J. Appl. Entomol.

Artemia cysts as an alternative food for the predatory bug pygmaeus B. Vandekerkhove1, L. Parmentier1, G. Van Stappen2, S. Grenier3, G. Febvay3, M. Rey3 & P. De Clercq1

1 Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium 2 Laboratory of Aquaculture & Artemia Reference Center, Department of Production, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium 3 UMR203 INRA/INSA-Lyon, Biologie Fonctionelle Insectes et Interactions, Villeurbanne, France

Keywords Abstract biological control, brine shrimp, carcass analysis, factitious food, mass rearing, The suitability of cysts of the brine shrimp Artemia sp. as a factitious food predator for the predator Rambur was investigated. The influence of decapsulation time and hydration of the cysts on the per- Correspondence formance of the predator were studied in the absence of plant material. B. Vandekerkhove (corresponding author), A longer time of decapsulation had a positive influence on the develop- Laboratory of Agrozoology, Department of ment of the predator. Hydration of cysts had a significant impact on Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links nymphal survival when cysts where non-decapsulated or poorly decap- 653, B-9000 Ghent, Belgium. sulated. An experiment in which nymphs were switched from a diet of E-mail: [email protected] hydrated cysts to non-hydrated cysts showed that in the absence of plant material the relative importance of hydrating the cysts decreased Received: April 18, 2008; accepted: August 4, with nymphal age. Especially, the first instar and to a lesser extent the 2008. second instar appear to be susceptible to water shortage. Effects of pro- longed rearing on development and reproduction on brine shrimp cysts doi: 10.1111/j.1439-0418.2008.01332.x from different origins were tested in the presence of plant material. Rearing M. pygmaeus on Artemia sp. (Jingyu Lake) cysts yielded similar survival, development, adult weight and fecundity in the fourth as in the second generation. In contrast, for Artemia franciscana cysts, an increase in nymphal development was notable. Biochemical analyses showed that total amino acid content and the concentration of the dif- ferent amino acids did not differ among diets and generations. There were, however, differences in total fatty acid content between the differ- ent diets and generations and in the concentration of certain fatty acids, indicating that fed brine shrimp cysts may show nutritional defi- ciencies compared to those reared on a diet of Ephestia kuehniella eggs. Our results indicate that decapsulated brine shrimp cysts are an eco- nomically viable alternative food source in at least part of the rearing process for M. pygmaeus.

Ephestia kuehniella Zeller as a food source. The mar- Introduction ket price of these eggs is currently around 500– Macrolophus pygmaeus Rambur is a polyphagous pred- 600 EUR/kg. Alternative, cheaper food sources may ator that is successfully used as a biological control reduce the production costs and thus stimulate the agent against several crop pests in European green- use of this mirid in biological control (De Clercq houses (Perdikis and Lykouressis 2000; Martinez-Ca- 2004). The brine shrimp Artemia spp. can be such an scales et al. 2006). One of its main drawbacks is its alternative. These branchiopod crustaceans are relatively high rearing cost, mainly due to the inten- routinely used as food sources in aquaculture sive use of eggs of the Mediterranean flour moth (Lavens and Sorgeloos 1996) and their cysts are an

ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 1 Artemia as a factitious food for Macrolophus pygmaeus B. Vandekerkhove et al. order of magnitude cheaper than E. kuehniella eggs in growth chambers at 23 Æ 1C, 60 Æ 5% RH and a (Arijs and De Clercq 2001). Cysts of Artemia sp. were 16L : 8D h photoperiod. successfully tested as a food for the anthocorid pred- ator Orius laevigatus (Fieber) (Arijs and De Clercq Effect of decapsulation time of Artemia franciscana cysts 2001 and De Clercq et al. 2005). Further, Callebaut on development and fecundity of M. pygmaeus in the et al. (2004), Riudavets et al. (2006) and Castan˜ e absence of plant material et al. (2006) also reported that the mirid predator Wagner can successfully Cysts of Artemia franciscana (San Francisco Bay, USA) develop and reproduce on a diet of Artemia cysts. were obtained from the Laboratory of Aquaculture & In above studies, the tested predators were offered Artemia Reference Center of Ghent University. An decapsulated or non-decapsulated cysts that were or initial experiment was carried out to test the effect of were not hydrated [by hydrating the cysts in water, decapsulation time on the value of brine shrimp cysts their water content increases up to 90% (De Clercq as a food for M. pygmaeus; this was performed for both et al. 2005)]. However, no attention was given to dry and hydrated cysts of A. franciscana. The hydration the degree to which the cysts were decapsulated. and decapsulation processes for Artemia cysts have Decapsulation is the process of removing the chorion been described by Lavens and Sorgeloos (1996). The that encysts the Artemia embryo; this can be made standard decapsulation procedure for A. franciscana by short-term exposure to a concentrated hypo- cysts consisted of hydration of the cysts, followed by chlorite solution (Lavens and Sorgeloos 1996). First, subjecting them to a decapsulation process of 150– this study investigated the effect of extent of decap- 180 s, at which time the colour of the cysts turned sulation on food value for M. pygmaeus. Second, the from brown to orange. At that moment, the chorion is importance of hydrating brine shrimp cysts when fully dissolved whereas the hatching membrane and supplied for use as a food during nymphal develop- embryo are not affected by the hypochlorite solution. ment was evaluated. In these experiments, no plant To test the effect of exposure time to the hypochlorite materials were offered throughout nymphal develop- solution on the quality of the cysts, the duration of ment to avoid masking effects of plant feeding. the decapsulation process was varied, with a shorter Finally, the developmental effects of prolonged rear- time (85 Æ 5 s) leading to a lower degree of decapsu- ing of M. pygmaeus on Artemia cysts from different lation (as observed under a stereomicroscope) and a geographic origins and with different physical and longer time (245 Æ 5 s) after which not only the cho- biochemical characteristics were assessed. Carcass rion is dissolved but also the hatching membrane is analyses were performed to compare predators affected. No plant material was used in this experi- reared on Artemia cysts with conspecifics reared on ment given that Callebaut et al. (2004) showed that E. kuehniella eggs in terms of amino acid and fatty plant material has a masking effect on nymphal acid composition. performances of M. caliginosus on different diets. First instars (<24 h old) were taken out of the stock cultures and were placed individually in plastic Materials and Methods cups (4 cm diameter, 2.5 cm high) on wax paper substrates (absorbent household paper drenched in colony beeswax). A parafilm capsule filled with tap water Individuals of M. pygmaeus were obtained from a lab- was offered to the insects as an alternative moisture oratory colony started with eggs supplied by Koppert source, according to the methods described by Van- B.V. (Berkel en Rodenrijs, The Netherlands). The dekerkhove et al. (2006). Predator nymphs were nymphs were reared in plastic containers subjected to one of five treatments: feeding on (24 · 16 · 8 cm), furnished with paper towels and E. kuehniella eggs; dry decapsulated cysts, with the covered with nylon netting. They were reared on decapsulation procedure stopped at 85 s; dry leaves of Nicotiana tabacum L. cv. Xanthi and were decapsulated cysts, with the decapsulation procedure fed frozen eggs of E. kuehniella, also supplied by Kop- stopped at 245 s; hydrated decapsulated cysts, with pert B.V.; the eggs were kept in the freezer for a the decapsulation procedure stopped at 85 s; maximum of 1 month prior to being fed to the pre- hydrated decapsulated cysts, with the decapsulation dators. Ephestia kuehniella eggs were replenished procedure stopped at 245 s. The foods were replen- every 2 days. A paper plug soaked with tap water ished on Mondays, Wednesdays and Fridays and old fitted into a plastic dish (4.3 cm diameter) was used foods were discarded weekly to avoid fungal growth as a water source. The containers were maintained in the containers. The water capsules were replaced

