Hereditas 126: 187-189 (1997)

Brief reportSupernumeraryheterochromatindoes not affect several morphologicaland physiological traits in the grasshopperEyprepocnemis ploransA. MARTÍN-ALGANZA**, J. CABRERO, M. D. LÓPEZ-LEÓN, F.PERFECTTIand J. P. M. CAMACHO*

Departmento de Genética, Universidad de Granada, E-ISD71 Granada, Spain. E-mail(jpmcama@goliat.ugr.es)

Supernumerary heterochromatin in the form of au-tonomous B chromosomes or A-linked supernumer-ary chromosome segments (SCSs) constitutes themost frequent genomic polymorphism in grasshop-pers (HEWITT 1979). Most B chromosomes are con-sidered selfish genetic elements because they showdrive mechanisms that enhance their maintenance innatural populations despite the negative effects theymay produce on carriers (JONES 1985; BEUKEBOOM1994). Even though SCSs have received less attentionin the literature, it is now c1ear that many show drive(LÓPEZ-LEÓNet al. 1992a) and should also be consid-ered selfish elements integrated in the standardgenome. The maintenance of these polymorphismsdepends mainly on a balance of positive (drive) andnegative (harmful effects) factors that influence theirfrequency in natural populations. In arder to revealtheir evolutionary role it is necessary to investigatethese two properties (drive and effects).

The grasshopper Eyprepocnemis plorans has bothpolymorphisms in natural populations from theIberian Peninsula. The three most widespread B types(B¡, B2 and Bs) lack drive (LÓPEZ-LEÓNet al. 1992b)and fitness effects (LÓPEZ-LEÓNet al. 1992c; CAMA-CHO et al. 1997a,b), so that they are expected to goextinct unless they are either replaced by a new selfishB variant (CAMACHOet al. 1997a,b) or they haveunknown beneficial effects on fitness. The SCS 10-cated proximally on the smallest autosome shows atransmission ratio significantly lower iban theMendelian one through females possessing B chro-mosomes (LÓPEZ-LEÓNet al. 1991, 1994). Therefore,it is also expected to become extinct unless it isbeneficial for carriers. Surprisingly, ibis polymor-phism seems to show frequency stability ayer time innatural populations (LÓPEZ-LEÓNet al. 1995), sug-gesting the existence of some unknown factors influ-encing SCS frequency. Therefore, it is important toanalyse the effects of both polymorphisms on asmany different traits as possible. Here, we report the

* Correspondence** Martín-AIganza ís now at the Max-Planck-Instítut für Verhal-tensphysíologíe, 0-82319 Seewíesen, Gennany, E-maíl<alganza@mpí-seewíesen.mpg.de).

results of such an analysis for several morphologicaland physiological traits. It has indicated the absenceof any significant effects of the B2 chromosome andthe SCS on these characters.

MA TERIALS AND METHODS

A total of 258 males and 258 females of the grasshop-per Eyprepocnemis plorans were collected at Salo-breña (Granada, Spain) during the 1992 season. Inthe laboratory, the insects were weighed and thel{.dissected to extract the gonads, which were alsoweighed. Testes were fixed in 3:1 ethanol-acetiq acid,where testis follic1es were individualized and countedunder a stereomicroscope (mean = 76.047, SE =0.792, N = 255). Females were injected with 0.05 %colchicine in insect saline solution 6 h prior to fixa-tion of ovario les in 3:1 ethanol-acetic acid. Ovarioleswere counted (mean = 74.328, SE = 0.550, N = 256)and the proportion of ovarioles with a developing egg(mean = 0.578, SE = 0.017, N = 201) was alsorecorded. Male and female bodies were sto red in70 % ethanol until measurements were performed.Cytological analyses to determine the number of Bchromosomes and SCSs in each individual were per-formed by the C-banding technique described in CA-MACHOet al. (1991).

The external traits measured were: thorax lengthand width, tegmina length, hind femur length, andhind tibia length. Due to allometry, these charactersare strongly correlated, so that they were combined ina single trait indicating body size by means of aPrincipal Component Analysis (PCA). In males, PCAfor the five traits measured gave rise to a single factorexplaining 66.59% of total variance, with factor load-ings ranging from 0.679 (tegmina length) to 0.897(thorax length). In females, the single factor obtainedexplained 64.3 % of variance, and factor loadingsranged from 0.703 (tegmina length) to 0.869 (thoraxlength). These factors were used as a measure of bodysize in both sexes.

