Nutritional regulation in nymphs of the German cockroach, Blattella germanica

Journal of Insect Physiology 47 (2001) 1169–1180 www.elsevier.com/locate/jinsphys

Nutritional regulation in nymphs of the German cockroach, Blattella germanica S.A. Jones, D. Raubenheimer

*

Department of Zoology, Oxford University, South Parks Road, Oxford OX1 3PS, UK Received 2 October 2000; received in revised form 5 April 2001; accepted 6 April 2001

Abstract Synthetic foods varying in protein–carbohydrate ratio and total nutrient concentration were used in food selection experiments to investigate the ingestive and post-ingestive regulation of macronutrients by male and female nymphs of the German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae). For both nutrient imbalance and food dilution, food ingestion varied between treatments with the effect that nutrient ingestion was regulated. However, this mechanism was insufficient to compensate for some food dilution treatments. In those cases, the regulation of protein intake was prioritised over that of carbohydrate intake, and two additional regulatory responses were seen. Firstly, cellulose digestion supplemented shortfalls in dietary soluble carbohydrates, and secondly the feeding period within the stadium was prolonged. These ingestive, post-ingestive and developmental responses were orchestrated in such a way that, in all treatments, nutrient gain approached similar levels, despite the variation in food properties.  2001 Elsevier Science Ltd. All rights reserved. Keywords: Nutrition; Regulation; German cockroach; Post-ingestive

1. Introduction The fitness consequences of insufficient or inadequate nutrient intake can be extreme, including effects upon growth and reproduction (McCaffery, 1975; Horie and Watanabe, 1983; Scriber and Slansky, 1981; Yarro, 1985; Clements, 1992) and survival (Smith and Northcott, 1951; Joern and Behmer, 1997; Raubenheimer and Simpson, 1999). There are thus strong selection pressures upon animals for effective mechanisms of regulating the amounts and balance of nutrients that they extract from their environment (Simpson and Raubenheimer, 1993). Cockroaches are of particular interest in the study of nutritional regulation, owing to the fact that they are extremely generalist, opportunistic scavengers (Schal et al., 1984), and thus the diversity of substances that they may treat as foods is greater than for any other insect order (Cornwell, 1968). For this reason, cockroaches are * Corresponding author. Tel.: +44-1865-271283; fax: +44-1865310447. E-mail address: [email protected] (D. Raubenheimer).

less able than any other group of insects to rely upon the nutritional composition of their foods as guarantor of a nutritionally adequate diet. Therefore, they might be expected to have evolved unusually efficient and accurate means of endogenously regulating their nutrient status. Cockroach biology in general, and nutrition in particular, has been studied consistently for a long period (e.g. for the German cockroach, Blattella germanica: Hummel, 1821; Sanford, 1918; Abbott, 1926; Melampy and Maynard, 1937; Noland and Baumann, 1951; Gordon, 1968; Cochran, 1983; Cooper and Schal, 1992a), on account of the ease of rearing and using cockroaches (Scharrer, 1951) and their economic importance as pests (Cornwell, 1968). German cockroaches have been used as model organisms for the study of insect digestive physiology, neurobiology, biomechanics and behaviour, but it is the study of cockroach nutrition which has recently intensified, following a shift towards the increased use of baits in domestic pest control (Reierson, 1995). Considerable progress has been made through such studies, which have revealed some unusual and important nutritional adaptations in cockroaches. For

0022-1910/01/$ - see front matter  2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 ( 0 1 ) 0 0 0 9 8 - 1

