Comparison of the fat allocation patterns in female pine sawflies ( Hymenoptera : Diprionidae )

The allocation of fat resources to somatic and reproductive tissues was studied in females of five species of pine sawflies (Diprion pini (L.), Diprion similis (Htg.), Gilpinia frutetorum (F.), Gilpinia pallida (Klug), Neodiprion sertifer (Geoffrey); Hymenoptera, Diprionidae). The soma and eggs of newly emerged females were separated and then put in ether to extract and deter­ mine their fat content. The fat allocation patterns differed between species. The gregarious outbreak species D. pini and N. sertifer allocated most of the fat to their eggs, whereas the gregarious non-outbreak species, G. pallida, allocated most to its somatic tissues. No modification of these basic allocation patterns was observed with increasing total fat content. In contrast, the solitary non­ outbreak species, D. similis and G. frutetorum, invested equal amounts of fat in soma and eggs, and the proportion allocated to the egg load decreased as total fat content increased. That is, they invested additional resources in somatic tissues. Apparently, outbreak species tend to allocate more fat to reproductive tissues than non-outbreak species.


INTRODUCTION
There are around 13 species of European pine sawflies (Dipri onidae) and some of them are important pests of pine forests (Larsson et al., 1993).The life histories of the different species appear to be similar: eggs are laid in pine needles, developing larvae feed on the needles, they pupate in cocoons and emerge as adults after a diapause.However, there are profound differ ences in the feeding behaviour of larvae as a consequence of particular oviposition strategies.Species with gregarious larvae lay their complete egg load more or less in one cluster (Escherich, 1942), whereas species with solitary living larvae dis tribute their eggs spatially (Pschorn-Walcher, 1982).The spe cific modes of oviposition should set particular demands for mobility and longevity of egg-laying females and be associated with a dissimilar investment of nutrient resources in reproduc tion and survival (Papaj, 2000).To test this hypothesis, we com pared the resource allocation patterns of several pine sawflies exhibiting different oviposition strategies.In particular, we con sidered the investment of fat in eggs versus soma, as fat is used as an important source for oogenesis as well as of energy in insects (Beenakkers, 1969;Ellers, 1996;Wheeler, 1996).Poten tial constraints arising from different allocation patterns are dis cussed in relation to the particular life histories and outbreak tendencies of the species studied.

Origin of insects
Five species of Diprionidae were studied.They exhibit dif ferent oviposition strategies and larval feeding behaviour.Moreover, they vary in their outbreak tendency (Table 1).The gregarious feeding Diprion pini (L.) and Neodiprion sertifer (Geoffrey) are severe pests showing frequent infestations, whereas outbreaks of the (semi-) solitary living Diprion similis (Htg.) and Gilpinia frutetorum (F.) are rare.The gregarious spe cies Gilpinia pallida (Klug) occurs only sporadically at high densities, at least in Central Europe (Pschorn-Walcher, 1982).Females were obtained from continuous laboratory mass cul tures (Herz, 1997).Laboratory stocks originated from field col lected larvae and cocoons from different European countries (Table 1) and were reared under constant laboratory conditions (22°C, 18L : 6D h) for several generations.However, it was not possible to rear N. sertifer continuously due to the diapause of the sawfly embryo inside the egg (Pschorn-Walcher, 1982) and the occurence of a highly virulent nucleopolyhedrosis.Conse quently, young instars of N. sertifer were collected in the field, reared under laboratory conditions, and the resultant adult females were used in this study.