ª 2008 The Authors 2 Journal compilation ª 2008 Blackwell Verlag, Berlin B. Vandekerkhove et al. Artemia as a factitious food for Macrolophus pygmaeus when needed. Nymphs that died on the first day of dissected after 1 week to count oocytes using the the experiment were replaced by new ones, as it methods described above. The experiment was car- was assumed that their death was probably due to ried out in a growth chamber at 23 Æ 1C, 60 Æ 5% handling rather than to treatment effects. Nymphal RH and a 16L : 8D h photoperiod. developmental time and survival were recorded on a daily basis. Nymphs that successfully reached the Prolonged rearing of M. pygmaeus on Artemia cysts from adult stage were sexed and weighed at emergence different origins on a Sartorius Genius balance type ‘ME215P’ with 0.01 mg precision (Sartorius, Goettingen, Germany). To assess the value of Artemia cysts for prolonged Adults were then paired and placed on a fresh rearing of M. pygmaeus, the predator was exclusively tobacco leaf disc; they were given the same diet as reared on this food during four successive genera- during their nymphal life. After 1 week, female tions. In this experiment, the predator was reared adults were dissected and oocytes were counted fol- either on E. kuehniella eggs (control), on A. francis- lowing the method described by Vandekerkhove cana cysts from San Francisco Bay, USA, or on et al. (2006): late vitellogenic to mature oocytes Artemia cysts originating from Jingyu Lake, Xinjiang, were scored 1, early to mid vitellogenic oocytes 0.5 China (ARC code 1524). The latter cysts belonged to and previtellogenic oocytes 0.25, mature oocytes a further non-determined Artemia , but were present in the oviducts were also scored 1. The selected because of their characteristics similar to A. scores for all ovarioles were then summed yielding a tibetiana, i.e. higher content in highly unsaturated weighted sum of oocytes. At the same time, the leaf fatty acids (HUFA) and their larger size than A. fran- discs were examined for oviposited eggs as described ciscana (San Francisco Bay) cysts (Van Stappen et al. by Ferran et al. (1996). The containers were main- 2003). All cysts were decapsulated for 150 s and tained in growth chambers at 23 Æ 1C, 60 Æ 5% were offered in non-hydrated form. First instars RH and a 16L : 8D h photoperiod. (<24 h old) were taken out of the stock culture and were placed in Plexiglas cylindrical containers (9 cm diameter, 3.5 cm high) provided with ventilation Effect of hydration of A. franciscana cysts on nymphal openings screened with fine mesh. In each development of M. pygmaeus in the absence of plant container, a small sweet pepper plant (Capsicum ann- material uum L., var. California Wonder) was placed, with its To evaluate the importance of hydrating cysts before roots immersed in water. Approximately 30 first presenting them to the predator nymphs, an experi- instars from the stock culture were transferred to ment was set up in which nymphs were switched at each of nine containers; these nymphs were allowed different ages from feeding on hydrated non-decap- to develop to adulthood on either of the tested sulated A. franciscana (San Francisco Bay) cysts to foods. This was called generation 1 (G1). The foods feeding on dry non-decapsulated cysts. Like in the were placed on the bottom of the cylinders and above experiment, no plant material was supplied to replenished on Mondays, Wednesdays and Fridays. the insects. First instars (<24 h old) taken from the When these nymphs had developed into adults, the stock cultures were placed individually in plastic cups sweet pepper plant was taken out of the cylinder (4 cm diameter, 2.5 cm high) on wax paper sub- and replaced by a new one every 3 days. The strates and offered a parafilm capsule with tap water. removed plant was then transferred to a new cylin- Predators were subjected to one of the following der to allow deposited eggs to hatch. To assess devel- treatments: dry cysts supplied from the first nymphal opmental and reproductive performance, about 30 instar up to dissection of adults (DN1); switching to newly hatched nymphs (G2) were collected from the dry cysts from the second nymphal instar on (DN2); rearing cylinders and placed individually on tobacco switching to dry cysts from the third nymphal instar leaf discs. The isolated nymphs were offered their on (DN3); switching to dry cysts from the fourth respective diet and developmental parameters were nymphal instar on (DN4); switching to dry cysts recorded. Nymphs that successfully reached the from the fifth nymphal instar on (DN5); and switch- adult stage were sexed, weighed and paired. Adult ing to dry cysts from the adult stage on (DAd). Food pairs were placed on a fresh tobacco leaf disc and water were supplied as described above. Nym- and were given the same diet as during their phal developmental time and survival were recorded nymphal stages. After 1 week, females were dis- daily and resulting adults were weighed at sected and oocytes were counted; the leaf discs were emergence. Adults were paired and females were examined for oviposited eggs as described above.

ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 3 Artemia as a factitious food for Macrolophus pygmaeus B. Vandekerkhove et al.

The remaining nymphs were maintained in the com- Nymphs reared on dry A. franciscana cysts that were munal rearing cylinders and reared up to a fourth incompletely decapsulated showed the lowest sur- generation of nymphs (G4). Then again, ca. 30 vival (32.1%). Almost half of these nymphs suc- nymphs were isolated to determine biological param- cumbed during the first days, suggesting that young eters as was performed in G2. In addition, newly nymphs have difficulties penetrating the remaining emerged females of G2 and G4 were subjected to chorionic structures in the incompletely decapsulat- biochemical carcass analyses. Amino acid analysis ed cysts. The other Artemia treatments resulted in and lipid analysis were performed according to the 71.4–75% survival. Developmental times were the methods described by Zapata et al. (2005). shortest for both males and females when offered flour moth eggs, averaging 17 and 17.5 days respec- tively (males: F = 24.2; d.f. = 4,47; P < 0.001; Statistical analysis females: F = 11.1; d.f. = 4,38; P < 0.001; anova). A fully randomized experimental design was used. A Development was prolonged when nymphs were one-way analysis of variance (anova) was performed offered A. franciscana cysts. Generally developmental to determine differences in nymphal development time increased with decreasing decapsulation time times, adult weights and fecundity data among treat- when cysts were not hydrated, from 18.6 to 24 days ments. Mean differences were distinguished by using for males and from 20.2 to 24.2 days for females. Duncan’s test or Tamhane’s T2 test for unequal vari- When using hydrated cysts, decapsulation time did ances. Mean values were significantly different when not affect developmental time of the predator. Adult P < 0.05. All data were statistically analysed using weights are the highest for the control diet and the spss 15.0 (SPSS Inc. 1989–2007). lowest for dry cysts that were incompletely decapsu- lated (males: F = 8.1; d.f. = 4,47; P < 0.001; females: F = 23.1; d.f. = 4,38; P < 0.001; anova). Results Females fed flour moth eggs had the highest weighted sum of oocytes (12.3), whereas those fed Effect of decapsulation time of A. franciscana cysts on cysts exposed shorter to decapsulation (85 s) had the development and fecundity of M. pygmaeus in the lowest oocyte count (6.2); however, differences were absence of plant material not statistically significant (F = 1.7; d.f. = 4,36; Nymphal survival of M. pygmaeus was the highest P = 0.17; anova). On the other hand, the number of when given E. kuehniella eggs (85.7%) (table 1). eggs deposited in the first week of adult life for

Table 1 Nymphal developmental time and survival, adult weight, oocyte counts and number of eggs laid per female in the first week of adult life of Macrolophus pygmaeus fed on eggs of Ephestia kuehniella or on dry or hydrated cysts of Artemia franciscana exposed to decapsulation for 85 or 245 s

Developmental time (days) Adult weight (mg) Nymphal Weighted Males Females survival, Males Females sum of No. eggs Diet (n) (n) % (n) (n) (n) oocytes (n) laid (n)