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instance, most insects store excess carbohydrate as fat for future metabolic use, but either void excess nitrogen to the exterior or store it in protein form (Telfer and Kunkel, 1991). Cockroaches, by contrast, have evolved the ability to store urates internally rather than excreting them (Cochran, 1985). Stored urates can be re-cycled, since specialised cells exist in the fat body which house bacterial endosymbionts capable of metabolising urates and releasing useable nitrogenous compounds into the cockroach haemolymph (Valovage and Brooks, 1979). This ability relaxes the requirement for regular nitrogen intake, enabling cockroaches to survive when nitrogenous foods are lacking in the environment (Mullins and Cochran, 1986). Further, numerous studies have demonstrated which nutrients are essential for growth and reproduction (Cooper and Schal, 1992a,b; Hamilton and Schal, 1988; Haydak, 1953; Gordon, 1959; Durbin and Cochran, 1985; Cochran, 1983; Silverman, 1986). These have established that more carbohydrate is needed than protein, leading to the speculation that, when cockroaches have the choice, foods high in carbohydrate are fed upon more often than foods high in protein (Ross and Mullins, 1995, p. 42). Many of these studies have, however, measured only long-term performance consequences of confinement to one food, and therefore have little to say about the dynamics or mechanisms of nutritional homeostasis. They have also tended to focus on adults, ignoring nymphs — yet B. germanica populations consist of up to 80% nymphs (Rivault, 1989; Sherron et al., 1982; Keil, 1981). In the present study, we quantified the regulatory responses of B. germanica nymphs to pairs of foods differing in the balance and concentrations of the essential macronutrients, protein and carbohydrate. In a first experiment, treatments differed in the degree of imbalance of two nutritionally complementary foods provided to each insect. In a second, degree of imbalance was held constant but one, both or neither food in each pairing was diluted using cellulose. Ingestive, post-ingestive and developmental responses were measured in order to evaluate the efficacy of homeostatic regulation in the face of food variation, and to partition this response across different levels of regulation.

2. Materials and methods 2.1. Insects, foods, and measurements The sixth nymphal stadium represents the last nymphal instar. Fifth instar nymphs were taken from the lab culture and kept individually in clear Perspex boxes (40×113×170 mm), with ad libitum access to rabbit pellets and fresh potato, but no harbourage site. The light regime throughout was 12:12. The insects were

inspected daily and entered into experiments on moulting into the sixth larval stadium (identified by pronounced wing buds). Treatments consisted of different synthetic foods, as developed by Dadd (1960) and further described by Simpson and Abisgold (1985). The nutrients used were all of ⬎95% purity and were obtained as follows: albumin — BDH, Poole, UK (Cat. No. 830083G); casein — Sigma Aldrich, St Louis, MO (C5890); peptone — BDH (440754K); sucrose — Sigma (S9378); α-cellulose — Sigma (C8002); Wesson’s salts — Sigma (W1374); agar — Oxoid, Basingstoke, UK (L11); linoleic acid — Sigma (L1626); ascorbic acid — BDH (44006). Three different proteins were used since a single protein source can sometimes lead to impaired development (e.g. Cooper and Schal, 1992a). Levels of protein, carbohydrate and cellulose were manipulated in the foods, which contained all other nutrients at satisfactory levels. Bacterial agar was dissolved in twice its mass of water at 60°C and added to the foods, which were then rolled out to a thickness of approximately 3 mm and dried at 30°C for 24 h, as per Warwick (1999) (Table 1). This method produces foods of a uniform, biscuit-like consistency. Throughout both experiments, each food was presented alone in small petri dishes (13×35 mm diameter), as a biscuit weighing 10–20 mg. The sides of these dishes prevented the insects from carrying the food out of the dish. Water was available ad libitum from an open dish. Two experiments were performed (see below). In both, foods were presented to the insects for 48 h before being replaced. They were weighed before and after presentation to the insects, having been allowed to equilibrate with relative humidity in the laboratory, and using Table 1 Constituents of treatment foods, by percentage weight, for Experiments 1 and 2. Foods 1, 2, 3 and 4 all had the same total nutrient content, but differed in protein–carbohydrate balance. Foods Pc, Pd, Cc and Cd contained either protein or soluble carbohydrate, at varying concentrations Nutrient

Experiment 1 1

Casein Peptone Albumin Sucrose Dextrin Cellulose Agar Linoleic acid Ascorbic acid Cholesterol Vitamin mix Wesson’s salts

2

Experiment 2 3

4

2.05 6.35 12.15 23.5 1.03 3.18 6.08 11.75 1.03 3.18 6.08 11.75 27.95 23.65 17.85 6.50 27.95 23.65 17.85 6.50 26.90 26.90 26.90 26.90 9.10 9.10 9.10 9.10 0.55 0.55 0.55 0.55 0.28 0.28 0.28 0.28 0.55 0.18 2.50

