Chromosomes of parasitic wasps of the genus Metaphycus ( Hymenoptera : Chalcidoidea : Encyrtidae )

Karyotypes of four species of the genus Metaphycus Mercet, 1917, namely, M. fl avus (Howard, 1881) and M. luteolus (Timberlake, 1916) (both have n = 10 and 2n = 20), M. angustifrons Compere, 1957 (n = 9 and 2n = 18) and M. stanleyi Compere, 1940 (n = 5 and 2n = 10) were studied. The latter chromosome number, n = 5, is the lowest known one for the family Encyrtidae. A karyotype with n = 10 is considered ancestral for the genus Metaphycus. Karyotype evolution in this genus is likely to have occurred through chromosomal fusions.


INTRODUCTION
The world fauna of the family Encyrtidae, a large group of the superfamily Chalcidoidea, contains more than 3700 species (http:// www.nhm.ac.uk/research-curation/research/ projects/chalcidoids/encyrtidae.html, on March 26, 2010).Chromosomes of only about 15 species of the family (i.e. less than 0.5% of described species) have been studied up to now (Gokhman, 2009).Moreover, karyotypes of the large genus Metaphycus Mercet, 1917 that includes many economically important parasitoids of soft scales (Homoptera: Coccidae) and related insects (Trjapitzin, 1989) remained fully unknown until recently.I have managed to study chromosomes of four species of this genus that are cultivated at the University of California at Riverside (USA) (UCR).Descriptions of these karyotypes are given below.

MATERIAL AND METHODS
Air-dried preparations of mitotic chromosomes were obtained in August 2009 from cerebral ganglia of prepupae extracted from parasitized individuals of Coccus hesperidum Linnaeus, 1758 (Coccidae).Both parasitoids and their hosts were kept as laboratory stocks at UCR.The preparations were made according to the technique used by Imai et al. (1988) with certain modifi cations.Images of metaphase plates were obtained using light microscope Zeiss Axioskop 40 FL fi tted with the digital camera AxioCam MRc (Table 1).Chromosomes were measured on digital micrographs of haploid metaphase plates using Zeiss AxioVision, their relative lengths (RL) and centromere indices (CI) were calculated (Table 2).However, since centromere position could not be precisely identifi ed on chromosomes of certain species, CI values for those species were omitted in the table (see below).All chromosomes were arranged on karyo- . Cytogenet., 2010 4(1) Comparative Cytogenetics grams according to the classifi cation provided by Levan et al. (1964).

RESULTS
Metaphycus fl avus (Howard, 1881) (Fig. 1, a-c).n = 10, 2n = 20.Most chromosomes gradually decrease in size, except for the fi rst pair (Fig. 1, b; or sometimes just one of the two homologues in the diploid set; Fig. 1, c) which is visibly longer than the others (see Table 2).All chromosomes appear to be either subtelocentric or acrocentric.
Metaphycus luteolus (Timberlake, 1916) (Fig. 1, d, e).n = 10, 2n = 20.The karyotype of this species is similar to that of the previous one.Nevertheless, chromosomes of the fi rst pair are only slightly longer than the others (Table 2).

Metaphycus angustifrons
Compere, 1957 (Fig. 1, f-h).n = 9, 2n = 18.Most chromosomes gradually decrease in size, except for the fi rst pair, which is apparently metacentric (Table 2).Chromosomes of the second pair are obviously acrocentric, all other chromosomes are subtelocentric.In this species, a few metaphase plates with the so-  Comp.Cytogenet., 2010 4(1) Comparative Cytogenetics considered ancestral not only for the Encyrtidae, but also for the superfamily Chalcidoidea as a whole (Gokhman, Gumovsky, 2009), the closest n value found in Metaphycus fl avus and M. luteolus, n = 10, is likely to be ancestral for the genus Metaphycus.If this is true, chromosome numbers of M. angustifrons and M. stanleyi, n = 9 and 5 respectively, must be derived, and they have therefore originated through apparently subsequent chromosomal fusions.The fact that all shorter arms of subtelocentrics are heterochromatic in the former species, also favours this hypothesis because those arms can easily be lost during the fusion process.On the other hand, strong intercalary heterochromatic segments are infrequent on chromosomes of parasitic wasps (Gokhman, 2009), and this aberrant feature may be correlated with massive insertions of certain DNA sequences recently found in the Encyrtidae (Gillespie et al., 2005).
Finally, the present study has potential practical implications.Immature stages of the above mentioned parasitoids of the genus Metaphycus can easily be sorted to sex and species by their karyotypes.Population sex ratios of the studied Metaphycus species can therefore be evaluated at the prepupal stage using this technique (see also Gokhman, 2009).

ACKNOWLEDGEMENTS
The author is very grateful to Prof. Robert F. Luck (UCR) for providing laboratory stocks of Metaphycus species, to Dr. Serguei V. Triapitsyn (UCR) for providing opportunity to work at the Entomology Research Museum, as well as to Vladimir V. Berezovsky (UCR) and Andrey P. Mikhailenko (Moscow State University, Moscow, Russia) for technical assistance.The present study was partly supported by a research grant no.10-04-01521 from the Russian Foundation for Basic Research.called spontaneous C-banding were found (Fig. 1, g).Shorter arms of all subtelocentrics are heterochromatic.Most chromosomes also carry visible intercalary (or sometimes pericentromeric) and weaker telomeric heterochromatic segments.
Metaphycus stanleyi Compere, 1940 (Fig. 1, i, j).n = 5, 2n = 10.All chromosomes of this species are obviously biarmed (mostly metacentric) and generally larger than those of the previous ones.Apart from karyotypes of preceding species, most chromosomes of M. stanleyi can easily be distinguished from the others by their length and centromere position.

DISCUSSION
Karyotypes of the studied Metaphycus species show a considerable variation in terms of both chromosome number and chromosomal morphology.Surprisingly, substantial differences regarding length of the fi rst chromosome have been found at least between the studied populations of M. fl avus and M. luteolus with n = 10, although these species are sometimes considered synonymous (Guerrieri, Noyes, 2000).The two other species also have different n values, i.e. 9 and 5.The latter chromosome number, n = 5, is the lowest known one not only for the genus Metaphycus, but for the whole family Encyrtidae as well (the second lowest n value, n = 8, has been recorded for the genus Copidosoma Ratzeburg, 1844; see Gokhman, 2009 for review).The data obtained therefore indicate further blurring the border between the so-called "high-numbered" and "lownumbered" chalcid families.This phenomenon has recently been observed also in the Eupelmidae, Eurytomidae and Torymidae (Gokhman, Gumovsky, 2009).

Table 1 .
Number of studied individuals and metaphase plates of Metaphycus species.

Table 2 .
RL and CI values of chromosomes of Metaphycus species (mean ± SD).Numbers of metaphase plates used for morphometric analysis are given in brackets.