Ephestia 17.0 Æ 0.2 a (13) 17.5 Æ 0.4 a (11) 85.7 (28) 0.81 Æ 0.02 a (13) 1.32 Æ 0.03 a (11) 12.3 Æ 1.3 a (10) 32.6 Æ 5.0 a (10) kuehniella Artemia dry 24.0 Æ 0.4 c (4) 24.2 Æ 1.3 c (5) 32.1 (28) 0.58 Æ 0.01 c (4) 0.87 Æ 0.02 c (5) 6.2 Æ 0.7 a (5) 21.2 Æ 2.9 ab (5) decapsulated for 85 s Artemia dry 18.6 Æ 0.4 b (14) 20.2 Æ 1.0 b (6) 71.4 (28) 0.70 Æ 0.02 b (14) 1.09 Æ 0.03 b (6) 8.8 Æ 1.6 a (6) 25.0 Æ 4.8 ab (6) decapsulated for 245 s Artemia 18.4 Æ 0.6 b (9) 20.0 Æ 0.4 b (12) 75.0 (28) 0.74 Æ 0.03 ab (9) 1.05 Æ 0.02 b (12) 9.0 Æ 1.5 a (11) 22.1 Æ 2.8 ab (11) hydrated decapsulated for 85 s Artemia hydrated 18.8 Æ 0.3 b (12) 19.4 Æ 0.6 ab (9) 75.0 (28) 0.72 Æ 0.03 b (12) 1.09 Æ 0.04 b (9) 11.2 Æ 2.2 a (9) 13.4 Æ 3.0 b (9) decapsulated for 245 s

Mean values (ÆSE) within a column followed by the same letter are not significantly different (P > 0.05; Duncan’s test).

ª 2008 The Authors 4 Journal compilation ª 2008 Blackwell Verlag, Berlin B. Vandekerkhove et al. Artemia as a factitious food for Macrolophus pygmaeus females fed hydrated cysts exposed longer to decap- the third instar (DN4), than when offered dry cysts sulation was significantly lower than that in the con- from the first instar. trol (F = 3.5; d.f.=4,36; P < 0.01; anova), despite Weights of adult males (F = 6.7; d.f. = 5,55; yielding a high weighted sum of oocytes. This is in P < 0.001; anova) and females (F = 9.9; d.f. = 5,38; contrast with earlier findings showing a good corre- P < 0.001; anova) tended to decrease as they were lation between weighted sum of oocytes and number switched to dry cysts earlier in nymphal develop- of eggs laid (Vandekerkhove et al. 2006). ment. Adult males weighed significantly less when they were solely fed on dry cysts or on hydrated cysts up to the fourth nymphal instar as compared Effect of hydration of A. franciscana cysts on nymphal with males that were exclusively fed on hydrated development of M. pygmaeus in the absence of plant cysts or switched to dry cysts from the fifth nymphal material instar on. Females had superior body weights when Figure 1 demonstrates the relative importance they were always offered hydrated cysts. of offering hydrated cysts for the survival of the Only females fed exclusively dry cysts as nymphs nymphal predators. 83.3% of the nymphs that only had significantly lower oocyte counts than those received dry cysts (DN1) never reached adulthood, who received hydrated cysts at least from the third with a mortality percentage during the first two instar on (F = 4.9; d.f. = 5,36; P = 0.002; anova). nymphal stadia reaching 60%. Mortality decreased as nymphs were allowed to feed longer on hydrated Prolonged rearing of M. pygmaeus on Artemia cysts from cysts before being switched to dry cysts. Mortality different origins was the lowest (20%) for nymphs that were fed exclusively on hydrated cysts. Table 3 shows the biological parameters for M. pygmaeus Table 2 shows that developmental time generally reared during successive generations on different increased as the nymphs were switched earlier in life diets. Nymphal survival was high on all diets varying from hydrated to dry cysts (males: F = 11.4; from 84.8% to 100% in G2 and from 90.0% to d.f. = 5,55; P < 0.001; females: F = 3.9; d.f. = 5,38; 93.7% in G4. Whereas in G2 there was no difference P = 0.006; anova). When dry cysts were offered from in developmental time between E. kuehniella eggs the first or the second nymphal instar on, this and A. franciscana cysts, nymphs took longer to resulted in significantly longer development of males develop on both Artemia cyst diets than on E. kuehniella than when offered dry cysts from the third instar on. eggs in G4 (males: F = 22.3; d.f. = 5,94; P < 0.001; Developmental time of females was significantly females: F = 12.1; d.f. = 5,67; P < 0.001; anova). shorter when fed on hydrated cysts from the first to Males and females took longer to develop on A. fran- fifth instar (DAd), to the fourth instar (DN5) or to ciscana cysts in G4 than in G2. Developmental times