0.55 0.18 2.50

0.55 0.18 2.50

0.55 0.18 2.50

Pc

Pd

Cc

Cd

30.00 15.00 0.00 0.00 15.00 7.50 0.00 0.00 15.00 7.50 0.00 0.00 0.00 0.00 30.00 15.00 0.00 0.00 30.00 15.00 26.90 56.90 26.90 56.90 9.10 9.10 9.10 9.10 0.55 0.55 0.55 0.55 0.28 0.28 0.28 0.28 0.55 0.18 2.50

0.55 0.18 2.50

0.55 0.18 2.50

0.55 0.18 2.50

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control foods that were not presented to insects to adjust for variation due to changing relative humidity. These measurements were used to calculate the amounts of food eaten. Faeces were carefully collected with fine forceps, dried at 60°C for 24 h, then allowed to equilibrate with relative humidity in the laboratory before being weighed. Insects were fresh-weighed at the start of the stadium, then re-weighed upon moulting into adults, and frozen. Carcasses were subsequently subjected to lipid extraction using chloroform (Simpson, 1982), and both the carcasses and faeces were analysed for nitrogen using the micro-Kjeldahl procedure. The amount of soluble nutrients undigested was estimated as the difference between the amount of bulk (cellulose+agar) ingested and the amount of faeces produced. Stadium duration was recorded to the nearest day. 2.2. Experiment 1 — nutrient balance Forty two nymphs of both sexes were allowed to compose their diet by switching between two nutritionally complementary foods, both of which contained 40% diluant (cellulose+agar) but varied in protein–carbohydrate ratio (Tables 1 and 2). Ratios were chosen which provided pairs of foods that were equally imbalanced in opposite directions, relative to the ratio self-selected by insects in a previous experiment, described here as Experiment 2. Two measures of divergence from the balanced food were used, which can be visualised using the geometrical approach of Raubenheimer and Simpson (1993) (see Fig. 1). Firstly, foods 1 and 3 formed a pair whose divergence from the previously self-selected balance was ‘absolutely’ matched, i.e. the angle between the line representing each food and the previously selfselected ratio was equal for both foods in the pair. In contrast, the degree of imbalance in foods 1 vs. 4 and foods 2 vs. 3 was relative, i.e. the angle between the food and the self-selected ratio was a constant proportion

Table 2 Factorial combinations of foods used in treatments in Experiments 1 and 2 Treatment

1 2 3 4

Experiment 1

Experiment 2

Fig. 1. Experiment 2: nutrient balance — cumulative amounts of protein and carbohydrate ingested (mean±SE) across treatments, after two days, four days, and at the end of the stadium. Treatments consisted of pairs of foods with different protein/carbohydrate ratios. Straight lines are used to represent foods with a fixed protein/carbohydrate ratio, since they indicate positions that could be reached by an insect starting at the origin and feeding solely on such a food. Dashed lines (1–4) represent the four treatment foods, and the dotted line represents the ratio that preliminary experiments led us to expect would be self-selected.

of the angle from the nearest axis to the self-selected ratio. The ambient temperature was 32±2°C. 2.3. Experiment 2 — nutrient dilution Eighty nymphs of both sexes were allowed to select their diet from a choice of two foods. One food contained protein but not soluble carbohydrates, and the other contained soluble carbohydrates but not protein (represented by the axes in Fig. 3). Additionally, protein and carbohydrate-containing foods contained either 40 or 70% diluants. Treatments differed in the nutrient concentration of the two foods, with nymphs being given either both concentrated foods, both dilute foods, or one of each (Table 2). The ambient temperature was 29±2°C.

Food A

Food B

Food A

Food B

2.4. Data analysis

1 1 2 2

3 4 3 4

Pc Pc Pd Pd

Cc Cd Cc Cd

Amounts of nutrients were analysed using ANOVA and ANCOVA in spss. Time series data were analysed using a Cox-regression, which is appropriate for failure time analysis (Fox, 1993).

