(Gyllenhal) (Coleoptera: Curculionidae)

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independent assortment and crossing-over. This may limit the potential of G. scutellatus to proliferate under changing conditions and increase its pest status in ...
RESEARCH ARTICLES Genetic recombination potential of Gonipterus scutellatus (Gyllenhal) (Coleoptera: Curculionidae), a pest of eucalyptus plantations in South Africa 1 2 1 T.M. Miles , A. Fossey & T. Olckers * 1

School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville, 3209 South Africa 2 Biotechnology, Faculty of Health and Environmental Sciences, Central University of Technology, Free State, Private Bag X20539, Bloemfontein, 9300 South Africa The eucalyptus snout beetle, Gonipterus scutellatus (Gyllenhal), introduced to South Africa from Australia, causes extensive damage to eucalyptus plantations in colder regions where biological control is less effective. A cytogenetic study was undertaken to assess the weevil’s genetic recombination potential and thereby its ability to adapt to changing environmental conditions. During this study we investigated: (i) the sequence of meiosis; (ii) the weevil’s karyotype; and (iii) the number and position of chiasmata on the bivalents. Squashes of male testes revealed a normal, chiasmate meiosis, a sex chromosome complement of Xyp and an autosome complement of three large, and two medium-sized, sub-metacentric pairs and seven small acrocentric pairs. The diploid chromosome number (2n = 26) is marginally higher than the ancestral chromosome number of the Curculionidae (2n = 22). Chiasmata were absent from the proximal regions of the bivalents, occurring mostly in the distal regions and less frequently in the interstitial regions. The mean of 15 chiasmata per cell was marginally higher than the number of bivalents (13); consequently, acrocentric and most sub-metacentric chromosomes displayed a single distal chiasma, while 2–3 sub-metacentrics also displayed a second distal or interstitial chiasma. The relatively low chromosome number and low frequency of chiasmata indicate that genetic recombination is controlled at independent assortment and crossing-over. This may limit the potential of G. scutellatus to proliferate under changing conditions and increase its pest status in the future. Key words: chiasmata, control mechanisms, crossing-over, cytogenetics, independent assortment, recombination, snout beetle, weevil.

INTRODUCTION The eucalyptus snout beetle, Gonipterus scutellatus (Gyllenhal), which belongs to one of the largest beetle families, the Curculionidae, with some 50 000 described species (Lachowska et al. 2006), defoliates commercial eucalyptus plantations in the colder regions of South Africa (Atkinson 1999). Native to Australia, the weevil was first detected in Cape Town in 1916 but rapidly became widespread throughout South Africa (Atkinson 1999; Tribe 2005). Both larvae and adults attack the leaves of several eucalyptus species, although severe infestations are rare due to biological control by the egg parasitoid Anaphes nitens (Girault) (Mymaridae) that was introduced from South Australia in 1926 (Atkinson 1999; Tribe *To whom correspondence should be addressed. E-mail: [email protected]

2005). However, the wasp is ineffective at high altitudes where cold winter temperatures inhibit oviposition which, together with a reduction in tree vigour during winter, results in severe foliar damage by first-instar larvae, thus necessitating chemical control (Atkinson 1999; Rivera et al. 1999; Tribe 2005). Cytogenetic studies of forestry insects are rare (Bione et al. 2005), which is also true of Curculionidae in general, where cytogenetic information is mostly limited to descriptions of chromosome numbers, sex chromosomes and chromosome banding patterns (Holecová et al. 2002; Lachowska et al. 2004; Roóek et al. 2004; Holecová et al. 2008). The diploid chromosome number of the Curculionidae ranges from 20 to 56 (Gill et al. 1990) with the chromosome complement African Entomology 19(3): 558–563 (2011)