100

90

80 3.3 10.0 70 10.0 60 N5 N4 50 6.7 N3 33.3 40 6.7 6.7

Mortality (%) N2

30 13.3 13.3 N1 10.0 3.3 6.7 20 3.3 3.3 3.3 13.3 3.3 3.3 3.3 26.7 10.0 10 6.7 6.7 6.7 10.0 6.7 6.7 6.7 6.7 0 DN1 DN2 DN3 DN4 DN5 DAd Treatment

Fig. 1 Mortality in consecutive nymphal instars (N1–N5) of Macrolophus pygmaeus when fed exclusively on non-decapsulated dry cysts (DN1) or non-decapsulated hydrated cysts (DAd) of Artemia franciscana, or when switched from non-decapsulated hydrated to non-decapsulated dry cysts from the second (DN2), third (DN3), fourth (DN4), or fifth (DN5) instar on.

ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 5 Artemia as a factitious food for Macrolophus pygmaeus B. Vandekerkhove et al.

Table 2 Nymphal developmental time, adult weight and oocyte counts of Macrolophus pygmaeus when fed exclusively on dry cysts (DN1) or hydrated cysts (DAd) of Artemia franciscana, or when switched from hydrated to dry cysts from the second (DN2), third (DN3), fourth (DN4), or fifth (DN5) instar on

Developmental time (days)* Adult weight (mg)* Weighted sum of Treatment Males (n) Females (n) Males (n) Females (n) oocytes (n)

DN1 24.3 Æ 0.9 c (3) 23.5 Æ 1.5 d (2) 0.58 Æ 0.02 b (3) 0.81 Æ 0.11 d (2) 0.6 Æ 0.1 b (2) DN2 22.0 Æ 0.7 b (9) 22.3 Æ 0.8 cd (6) 0.61 Æ 0.02 b (9) 0.84 Æ 0.04 cd (6) 8.3 Æ 1.4 ab (5) DN3 20.2 Æ 0.5 a (11) 21.7 Æ 0.6 bcd (6) 0.62 Æ 0.02 b (11) 0.93 Æ 0.04 bc (6) 9.3 Æ 1.0 a (6) DN4 18.7 Æ 0.5 a (10) 20.5 Æ 0.6 abc (11) 0.63 Æ 0.02 b (10) 0.93 Æ 0.02 bc (11) 8.7 Æ 1.0 a (11) DN5 19.0 Æ 0.4 a (11) 20.1 Æ 0.4 ab (12) 0.70 Æ 0.02 a (11) 0.96 Æ 0.02 b (12) 12.8 Æ 1.5 a (11) DAd 18.7 Æ 0.4 a (17) 19.4 Æ 0.4 a (7) 0.71 Æ 0.01 a (17) 1.14 Æ 0.04 a (7) 15.9 Æ 2.8 a (7)

Mean values (ÆSE) within a column followed by the same letter are not significantly different (P > 0.05; *Duncan’s test or Tamhane’s T2 test for unequal variances).