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3. Results 3.1. Experiment 1: nutrient balance

3.1.1. Amounts of nutrients ingested Fig. 1 shows the amounts of nutrients ingested in Experiment 1. By feeding on a pair of foods, nymph nutrient intake could reach any point in the space between the two lines which represent the relevant pair of treatment foods. The amount of protein ingested was significantly affected by sex, but the amount of carbohydrate ingested was not. Neither was significantly affected by start weight or food choice treatment (Fig. 1, Table 3). This latter result shows that the nymphs were able to compensate fully for the nutritional imbalances of the available foods by altering food intake. Interestingly, the amounts of nutrients self-selected by the insects differed from those self-selected in a preliminary experiment (Fig. 1). While the reason for this is uncertain, the start weight of insects in this experiment was significantly greater than that of insects in the preliminary experiment (males: 27.29±0.49 mg cf. 21.51±0.56 mg; females 32.59±0.84 mg cf. 26.53±0.61 mg). We have casually observed that nymphs are often smaller in longer established cultures than in recently established cultures, perhaps because the latter cultures are at a lower population density. The nymphs used in the preliminary experiment were taken from a longer established culture than was used in this experiment, and this may explain the differences in amounts of foods ingested. 3.1.2. Amounts of nutrients digested The difference between the amount of faeces produced and the amount of bulk (cellulose+agar) ingested by each insect was calculated. Neither this measure (‘undigested soluble nutrients’) nor the amount of nitrogen in the faeces was significantly affected by either start weight or food choice treatment, but both measures were

significantly affected by sex (Table 3, Fig. 2c, d). The amount of undigested soluble nutrients was also affected by the amount of protein eaten, with insects that ingested more protein having less residual nutrient in the faeces. The lack of treatment effects suggests that differences in the digestion and/or absorption of nutrients were not used as mechanisms of nutrient regulation in this experiment. This is unsurprising, since nymphs were successful in accurately regulating their nutrient intake (Fig. 1). 3.1.3. Amounts of nutrients assimilated into the body The lipid content of carcasses was significantly affected by the amount of carbohydrate ingested, but not by sex, food choice treatment or the amount of protein ingested (Table 3, Fig. 2a). The nitrogen content of carcasses was significantly affected by sex and start weight, but not by food choice treatment or the amounts of protein or carbohydrate ingested (Table 3). Female carcasses had significantly greater nitrogen weight (Fig. 2b), paralleling the greater amount of protein that they ingested in the sixth stadium (9.814±0.395 mg cf. 6.136±0.285 mg for males). 3.2. Experiment 2: food dilution 3.2.1. Amounts of nutrients ingested Fig. 3 shows the amounts of nutrients eaten from food dilution treatments. Since treatment foods in this experiment contained either protein or carbohydrate, all points in the space shown could have been reached by nymphs. Nymphs given both concentrated foods (Pc+Cc) ate an average of 9.46 mg of the protein food and 27.10 mg of the carbohydrate food. These quantities of food intake resulted in the nutrient intakes shown in Fig. 3 — 5.67 mg of protein and 16.26 mg of carbohydrate. Had the animals not responded to protein dilution by increasing consumption, those given the dilute food would have eaten half as much protein (i.e. 2.84 mg) as those given the concentrated protein food, as shown Fig. 3 by the dotted line. Insects fed dilute protein foods that fell on this line would thus be considered not to have

Table 3 F-values from ANCOVA’s predicting nutritional measures for sixth instar B. germanica (all in mg) from Experiment 1, using sex, weight at the start of the stadium and treatment food as factors, and the total amount of protein ingested in the stadium and the total amount of carbohydrate ingested in the stadium as an independent variable. Each independent variable was omitted from the model when it was also the predicted variable. All ANCOVA’s had 33 error d.f. Key: * p⬍0.05; ** p⬍0.01; *** p⬍0.001 Factors

d.f.

Protein ingested Carbo ingested

Nitrogen in faeces

Undigested Carcass lipid soluble nutrient

Carcass nitrogen

Sex Start weight Treatment Sex×treatment Protein ingested Carbohydrate ingested

1 1 3 3 1 1

10.418** 0.106 0.844 0.079 – 2.471

6.972* 0.611 0.196 0.592 1.333 1.463

6.983* 0.315 0.684 1.111 7.830** 0.579

35.962*** 16.832*** 1.532 0.809 0.002 0.345

3.848 2.272 3.071 1.531 2.471 –

1.377 0.154 2.531 0.933 1.437 26.999***

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Fig. 2. Experiment 1: (a) amounts of lipid in the carcass after the imaginal moult; (b) amounts of nitrogen in the carcass after the imaginal moult; (c) amount of nitrogen in the faeces collected throughout the stadium; and (d) estimated amount of undigested soluble nutrients through the stadium. All given as mean±SE, and by food pair treatment and sex (solid bars, males; hollow bars, females).

