed by 1h of chemical digestion with proteinase K and then a salt and ethanol precipitation.PCR products were amplified using the following ITS2 ribosomal primers (Porte & Collins, 1991): 5.8SF: 5'-TGTGAACTGCAGGACACATGAAC and 28SR: 5'-ATGCTTAAATTTAGGGGGTA.These primers anneal to highly conserved sequences in the 5.8S and 28S rDNA genes flanking the ITS2 region.The ITS2 PCR protocol was 40 cycles of 94°C for 45 s, 54°C for 45 s and 72°C for 60 s.The PCR products were cloned in a Blue Script KS+ plasmid vector (Agilent Technologies-Stratagene Products Division, La Jolla, CA, USA).Samples were sent for direct sequencing in both directions.

Sequence alignment and phylogenetic analysis
The boundaries of the ITS2 region were defined by comparison with previously determined Braconidae 5.8S and 28S rDNA sequences submitted to the GenBank database at the US National Centre for Biotechnical Information (NCBI) (http://www.ncbi.nlm.nih.gov).Nucleotide sequences were aligned using the ClustalW program (http://www.ebi.ac.uk/clustalw/) under default parameters.Mean sequence divergence of ITS2 variants within and between each Aphidius population was estimated using PAUP* by uncorrected P, which is the propor-tion of nucleotide sites differing between two compared sequences.The multiple alignment resulting from the ClustalW analysis was the input for BOXSHADE program (http://www.ch.embnet.org/software/BOXform.html),which was used to indicate residue similarity based on ITS2 sequences from various insects.All sequences were retrieved from Gen-Bank.The retrieved sequences were aligned using ClustalX program and the multiple alignment method (Thompson et al., 1997).Gaps and missing data were excluded from the data analysis.The program package PHYLIP 3.6a2 (Felsenstein, 2001) was used for calculating trees.The tree was constructed using the Neighbour-Joining method.
The offspring were separated into three groups based on the morphological characters used to separate A. colemani and A. transcaspicus.The individuals with 15 antennal segments and 2-segmented labial palps were assigned to A. colemani.Those with more than 15 antennal segments were assigned to A. transcaspicus.Finally, individuals with 15-segmented antennae but 3 segmented labial palps were also grouped under A. transcaspicus, but in a different category following Kavallieratos & Lykouressis (1999).The 3 categories are referred to as A.c., A.t.1, and A.t.2, respectively.Crossing did not significantly affect the percentage of offspring assigned to A.c. or A.t. (F1,56 = 0.16, p > 0.89).However, a significantly higher percentage of the offspring were assigned to A.t. than A.c. (F1,56 = 370.78,p < 0.001).When A.c. mothers were used the percentage of the offspring assigned to A.c. was 31 ± 3% and to A.t. 69 ± 4%.In the case of A.t. mothers the percentages were 25 ± 1% and 75 ± 2%, respectively.
The percentage of the offspring assigned to each morphological type (A.c., A.t.1 and A.t.2), was not affected by the crossing (F1,84 = 0.82, p > 0.36).However, there were significant differences among the percentages of 493 Values within columns followed by the same lower case letter are not significantly different .offspring assigned to each of the three morphological types (F2,84 = 194.14, p < 0.0001).The interaction between these two factors was significant (F2,84 = 35.63,p < 0.0001).Significantly more of the offspring of A.c.& × A.t.% were assigned to A.t.1 than of the A.c.% × A.t.& (Fig. 1).The percentages of the other categories (A.c.and A.t.2) were similar when the maternal female was A. transcaspicus.However, in the case of A. colemani females the percentage of the offspring assigned to A.t.2 was much reduced compared to A.c. (Fig. 1).
The sex ratio of the offspring within each morphological category is shown in Table 2. Statistical analyses revealed that the effect of the crossing, the effect of the morphological category of the offspring and their interaction were insignificant (F1,84 = 0.52 p > 0.47; F2,84 = 1.72, p > 0.18 and F2,84 = 0.63, p > 0.53, respectively).The sex ratios ranged between 0.53 ± 0.02 and 0.59 ± 0.05.
All the female offspring used in fertility tests produced offspring.The average sex ratio of the offspring was 0.54.

Molecular analysis
Analysis of the 5.8S, ITS2, and 28S regions The primer pair (5.8SF and 28SR) amplified the ribosomal ITS2 of A. colemani and A. transcaspicus.Sequences were confirmed as ribosomal ITS2 by searching the GenBank database of the NCBI using BLAST protocol.Each sequence is available from Gen-Bank (Accession number for A. colemani = DQ504298 and for A. transcaspicus = DQ504299).
Variability in the length of 5.8S-ITS2-28S was recorded both within and between populations of the two Aphidius examined.Lengths ranged from 739 to 745 bp for the five individuals of A. colemani and from 639 to 645 bp for the five of A. transcaspicus (Table 3).
Aligned ITS2 sequences of A. colemani (DQ504298) and A. transcaspicus (DQ504299), contained several inferred insertions or deletions (indels), the largest being 60 bp (Fig. 2).Three different regions of the rRNA gene can be distinguished.Region I (~90 bp in length) corresponds to the 5.8S rRNA coding sequence.ITS2 covers Region II (~450 bp in A. colemani and ~385 bp in A.