Table 3 Nymphal developmental time, survival, adult weight, oocyte counts and number of eggs laid per female in the first week of adult life of Macrolophus pygmaeus fed during four consecutive generations on eggs of Ephestia kuehniella, dry decapsulated cysts of Artemia franciscana or dry decapsulated cysts of Artemia sp. (Jingyu Lake)

Diet/generation Developmental time (days) Nymphal Adult weight (mg)* Weighted sum Oviposition per survival, of oocytes* (n) female* (n)

Males Females* %(n) Males Females (n) (n) (n) (n) Ephestia 15.9 Æ 0.1 a (19) 16.1 Æ 0.2 ab (11) 96.8 (30) 0.78 Æ 0.02 a (19) 1.34 Æ 0.04 a (11) 14.4 Æ 1.5 a (11) 21.5 Æ 3.8 a (11) kuehniella/G2 Artemia 16.4 Æ 0.2 ab (16) 16.8 Æ 0.2 bc (14) 100 (31) 0.78 Æ 0.02 a (16) 1.13 Æ 0.03 c (14) 11.7 Æ 0.7 a (11) 16.7 Æ 4.0 a (11) franciscana/G2 Jingyu 16.8 Æ 0.2 bc (13) 17.2 Æ 0.2 cd (14) 84.8 (33) 0.79 Æ 0.02 a (13) 1.21 Æ 0.03 bc (14) 12.3 Æ 3.4 a (12) 18.5 Æ 1.8 a (12) Artemia/G2 Ephestia 15.5 Æ 0.2 a (15) 15.7 Æ 0.2 a (12) 93.7 (32) 0.78 Æ 0.03 a (15) 1.26 Æ 0.04 ab (12) 14.5 Æ 0.5 a (11) 25.5 Æ 2.8 a (11) kuehniella/G4 Artemia 18.1 Æ 0.3 d (21) 18.2 Æ 0.3 e (11) 91.5 (31) 0.78 Æ 0.01 a (21) 1.26 Æ 0.04 ab (11) 11.1 Æ 1.3 a (11) 19.0 Æ 3.6 a (11) franciscana/G4 Jingyu 17.8 Æ 0.3 cd (16) 17.6 Æ 0.4 de (11) 90 (30) 0.81 Æ 0.02 a (16) 1.29 Æ 0.03 ab (11) 11.4 Æ 1.0 a (10) 23.5 Æ 3.6 a (10) Artemia/G4

G2, generation 2; G4, generation 4. Mean values (ÆSE) within a column followed by the same letter are not significantly different (P > 0.05; *Duncan’s test or Tamhane’s T2 test for unequal variances). of predator nymphs on Artemia sp. (Jingyu Lake) differences in development and reproductive perfor- cysts were, however, similar in G2 and G4. Male mances in the same generation of the predator on weights were not affected by treatment (F = 0.62; either Artemia cyst type. d.f. = 5,94; P = 0.69; anova). For females, adult Contents of fatty acids and amino acids deter- weights showed only differences in the second gen- mined by carcass analysis of females reared on differ- eration, with the control diet yielding higher body ent diets in G2 and G4 are reported in table 4. Total weights than either Artemia cyst diet (F = 4.3; fatty acid content was significantly higher for preda- d.f. = 5,67; P = 0.002; anova). The number of eggs tors fed E. kuehniella eggs than on the Artemia diets oviposited in the first week of adult life did not differ (F = 14.9; d.f. = 4,16; P < 0.001; anova). Compared among treatments (F = 1.0; d.f. = 5,59; P = 0.42; to the diet of E. kuehniella eggs, insects fed brine anova), nor did oocyte counts upon dissection shrimp cysts showed deficiencies in oleic (C18:1), (F = 2.1; d.f. = 5,60; P = 0.78; anova), although the linoleic (C18:2) and, to a lesser extent, palmitic acid A. franciscana cysts resulted in somewhat lower (C16:0) in both generations tested (fig. 2). oocyte counts and oviposition compared to the other Table 4 shows that the total amino acid content of diets in both generations. There were no significant M. pygmaeus females did not differ among diets and

ª 2008 The Authors 6 Journal compilation ª 2008 Blackwell Verlag, Berlin B. Vandekerkhove et al. Artemia as a factitious food for Macrolophus pygmaeus