compensated for food dilution, whilst those that fell to the right of this line would have shown some degree of ingestive compensation. Similarly, the dashed line represents the ‘line of no compensation’ for insects fed dilute carbohydrate foods, and insects falling above this line would be considered to have compensated ingestively for carbohydrate food dilution. The amount of protein food eaten was significantly affected by the protein food’s concentration, by insect sex and by their interaction, but not by the carbohydrate food’s concentration or insect start weight (Table 4). Insects fed the dilute protein food (‘Pd’) consumed 81% more food than insects fed the concentrated food (‘Pc’), and thereby largely compensated for protein food dilution (Fig. 3). The amount of carbohydrate food eaten was significantly affected by insect sex and start weight, and by both the carbohydrate food’s concentration and the concentration of the protein food with which it was paired (Table 4). No higher order interaction terms were significant. Insects fed the dilute carbohydrate food ate more carbohydrate food than insects fed the concentrated carbohydrate food, but the increase in consumption on dilute foods was modest (21.18%), such that compensation for carbohydrate food dilution through increased ingestion was far from complete (Fig. 3). Turning to the amounts of actual nutrients ingested,

the amount of protein ingested was affected significantly by the protein food’s concentration and also by sex, but not by insect start weight, carbohydrate food concentration or any higher order interactions (Table 4). Females ingested 6.92 mg on average, compared to 4.23 mg by males. In contrast, the amount of carbohydrate ingested was unaffected by the concentration of the protein food, but was significantly affected by insect start weight, the carbohydrate food’s concentration, the interaction of that factor with sex and the amount of protein ingested (Table 4). Insects that ate less protein also ate less carbohydrate, whilst larger insects ate more soluble carbohydrate. Females presented with the concentrated carbohydrate food ingested more carbohydrate, but males did not. 3.2.2. Amounts of cellulose digested The amount of faeces collected weighed significantly less than the combined weight of cellulose and agar ingested over the stadium for all treatments except treatment 1 (2-tailed t-tests: treatment 1, p=0.66; treatment 2, p⬍0.001; treatment 3, p=0.005; treatment 4, p⬍0.001; see Table 2 for treatment foods). Thus, some of the cellulose or agar must have been digested. Cellulases have been found in a range of cockroach species (Wharton and Wharton, 1965; Slaytor, 1992), but no agarase has yet been found. We therefore conclude that

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Fig. 3. Experiment 2: nutrient dilution — amounts of protein and carbohydrate ingested (mean±1 SE) across treatments varying in food nutrient concentration, after (a) two days (b) four days (c) six days, and (d) at the end of the stadium. Foods contained only protein or carbohydrate, and are therefore represented by the axes. The dotted and dashed lines in (d) are drawn with reference to the amounts ingested by insects given both concentrated foods (i.e. ‘Pc+Cc’). The dotted line represents the amount of protein that would have been ingested by insects fed dilute protein foods, had they eaten the same amount of the protein food as insects given both concentrated foods over the whole stadium. The dashed line similarly shows this amount for carbohydrate. Any intake above these lines by insects fed dilute foods represents ingestive regulation over the whole stadium.

cellulose digestion occurred facultatively on dilute foods. Since we have estimated cellulose digestion as the difference between ingested and egested cellulose, it remains a possibility that this quantity might be underestimated to the extent that non-cellulose wastes are present in the faeces. We believe, however, that this would at worst be minimal. Firstly, insects fed dilute foods tend to have very high digestive efficiencies (Slansky and Scriber, 1985; Zanotto et al., 1993). Secondly, insects generally do not egest excess carbohydrate in the faeces, but rather burn it off through increased

respiration rate (e.g. Zanotto et al., 1993) or store it as fat. Thirdly, German cockroaches may excrete nitrogen as ammonia (Cochran, 1985), but this would have been volatilised in the drying process before faeces were weighed. Finally, other food constituents (i.e. salts and vitamins) only made up a small percentage of food weight, and so would only be present in trace quantities in the faeces. This measure of cellulose digestion was unaffected by insect sex or start weight, but was significantly affected by both carbohydrate food dilution and protein food dilution (Table 4).