Miles et al.: Genetic recombination in Gonipterus scutellatus, a pest of eucalyptus

of 22 chromosomes being ancestral for the group, since a large percentage of the species surveyed displayed this karyotype (Smith & Virkki 1978; Sharma et al. 1980; Lachowska et al. 1998; Holecová et al. 2008). The sex chromosome system is mostly similar to that of humans, namely XX:XY, although multiple sex chromosome systems have been reported (Vidal 1984; Postiglioni et al. 1987). The Y chromosome is usually one of the smaller sized chromosomes, usually described as yp, while the X is usually medium sized (Holecová et al. 1999; Roóek et al. 2004). An understanding of the cytogenetics of G. scutellatus, in particular the frequency of crossingover, could provide some insight into its potential to create genetic variation and thereby adapt to changing environmental conditions, such as changes in climate and insecticide regimes. The aim of this study was thus to assess the recombination potential of G. scutellatus by: (i) describing the sequence of meiosis; (ii) determining the karyotype; and (iii) analysing the bivalents in terms of the frequency and position of chiasmata during crossing-over. In doing so, we tested the hypothesis that crossing-over in G. scutellatus occurs randomly in all bivalents, thus indicating the absence of a cytogenetic control mechanism on recombination. If this is true, G. scutellatus may be unrestricted in the creation of genetic variation which could have implications for its future pest status. MATERIAL AND METHODS Adult weevils were collected in eucalyptus plantations around Hilton (29°33.021’S 30°20.303’E) in KwaZulu-Natal. Males were killed with ethyl acetate and their testes dissected under a stereomicroscope and placed in vials containing a fixative of two parts methanol: one part acetic acid (Fossey et al. 1989). The vials were sealed and left at room temperature for 24 h to allow effective fixation of the chromosomes. Thereafter, the fixative was poured off and replaced with 100 % ethanol and the material was stored in a freezer until required (Fossey et al. 1989). Squashes of one third of a testis at a time, for each of 12 male individuals, were prepared in a 1 % acetocarmine stain of 1 % carmine in 45 % acetic acid (Sharma & Sharma 1980). A trace of ferric acetate was added with a needle to intensify the staining of the chromatin. After maceration of the material, an albumin-layered cover slip, dried over

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an alcohol flame, was placed on the material on a slide and the preparation was squashed between layers of filter paper. The preparation was gently heated over an alcohol flame to flatten the cells and facilitate sticking to the albumin layer on the cover slip. Cells were viewed briefly under a light microscope to confirm adequate staining and the presence of the desired divisions. Permanent mounts were then prepared by separating the cover slips from the slides in 45 % acetic acid, dehydrating them in a series of alcohol solutions (70 %, 80 %, 95 % and absolute alcohol) for two minutes each and finally mounting them in Euparal. Meiosis was studied and photographed using oil immersion at ×1000 magnification with an Olympus photomicroscope. Although it is generally accepted that chiasma analysis is undertaken at the diakinesis stage, the paucity of these cells necessitated the recording of the frequencies and positions (i.e. proximal, interstitial and distal) of chiasmata of bivalents at metaphase I. Fifteen metaphase I cells, from each of the 12 individuals were examined (i.e. 180 cells). Thereafter, the mean number of chiasmata per bivalent per individual and the mean number of proximal, interstitial and distal chiasmata per cell per individual were determined. The small size of the chromosomes did not permit measurements of the arms at anaphase I or anaphase II; therefore the karyotype was deduced through inspection of metaphase I and anaphase I cells. The position of larger heterochromatic regions could also be identified as darker staining areas on prophase I (pachytene) bivalents. Statistical analyses using SPSS Version 13.0 for Windows (2002) were conducted to ascertain whether crossing-over in G. scutellatus is limited in number and/or position. One-way ANOVA was used to determine whether chiasmata frequency varied between individuals. A generalized linear model, using the repeated measurements option of the SPSS software, was also used to determine whether chiasmata were positioned randomly in the different bivalents. RESULTS AND DISCUSSION Meiosis As expected, G. scutellatus displayed a normal, chiasmate meiosis similar to that of other insect species, including Curculionidae (Roóek et al.