Treatment of females
Diprionid females are proovigenic and contain their entire supply of mature eggs when they emerge from the cocoon (Schedl, 1991).Therefore, females were killed directly after emergence by freezing at -25°C.The amount of fat was deter mined gravimetrically for individual females following the pro cedure described by Ellers (1996).Two cover slips (18 x 18 mm) were weighed, a female was put on one of them and its abdomen was cut open.After adding one drop of deionised water, the eggs were removed from the ovarioles, transferred individually to the second cover slip and counted.The cover slips plus insect parts were dried until of constant weight (80°C, 6 days for all sawfly species) and the dry weight of the soma (cover slip 1) and the egg load (cover slip 2), respectively, was determined.Then 3 ml of diethylether was added to each cover slip and the fat extracted for 24 h at room temperature.Then the diethylether was carefully changed and the extraction continued for another 24 h.After this, the insect parts were dried for 6 days at 80°C and the dry weight determined again.The loss in weight corresponded to the amount of fat in soma and egg load, respectively.Preliminary investigations on D.pini, the largest of the species studied, were used to establish this procedure.Con stant dry weight was achieved after 5 days.There were no dif ferences in the fat content of whole bodies (abdomen open, but still containing all the eggs), extracted for 24 h, 48 h or 96 h, so we chose 48 h as a sufficient period for fat extraction.
In addition, the average egg size of each species was esti mated by measuring the lengths and middle widths of 10 eggs/female and 5 females/species.The shape of the eggs of the different species is similar ("banana"-like) and the volume (V) of an egg was calculated using V = ^(width/2)2 length.Evaluation of data The following variables were determined: The statistical analysis was performed using linear regression and ANOVA by adopting General Linearized Models.Propor tions were arcsine-transformed where appropriate.Means were compared by t-tests on pairwise differences between parameter estimates in the General Linearized Model (Crawley, 1993).

RESULTS
The sawfly species studied differed in several somatic and reproductive traits (Table 2).The highest egg load was found in D. pini, which was also the largest of the species (as indicated by total BW).G. pallida and N. sertifer were significantly smaller than the other species and had fewer eggs.However the eggs of N. sertifer were larger than those of the other sawflies (Table 2: column 5).The sawflies also varied significantly in their total fat content, with females of G. frutetorum accumu lating the most fat (Table 2: column 6).The fat content (fat/unit [BW]) of the egg load was significantly different from that of the soma in the gregarious species D. pini, N. sertifer and G. pallida, whereas the (semi-)solitary species D. similis and G. frutetorum invested equal amounts of fat in eggs and soma (Table 2: column 7).
The allocation of fat to the eggs differed between species (Fig. 1, ANOVA: F = 24.53,d.f.= (4, 207), P < 0.01).In princi ple, three species groups can be distinguished: (1) the gregarious species N. sertifer and D. pini allocated most of their fat (about 60%) to their eggs, (2) the solitary species D. similis and G. fru-SE) in females of five pine sawfly species.Significant differ ences between means are indicated by different letters (P < 0.05, t-tests on pairwise differences between parameter esti mates in the General Linearized Model).
tetorum invested about half of their fat in their eggs and (3) the gregarious species, G. pallida, less than half of its fat (43%).
Plasticity in the allocation of fat with increase in total fat was tested by the regression of the total fat content (x = total fat/[unit BW]) on fat allocation (y = proportion of fat allocated to eggs) in a particular species.The basic allocation pattern remained unchanged in the gregarious species.However, in the (semi-)solitary living D. similis and G. frutetorum the propor tion in the egg load decreased with increasing fat content (D. similis: y = -1.299x+ 0.824, F = 10.16,(1, 23), P < 0.005; G. frutetorum: y = -0.701x+ 0.679, F = 3.34, (1, 51), P = 0.072).