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Table 4 F-values from ANCOVA’s predicting nutritional measures for sixth instar B. germanica (all in mg) from Experiment 2, using sex, weight at the start of the stadium, concentration of protein treatment food, concentration of carbohydrate treatment food as factors. In addition, the total amount of protein ingested in the stadium was included as an independent variable specifically to test whether or not the amounts of protein and of carbohydrate ingested were independent of each other. All ANCOVA’s had 72 error d.f. Factors

d.f.

Protein food ingested

Carbo food ingested

Protein ingested Soluble carbo ingested

Cellulose digested

Carcass nitrogen

Carcass lipid

Sex Start weight Carbo food concentration Protein food conc. Carbo conc.×sex Prot conc.×sex Total protein ingested

1 1 1

95.105*** 0.095 0.126

26.552*** 6.639* 19.442***

110.100*** 0.067 0.430

1.279 8.710** 287.534***

0.247 0.123 37.076***

201.459*** 22.263*** 0.902

11.698** 6.379* 67.754***

1

153.348***

7.503**

28.639***

0.130

13.354***

5.345*

3.253

1

0.283

0.088

0.578

15.243***

0.092

0.677

7.667*

1 1

11.911** –

0.029 –

0.822 –

0.276 13.763***

0.357 –

0.448 –

0.228 –

Cellulose is a polysaccharide that is roughly energetically equivalent to sucrose and dextrin weight for weight (Macrae et al., 1993). The amount digested can therefore be added to the amount of soluble carbohydrate extracted, to give a measure of the total amount of carbohydrate extracted from the food, as shown in Fig. 4. Comparison with Fig. 3d shows that more cellulose was digested by cockroaches fed dilute carbohydrate foods, and thus had the effect of regulating the amount ofcarbohydrate digested.

3.2.3. Amounts of nutrients assimilated into body Both nitrogen and lipid contents of insects at the end of the stadium were significantly affected by both sex (Fig. 5a and b) and start weight (Table 4). Further, carcass nitrogen weight was not affected by the treatment carbohydrate food, but was affected by the treatment protein food, with the carcasses of insects given the high protein food containing more nitrogen (Table 4, Fig. 5b). Conversely, carcass lipid weight was not affected by the treatment protein food, but was significantly affected by the treatment carbohydrate food and its interaction with sex (Table 4). The carcasses of insects given the high carbohydrate food containing more lipid, especially in females. Though food dilution significantly affected amounts of nutrient stored for both nutrient types, the effects for protein and carbohydrate were of different magnitudes. The overwhelming determinant of carcass lipid content was sex, with females containing more lipid (Fig. 5a). In contrast, carcass nitrogen content was affected by both the treatment foods presented and insect sex (Fig. 5b). 3.2.4. Stadium duration Stadium duration was not significantly affected if only one food was diluted, but was longer and more variable for insects presented with both dilute foods (Cox regression: sex p=0.013, diet p=0.029; Levene’s test for homogeneity of error variance between food choices: p⬍0.001) (Fig. 5c).

Fig. 4. Experiment 2: nutrient dilution — amounts of protein and carbohydrate digested (mean±1 SE) across treatments varying in food nutrient concentration, incorporating the digestion of cellulose. For further explanation, see Fig. 3. Any intake above the dotted and dashed lines by insects fed dilute foods represents the sum of ingestive and digestive regulation, for protein and carbohydrate, respectively.

3.2.5. Weight gain An ANCOVA with weight gain as the dependent variable, sex as a factor and start weight as the independent factor showed a significant sex×start weight interaction (sex: F=0.1011,72, p⬎0.05; start weight: F =19.5111,72, p⬍0.001; sex×start weight: F=4.0971,72, p⬍0.05).

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Fig. 5. Experiment 2: (a) amounts of lipid in the carcass after the imaginal moult; (b) amounts of nitrogen in the carcass after the imaginal moult; and (c) stadium duration. All given as mean±SE, and by food pair treatment and sex (solid bars, males; hollow bars, females).

Insects that were heavier at the start of the stadium put on less weight, with the effect that weight at the imaginal moult was regulated, an effect which was more pronounced in males than in females (Fig. 6). Further, the weight of female insects at the end of the last nymphal stadium were more variable than at the start (Levene’s test for homogeneity of error variance: p=0.009), whereas weights of male insects were no more variable at the end of the stadium than at the start (Levene’s test for homogeneity of error variance: p=0.356). Together, these data suggest that long-term regulation of adult male weight was occurring.