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a

b

c

d

Fig. 1. Cell division in Gonipterus scutellatus. a, Pachytene (prophase I), showing the heteropycnotic sex bivalent (arrow) and heterochromatin segments (arrow heads); b, late segregation of bivalents at anaphase I (arrow); c, polar view of mitotic anaphase; d, metaphase I showing ring- and rod-shaped bivalents, as well as the parachute-shaped heteromorphic sex bivalent (arrow). Scale bars = 10 µm.

2004). Meiosis appeared to proceed quickly as most of the stages were rarely detected, except the early prophase I stages, leptotene and zygotene, and the final stage of telophase II. At pachytene, the Xyp bivalent was present as a heteropycnotic body (Fig. 1a), similar to other Curculionidae species (Lachowska et al. 2006; Holecová et al. 2008). Although the incidence of anaphase I cells was rare, the few cells that were detected were used to confirm the weevil’s karyotype (see below). Synapsis appeared to be accurate, as asynaptic homologous univalents were rare and noticed in only one of the 180 cells studied. Two, sometimes three, of the larger bivalents displayed anomalous segregation by segregating later than the other bivalents at anaphase I (Fig. 1b).

Karyotype The diploid chromosome number of male weevils (2n = 26) was determined by the number of bivalents at metaphase I and confirmed by the number of chromosomes at the anaphase II and mitotic anaphase poles as demonstrated in the anaphase spread (Fig. 1c). The diploid chromosome number is marginally higher than the ancestral chromosome number for the Curculionidae (2n = 22) and falls within the lower range of weevil chromosome numbers (2n = 20–56) (Gill et al. 1990). Only a few other weevil species, namely Apotomorhinus cribratus Sch., Curculio ficusi P. & S., Curculio longirostris P. & S. (Gill et al. 1990), Curculio pellitus (Boheman) (Lachowska et al. 2001) and Curculio venosus Grav. (Lachowska et al.1999) have so far

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Table 1. Mean (±S.D.) number of chiasmata per cell, recorded from 15 cells in each of 12 males of Gonipterus scutellatus. Male

1 2 3 4 5 6 7 8 9 10 11 12 Sample mean

Total number of chiasmata

Number of distal chiasmata

Number of interstitial chiasmata

14.80 ± 0.94 15.53 ± 1.06 15.93 ± 1.03 15.00 ± 1.07 15.27 ± 0.70 14.93 ± 0.70 15.47 ± 0.99 16.00 ± 0.85 16.07 ± 0.88 15.40 ± 0.99 16.20 ± 0.56 15.53 ± 0.64 15.51 ± 0.47

12.67 ± 1.99 14.53 ± 1.68 14.60 ± 1.55 13.53 ± 1.25 12.87 ± 1.19 12.20 ± 2.04 12.73 ± 1.87 13.73 ± 1.62 13.07 ± 1.42 12.80 ± 1.42 13.13 ± 2.00 13.00 ± 1.31 13.24 ± 0.70

2.13 ± 1.88 1.00 ± 1.20 1.33 ± 1.29 1.47 ± 1.30 2.40 ± 0.99 2.73 ± 1.79 2.73 ± 1.39 2.27 ± 1.16 3.00 ± 1.36 2.60 ± 1.30 3.07 ± 2.19 2.53 ± 1.41 2.27 ± 0.64