494
Values followed by the same lower case letters are not significantly different.0.56 ± 0.02a 0.54 ± 0.01a 0.53 ± 0.02a Morphological category of the offspring Crossing TABLE 2. The sex ratio (mean ± SE) of offspring of crosses between A. colemani and A. transcaspicus assigned to each morphological category.The offspring with 15 antennal segments and 2-segmented labial palps were assigned to A. colemani (A.c.) and those with more than 15 antennal segments to A. transcaspicus (A.t.1).Finally, offspring that had 15-segmented antennae and 3 segmented labial palps were assigned to A. transcaspicus (A.t.2).  transcaspicus).Region III covers a ~205 bp sequence at the 5' end of the 28S rRNA gene.
Mean sequence divergence of 5.8S-ITS2-28S within and between A. colemani and A. transcaspicus populations was estimated using PAUP* by uncorrected P (Table 3).The mean GC content percentage of A. colemani was 22.8 (SD = 0.19), whereas that of A. transcaspicus was 23.5 (SD = 0.21).Sequence divergence recorded between individuals of A. colemani was 0.087, the same as between A. transcaspicus.Sequence divergence (uncorrected P) between populations was 0.16.

ITS2 phylogenetic analysis
Complete ITS2 sequences of the ribosomal DNA of six specimes/species of Hymenoptera (Braconidae) were determined using a BLAST search of GenBank.Namely: A. colemani (Greece) (DQ504298), 2-A.colemani (USA) (EU561659), A. transcaspicus (DQ504299), Lysiphlebus testaceipes Cresson (Hymenoptera: Braconidae) (AY498555) and Leptopilina boulardi (Hymenoptera: Figitidae) (AY124568).ITS2 sequences were phylogenetically informative in separating Braconidae species (Fig. 3).Our results indicate that Aphidius populations (A.Cross-mating is the main criterion for species separation (cryptic or complex species) (Mackauer, 1969;Starý et al., 1980).However, more recent research has shown that interspecific crosses may occur in the laboratory as in this artificial mating environment the ecological or behavioural barriers active in nature are not effective (Smith et al., 1993).In fact, neither the sterility or viability of offspring of cross matings are useful criteria of species status (Paterson, 1988).Even when interbreeding occurs the hybrid population might be viable only for a few generations or hybridization might result in a significant post mating fitness cost such as a reduction in offspring fecundity.According to the Haldane's rule, in haplodiploid species, the fertility of F2 males must be assessed since F1 males are not hybrids (Koevoets & Beukeboom, 2009).These shortcomings weaken the reliability of mating assays for identifying species, when done in the laboratory.
However, if the offspring of the crosses are classified according to their morphology, then most of them are A. trascaspicus (Fig. 1).In addition, 26% of the offspring of A.t.& × A.c.% crosses and 9% of those of the A.c.& × A.t.% crosses had 15 antennal segments and 3-segmented labial palps and were assigned to A. transcaspicus (A.t.2).According to Kavallieratos & Lykouressis (1999) individuals of A. transcaspicus reared from Hyalopterus spp.collected from several areas in Greece, only very rarely have 15 antennal segments (1 female specimen in a total of 49 specimens collected).Therefore, a relatively high percentage of offspring of the crosses belong to a morphological type rarely occurring in nature.This indicates the potential for producing different morphological types, which could be used to evaluate the results of field surveys of variability in A. colemani and A. transcaspicus.
Based on morphological characters and cross mating it is difficult to determine conclusively the exact taxonomic association between A. colemani and A. transcaspicus.Therefore, a genetic approach was adopted based on quantifiable data obtained by characterization of rDNA sequences, including the second internal transcribed spacers, the inverting 5.8S and adjacent portions of the 28S coding regions.An advantage of this genomic target is that it includes highly conserved segments in the coding regions as well as hypervariable spacer sequences.The latter have repeatedly proved useful in distinguishing between taxonomic entities at and below the species level.
ITS2 sequences of A. trancaspicus were shorter than those of A. colemani and the difference in length was due to a deletion of ~60 bp at position 235 in the ITS2 fragment of A. trancaspicus (Fig. 2).The placement of indels was, in general, unambiguous and easily identifiable because they were sufficiently infrequent that they generally did not overlap (Lopez-Vaamonde et al., 2001).These consistent differences in length could be used to separate the two populations without the necessity of sequencing.
The ITS2 in the A. colemani and A. transcaspicus populations studied differed by 16%.This level of divergence is quite high for the same species or sub-species.In similar studies it was difficult to confirm the species level when the variability in ITS2 sequence between populations is of this order (Onyabe & Con, 1999;Romi et al., 2000;Lehr et al., 2005).Thus, we suggest that the DNA sequence divergence between A. colemani and A. transcaspicus populations might indicate a species-complex with different morphological and biological characters attacking different host aphids.Our results indicate that the A. colemani, A. transcaspicus-species complex is very complicated and requires further study.Future studies, including more nuclear and mtDNA genes, might further verify the species status of these two closely related aphid parasitoids.

Fig. 1 .
Fig. 1.Percentage (%) (mean ± SE) of the offspring of each cross that were assigned to the different morphological categories.The individuals with 15 antennal segments and 2-segmented labial palps were assigned to A. colemani (A.c.) and those with more than 15 antennal segments were assigned to A. transcaspicus (A.t.1).Finally, individuals that had 15segmented antennae and 3 segmented labial palps were assigned A. transcaspicus (A.t.2).Columns topped with different letters indicate significantly different values.
colemani and A. transcaspicus) do not cluster in the same clade as Lysiphlebus, within the Aphidiinae.DISCUSSIONAccording to our results, A. colemani and A. transcaspicus can interbreed.The F1 generation consists of both females and males and the F1 females are fertile.Thus the A. colemani and A. transcaspicus populations used in this study are compatible and genetically very similar.

TABLE 1 .
Mean (± SE) number of mummies, mean number of offspring, emergence rate, and sex ratio of offspring resulting from cross mating A. colemani and A. transcaspicus.

TABLE 3 .
t.2).Columns topped with different letters indicate significantly different values.Sequence and length diversity of 5.8S-ITS2-28S variants from A. colemani and A. transcaspicus populations.