4. Discussion Our data show that cockroach nymphs use several mechanisms to minimise the difference between their nutritional intake and their optimal nutritional target (i.e. they ‘defend’ that target) in the face of variation in food quality, including regulation of intake, cellulose digestion and flexible stadium duration. While each of these responses has been observed previously in insects (e.g. intake: Chambers et al., 1995; cellulose digestion: Bignell, 1978; stadium duration: Kunkel, 1966), the main aim of the present study was to measure them simultaneously and so to determine how they are prioritised and orchestrated towards the end of achieving nutritional requirements in the face of different nutritional challenges. Also central to our study is the simultaneous

Fig. 6. Experiment 2: regression of start weight against the weight gained during the last nymphal stadium, by sex.

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manipulation of both protein and carbohydrate, which enabled us to measure compensatory responses to both macronutrients, as well as interactive effects of specific combinations of the two. 4.1. Regulation of ingestion The regulation of ingestion was seen in the fact that amounts of foods consumed varied with the foods’ nutritional content in such a way that nutrient intake across treatments was either constant (Fig. 1), or varied less than did the nutrient content of the foods (Fig. 3). In insects fed dilute foods, ingestive regulation alone did not suffice to compensate for both protein and carbohydrate, with only protein intake reaching the target level (Fig. 3). Evidently, the insects on dilute foods were under some constraint which prevented them from reaching the optimal intake of both nutrients, the most likely being the upper limit on the rate of food consumption (Van Herrewege, 1974). Whatever the constraint, the data suggest the interesting conclusion that when cockroach nymphs were prevented from ingesting both nutrients at the optimal rate, carbohydrate intake was compromised in order to allow the optimal level of protein to be ingested. Further, in Experiment 2, the amount of carbohydrate food eaten was significantly affected by the total amount of protein ingested (Table 4), independently of insect sex or size, with higher protein intake corresponding to higher carbohydrate intake. This indicates that protein intake and carbohydrate intake were not simply regulated independently, but that the balance of intake of these macronutrients was itself regulated. That is, mechanisms of nutritional regulation did not only act to minimise the discrepancy between the amounts of each macronutrient ingested and a fixed, regulatory ‘target’ amount for that macronutrient. They also changed those regulatory targets in response to the amounts of other nutrients ingested. The amount of protein ingested affected the amount of the carbohydrate selected, and the amount of carbohydrate ingested affected the amount of protein selected. Similar effects have been observed in locusts, and a framework has been suggested for describing them (Raubenheimer and Simpson, 1999). 4.2. Cellulose digestion When the protein food and/or the carbohydrate food were diluted, our data suggest that insects digested more cellulose, with the effect that carbohydrate absorption was increased (Fig. 4 cf. Fig. 3d). These data conflict with the conclusions of previously reported studies. It has been established that cockroach cellulases are produced endogenously rather than by symbionts (Slaytor, 1992), and B. germanica was the only cockroach species investigated by Wharton and Wharton (1965) not to pro-

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duce an endogenous cellulase. However, the cellulose digestion observed in our study was facultative and did not occur when treatment foods contained adequate soluble carbohydrate. It may be that Wharton and Wharton’s cockroaches were fed a relatively high-carbohydrate food, and for this reason showed no cellulytic activity; unfortunately, they did not publish sufficient information on the composition of the foods they used to verify this. Our data also explain the difference between Van Herrewege’s (1974) study of B. germanica, which showed that ingestive compensation for carbohydrate food dilution was almost complete, and that of Gordon (1968), who found such compensation to be far from complete. Neither study measured post-ingestive effects, but whilst the former used aluminium oxide as a diluant, which is truly indigestible, the latter used cellulose. Results of the present study suggest the possibility that cockroaches in both these studies regulated their carbohydrate status, but that Gordon’s did so primarily postingestively, through the digestion of cellulose.