DISCUSSION
The results of this study confirm that pine sawfly species differ in their pattern of fat allocation to eggs and soma.How ever, there is no clear relationship between the allocation pat terns and the dichotomy in sawfly oviposition.D. pini and N. sertifer are gregarious and lay their eggs in one large cluster (Pschorn-Walcher, 1982;Blumke & Anderbrant, 1997).Sur vival of larvae increases with colony size (Lyons, 1962), probably because the feeding of the early instars is enhanced or large colonies are better able to defend themselves so reducing the mortality risk per capita (Heitland & Pschorn-Walcher, 1993;Codella & Raffa, 1995;Hunter, 2000).The data presented indicate that these sawfly species invested most of their fat in their egg load, producing more or larger eggs.D. pini had the highest number of eggs (especially in comparison to the nearly similar sized D. similis).N. sertifer had the least, but the largest eggs of all the species.In contrast to the other European sawflies, this species overwinters in the egg and presumably has to optimize its egg size in terms of energy reserves.
Distributing eggs singly or in several small clusters spatially can be seen as a risk-avoiding strategy, which is often associ ated with cryptic colouration and behaviour of solitary feeding larvae (Prop, 1960).These species, in contrast to egg-clustering species, are likely to spend more time travelling and ovipositing, which are energetically costly.The results of this study lend to this contention, as the (semi-)solitary species G. frutetorum and D. similis allocated comparatively more fat to soma than D. pini and N. sertifer.Moreover, with increasing fat content, both (semi-)solitary species increased their investment of fat in soma.In addition, in the laboratory the longevity of the solitary G. frutetorum (10 days) was greater than that of the gregarious D. pini (7 days) (Herz, unpublished data).
Table 2. Average somatic and reproductive traits of newly emerged females of five pine sawfly species.Values are means ± SD. n: number of females examined per species, [u BW]: unit body weight.Means followed by different letters are significantly dif ferent (P < 0.05, estimated by t-tests on pairwise differences between parameter estimates in the General Linearized Model).2:Sig nificant differences between the fat content of soma and egg load for a particular sawfly species at P < 0.001 are indicated by ***.The fat allocation found in the gregarious species G. pallida did not conform to the relationship outlined above.This species distributed significantly more fat to soma than to eggs and did not change this pattern with increase in total fat content.It is possible that the egg-clustering G. pallida might spend more energy selecting favourable oviposition sites than other similar sawfly species.For N. sertifer and D. pini it has been demon strated that it is mainly the exposed position of shoots to sun light (Blumke & Anderbrant, 1997), the presence of oviposition deterrents (Hilker & Weitzel, 1991) and needle quality (Bjorkman et al., 1997) that affects the selection of pine shoots by females.Unfortunately, nothing is known about the oviposi tion preference of other pine sawflies and therefore it is difficult tojudge how fastidious G. pallida is in its choice of oviposition sites.
Several comparative studies of pest insects and their non outbreak relatives have suggested an association between egg clustering/gregariousness of larvae and outbreak frequency (Hanski, 1987;Wallner, 1987;Larsson et al., 1993).For the species considered in this study, this is only partially true.The pattern of fat allocation to somatic and reproductive tissues seems to be a better predictor: outbreak tendency increases with an increase in the investment of fat in the egg load.In the case of pine sawflies, outbreaks most likely occur when populations are released from top-down control (Hanski & Parviainen, 1985;Hanski, 1987).When this happens, species with a high potential fecundity may exhibit a rapid population build-up (Hanski, 1987;Wallner, 1987), whereas species with a low fecundity but high power of dispersal may be comparatively restricted in their population increase.This strategy may be more advantageous in non-outbreak situations, as particular mortality risks for the progeny (e. g. attack by egg parasitoids) will be minimized by dispersing spatially or carefully selecting oviposition sites.In fact, there is evidence that densities of some non-outbreak spe cies are at least as high as those of the typical outbreak species during latent periods (Larson & Tenow, 1980;Simandl, 1989).
Taking into account the small sample size we analysed, the proposed relationship between fat allocation and outbreak ten dency in pine sawflies should be regarded as a preliminary conclusion.For instance, the outbreak tendency of species, such as G.pallida, can vary geographically (Pschorn-Walcher, 1982).Moreover, fat allocation may be influenced by diapause or nu trition.We are also aware of the fact that our study was done mainly on insects reared in laboratory cultures and we cannot exclude that the sawflies were subject to an artificial selection.Ideally, fat allocation patterns and other life history traits of field collected specimen from different geographical origins and of different pest status should be compared to determine wether there is a link between resource allocation and population dynamics in pine sawflies.
1. Number of eggs per female and average volume of eggs.2. Total body weight (BW) [mg]: dry weight of soma + dry weight of egg load before fat extraction.BW was used as an estimate of body size.3. Fat [mg]: absolute amount of fat in soma or egg load.4. Fat content (Fat/[unit BW]): relative amount of fat in soma or egg load in relation to BW.Total fat/[unit BW] is the sum of these values.5. Fat allocation: Proportion or percentage of total fat allocated to the egg load or soma, respectively.

Table 1 .
Life history traits, outbreak tendency and origin of the laboratory cultures of pine sawfly species.