As a measure of body condition, we have usedweight-based somatic condition. To define ibis, weobtained the somatic weight of each individual bysubtracting gonadal weight from total weight, and

188 A. Martín-AIganza et al. Hereditas 126 (1997)

Table 1. Comparison of three morphological traits between males with different constitutions for the Blchromosome and the SCS, by means of a parametric ANOVA. In the case of B effect on soma tic condition,however, a Levene test indicated that variances were significantly different, for which a non-parametric Kruskal-Wallis ANOVA was used

then performed a regression analysis of this variableon body size (represented by the factor previouslydefined by PCA). Somatic weight has the advantageof being independent of the physiological state of thegonads. This is especially important in females wherea new pod cycle is started about each week, so thattotal female weight is heavily dependent on whetheran egg-laying cycle was beginning or ending. Theresults of the regression analyses were highly signifi-cant in both sexes, thus providing a good definitionof somatic condition as the standardized residuals ofthis regression (males: r = 0.788 :t 0.041, F = 365.04,df=l, 223, p<O.OOOl; females: r=0.765:t0.041,F=353.14, df= 1,250, p<O.OOOl).

Since individual s with three or more Bs were rarelyfound, they were included in the same class as 2Bindividual s (noted as 2B+).

RESUL TS AND DISCUSSION

Table 1 shows the absence of significant differencesbetween males with OB, lB and 2B+ for the numberof testis follicles, body size, or somatic condition,although the latter showed a significantly higher vari-ance in lB males (1.313) than in the other two classes(0.824 in OB males and 0.681 in 2B+ ones). In fe-males, no significant differences were observed as wellfor the four characters analysed: the number of ovar-ioles, the proportion of ovarioles developing an egg,body size, and somatic condition (Table 2). Similaranalyses also indicated absence of any effect of a SCSproximally located on the smallest autosome in eithersex (Tables 1 and 2).

The absence of effects of supernumerary hete-rochromatin on morphological traits such as bodysize and the number of testis follicles or ovarioles isconsistent with most previous studies, in which it hasbeen apparent that both B chromosomes and SCSsrarely produce visible effects on the external pheno-type of its carriers (for review, see JONESand REES1982). On the other hand, it is not infrequent thatsupernumerary heterochromatin causes diverse physi-

ological effects, many detrimental to carrier fitness(for review, see JONESand REES 1982).

The most plausible evolutionary scenario for thepresence of B chromosomes in E. plorans is that theywere selfish (showed drive) when they first invadednatural populations, but the drive has been neutral-ized by the appearance of supressor genes in the Achromosomes (CAMACHOet al. 1997a,b). The ab-sence of significant effects on several fitness compo-nents (LÓPEZ-LEÓNet al. 1992c; CAMACHOet al.1997b), and the morphological and physiologicaltraits analysed here, suggests that the neutralizationprocess not only involves a decrease in the B-trans-mission ratio but also phenotypic selection forgrasshoppers less affected by the presence of Bs. Thisscenario is clearly reminiscent of host-parasite coevo-lutionary processes (FUTUYMA 1986). In fact, theonly B variant showing drive hitherto known in E.plorans, a Bl-derivative named B14located in Torrox(Málaga, Spain), showed drive through females anddecreased female fertility, an effect not caused by Blin the nearby populations at Jete and Salobreña(Granaqa, Spain) (CAMACHOet al. 1997b). Accord-ingly, it seems that selfishness (drive)and parasitism(harmful effects on carriers) are parallel conditions.

The case of the SCS is slightly different because wehave never observed drive for this heterochromaticelement, although it cannot be ruled out that driveoccurred at the beginning when it appeared in thepopulation. On the contrary, heterozygous femalescarrying B chromosomes do tend to eliminate theSCS chromosomes (LÓPEZ-LEÓNet al. 1991, 1994),perhaps as result of genomic interaction between thetwo genome parasites. At present, however, we haveno more information about the cause of the SCSundertransmission. In any case, the result is that,other factors being neutral, the SCS is doomed to goextinct. But its frequency is not decreasing in naturalpopulations, at least in the short term, and the fitnesscomponents hitherto analysed are not affected by thepresence of the SCS (LÓPEZ-LEÓNet al. 1995; thispaper). Therefore, future efforts should be focused to

Item Bl chromosome SCS-

F df p F df p

Number of testis follicles 1.05 2,232 0.351 1.70 2,232 0.185Body size 2.19 2,234 0.114 1.08 2,234 0.341Somatic condition 4.81 2,206 0.090 0.50 2,206 0.609

Hereditas 126 (1997) Brief report 189

Tab1e 2. Comparison of four morphological traits between females with different constitutions for the B2chromosome and the SCS, by means of a parametric ANO VA

Item

Number of ovariolesEggsjovariolesBody sizeSomatic condition

","

.~ analyse other fitness components that might explainhow this polymorphism persists in natural popula-tions.

ACKNOWLEDGMENTS

This study was partially supported by grants from theDirección General de Investigación Científica y Técnica(no. pB93-1108) and the Plan Andaluz de Investigación,Grupo no. 3122 (Spain).

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B2 chromosome SCS

F df p F df P

2.97 2,253 0.053 0.27 2,253 0.7620.08 2, 198 0.925 0.03 2, 198 0.9751.64 2, 251 0.197 2.92 2, 251 0.0561.09 2,249 0.337 0.13 2,249 0.881