4.3. Developmental responses

One developmental response observed in our experiments was a negative correlation between the mass at which nymphs entered Experiment 2 (i.e. the final larval stadium) and the level of growth in that stadium (Fig. 6). This indicates that weight at the imaginal moult was being regulated, with insects in the penultimate nymphal stadium using nutrient ingestion and/or the efficiency of utilisation of ingested nutrients to compensate for deficiencies that arose in previous stadia. Therefore, nutritional regulation occurred not only within the stadium with respect to a regulatory target for intake during that stadium, but also across stadia with respect to a regulatory target for the whole nymphal period. A second developmental response was the increased stadium duration observed for nymphs given two dilute foods. While incurring a time cost, extended stadium duration would have the advantage of increasing feeding time available and thus relaxing the constraint on the total amount of food that can be eaten during the stadium (see Raubenheimer and Simpson, 1993, for a similar observation in locusts fed nutritionally imbalanced foods). Previous experiments have found links between feeding and the moulting cycle in B. germanica (Kunkel, 1966), and also between the amount (and quality) of protein ingested and juvenile hormone levels in adult females (Schal et al., 1993; Gadot et al., 1989). Therefore, it may be that the amount (and quality) of nutrients ingested affects levels of moulting hormone also, such that there is a direct integration of protein status, carbohydrate status and moulting.

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4.4. The co-ordination of regulatory mechanisms — trade-offs and functional implications

While ingestive, post-ingestive and developmental responses demonstrated by B. germanica are interesting in their own right, we were most interested in the ways that these responses were prioritised and co-ordinated to buffer nutrient gain against variation in the insects’ nutritional environment. Not only are biological processes and behaviours themselves the result of natural selection, but so is the manner in which they are coordinated. The details of such coordination may, therefore, provide information concerning the function of regulation and the relative importance of each mechanism. For example, the complex and redundant neuronal and endocrinological control of egg production in Rhodnius prolixus has been interpreted to suggest that precise control over reproduction is important in this species (Davey, 1993). In our experiments, we found that regulation of ingestion occurred in insects on all treatments in both experiments, but both cellulose digestion and changes in stadium duration were seen to be secondary mechanisms of nutritional regulation, invoked only when the regulation of ingestion alone failed to compensate for food nutrient content. Ingestion was thus the primary level of nutritional regulation but, when this reached its limits, two auxiliary mechanisms came into play and reduced the shortfall between the insects’ nutrient intake and their nutritional requirements. Other interplays between ingestive and postingestive regulation have been described for a wide range of insects (Slansky and Scriber, 1985) and other animals (Raubenheimer and Simpson, 1998). Each of the three mechanisms has costs and benefits. Firstly, to regulate ingestion means finding foods, sampling them and then sometimes rejecting them for meals and continuing foraging (Chambers et al., 1995). This will increase energetic costs and may increase predation risk (Krebs and Kacelnik, 1991), but enables diet to be accurately regulated despite tremendous variation in available foods. Secondly, the beneficial regulatory effect of cellulose digestion is limited to one nutrient type, and might well carry significant costs (e.g. the time and energy required to hydrolyse refractory cellulose molecules). This might be why the American cockroach, Periplaneta americana (L.) only uses cellulose digestion as a ‘last-option’, when all available foods contain very little soluble carbohydrate or protein (Mira, 1999). Thirdly, any benefit of delayed moulting is indirect, since it does not directly affect nutritional state, but simply gives more time for other mechanisms to operate. However, it has direct demographic costs in delaying reproductive maturity. Further, it increases mortality risk due to predation and also cannibalism by larger individ-

uals, which is common in B. germanica (Gordon, 1959; Mu¨ ller, 1978). The primacy and accuracy of ingestive regulation in cockroaches highlights the fact that a huge range of substances serve as potential foods (Schal et al., 1984), such that tight control must be exercised over which foods enter the insect and in what quantity. Further, the existence of apparently costly, post-ingestive mechanisms indicates the importance of nutritional regulation to German cockroaches, and it no doubt reflects the fact that German cockroaches are adapted to environments in which the available foods are highly unfavourable to growth and development. These findings indicate that our understanding of nutritional regulation will be limited if only one level (i.e. behaviour, physiology or development) or nutrient type is investigated, or if several are investigated in isolation. The multi-faceted nature of nutritional regulation is central to the biology of cockroaches, and should thus feature in experimental attempts to understand the nutritional adaptations of these most adaptable of insects.

Acknowledgements This work was supported by a BBSRC Industrial CASE studentship to S.A. Jones, in association with Rentokil Initial plc (Research and Development Division). We would like to thank John Castle for assistance with Kjeldahl analysis.

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