been reported to have the same chromosome number. The sex chromosome system (XX:Xyp) is of the more common type with the yp chromosome being one of the smaller-sized chromosomes and the X being medium-sized (Roóek et al. 2004). The karyotype can thus be described as 2n (8) = 24 + Xyp = 26. From the male chromosome complement, it can be deduced that the female complement is 2n (9) = 24 + XX = 26. The karyotype of G. scutellatus was partially asymmetrical, in terms of chromosome length and centromere position. Most chromosomes appeared to be of different lengths and none were metacentric. The autosome complement of 12 chromosome pairs consisted of three large, and two medium-sized, sub-metacentric pairs and seven small acrocentric pairs. The X chromosome was similar in size to the largest acrocentric autosome, while the yp chromosome was similar in size to the smallest autosome. The configuration of the heteromorphic Xy p bivalent at metaphase I, containing a single distal chiasma, was parachute shaped (Fig. 1d). Early prophase I (pachytene) cells also revealed the presence of heterochromatic segments (Fig. 1a) in the vicinity of the centromeres, which is typical for many species of Curculionidae (Holecová et al. 2008). Chiasma frequency and position Chiasmata were present in the interstitial and distal regions of the bivalents, but were absent from the proximal regions, i.e. in the immediate

vicinity of the centromere (Fig. 1d). The small acrocentric bivalents displayed a single distal chiasma, while the larger sub-metacentric bivalents displayed both interstitial and distal chiasmata. The mean number of chiasmata per cell differed significantly between individual males (P < 0.001) and ranged from 14.8 to 16.2 per cell (Table 1), which were approximately two to three chiasmata more than the number of bivalents (13). These limitations in the positioning of chiasmata resulted in a limited number of bivalent configurations (types); these were mainly ring-shaped and rod-shaped structures (Fig. 1d). For the 12 individuals sampled, the mean number of distal chiasmata per cell ranged from 12.2 to 14.6, which was significantly higher (P < 0.001) than the mean number of interstitial chiasmata per cell, which ranged from 1 to 3.1 (Table 1). CONCLUSION The partially asymmetrical karyotype, chromosome number and chiasmata data revealed that genetic recombination in G. scutellatus is controlled by crossing-over as well as by independent assortment. Genetic recombination restriction is a feature that is typical of many insects, particularly grasshoppers (White 1973). In G. scutellatus, crossingover does not occur randomly in the bivalents but is restricted largely to the distal regions, probably due to the presence of centromeric heterochromatin that tends to repel chiasmata (De Jager & Fossey 1993). This results in a low mean number of

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chiasmata per cell (around 15), which is marginally more than the number of bivalents. In terms of independent assortment, the number of bivalents in G. scutellatus is lower than in most other species of Curculionidae, thereby influencing its recombination potential by also controlling the number of bivalent arrangements on the metaphase I equator. For example, the weevil, Lixus iridis Olivier, has 21 autosomal bivalents (Holecová et al. 2000) permitting a much higher number of possible bivalent arrangements at metaphase I. A reduced recombination potential has little impact on a large random mating population with a broad inherent genetic variation. However, if a population arose from a small founder population, as is often the case with introduced species, the inherent genetic variation is expected to be limited (White 1973; Fossey et al. 1989) which, together with a limited recombination potential, could negatively affect its ability to proliferate in changing environments. Gonipterus scutellatus was accidentally introduced into the Western Cape around 1916 (Atkinson 1999; Tribe 2005), but the size of the founder population and the extent of its inherent genetic variation is unknown. However, if G. scutellatus indeed arose from a small founder

population, its rapid increase and wide distribution in South Africa may be explained by the lack of natural enemies at the time. Low genetic variation may also explain the success of biological control because of a lower possibility of the weevil evolving strategies to cope with the parasitoid. Consequently, it can be hypothesized that in the event of sustained selection pressure, such as insecticide applications, the ability of G. scutellatus to adapt and proliferate (e.g. develop resistance) could be restricted, ensuring that it is unlikely to pose a substantially greater threat to eucalyptus plantations in the future. To substantiate this argument, weevil populations from other parts of South Africa should be tested to confirm that limited genetic recombination is indeed the norm for G. scutellatus. ACKNOWLEDGEMENTS We thank P.E. Hulley (Rhodes University) and anonymous referees for comments on the manuscript, S. Mackellar (University of KwaZulu-Natal) for her assistance with the photography and the School of Biological and Conservation Sciences (University of KwaZulu-Natal) for financial support.

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