Table 4 Mean total fatty acids and total amino acids in carcasses of Discussion Macrolophus pygmaeus fed with eggs of Ephestia kuehniella, or with cysts of Artemia franciscana or Artemia sp. (Jingyu Lake) during con- When testing the effect of decapsulation time on the secutive generations value of brine shrimp cysts as a food for M. pygmaeus, survival was similar to that reported by Castan˜ e Diet/ Total fatty acids, Total amino acids, et al. (2006), who offered dry or hydrated non- generation lg/mg fresh nmol/mg fresh decapsulated Artemia cysts to M. caliginosus nymphs weight (n) weight (n) provided with Sedum rubrotictum R.T. Clausen (Crassulaceae) leaf cuttings as plant material. Ephestia 85.1 Æ 2.6 a (5) 1146 Æ 29 a (5) kuehniella/G2 Mahdian et al. (2006) found that hydrated decapsu- Artemia 53.2 Æ 3.1 c (4) 1128 Æ 33 a (5) lated A. franciscana cysts were an inadequate food for franciscana/G2 the predatory stinkbugs Podisus maculiventris (Say) Jingyu 53.5 Æ 7.2 c (3) 1111 Æ 97 a (5) and bidens (L.). Arijs and De Clercq (2001) Artemia/G2 reported that it was not possible to rear O. laevigatus Artemia 67.3 Æ 3.0 b (5) 1144 Æ 112 a (5) on dry cysts, as nymphs of this predator failed franciscana/G4 to reach the second instar on this food. They con- Jingyu 58.11 Æ 3.0 bc (4) 1132 Æ 90 a (5) Artemia/G4 cluded that cysts had to be hydrated for successful development of this predator. Castan˜ e et al. (2006) G2, generation 2; G4, generation 4. recorded a similar development for M. caliginosus Means (ÆSE) within a column followed by the same letter are not sig- given E. kuehniella eggs (ca. 18 days) as in our study nificantly different (P > 0.05; Duncan’s test). for M. pygmaeus, even though in their study the experiment was carried out at 25C and the insects generations (F = 0.13; d.f. = 4,19; P = 0.97; anova). were provided with plant material. For M. caliginosus The values for each amino acid (fig. 3) also showed nymphs fed brine shrimp cysts, latter authors no differences between the different diets and gener- recorded developmental times ranging from 17.5 to ations. The main amino acids found in the carcasses 19.5 days. Comparison of the results is complicated of nymphs on the different diets were (expressed as by the fact that different species were tested at % of total amino acid content) aspartate (8.3–8.5%), different climatic regimes and in the presence or glutamate (9.8–10.4%), glycine (8.6–8.9%), alanine absence of plant material. (10.1–10.8%) and to a lesser extent leucine (7.6– From our study, it appears that the longer the 7.9%), valine (6.5–6.6%), serine (6.0–6.2%), lysine decapsulation time of the Artemia cysts, the better (5.8–6.5%) and proline (5.8–6.1%). the developmental performance of the nymphs is.

40 E. kuehniella A. franciscana G2 Jingyu artemia G2 A franciscana G4 Jingyu artemia G4 a 35

30

25 a b

20 b a bc c c c c 15 c a

Concentration (µg/mg) ab ab b abc a bc 10 bc c c c

b 5 bc a a c b b b 0 16:0 16:1 18:0 18:1 18:2 n-6 18:3 n-3 Fatty acids

Fig. 2 Fatty acids in carcasses from females of Macrolophus pygmaeus reared on eggs of Ephestia kuehniella, cysts of Artemia franciscana or cysts of Artemia sp. (Jingyu Lake) during consecutive generations; G2: generation 2, G4: generation 4; within fatty acids, bars with the same letter are not significantly different among treatments (P > 0.05; Duncan’s test).

ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 7 Artemia as a factitious food for Macrolophus pygmaeus B. Vandekerkhove et al.

140 E. kuehniella A. franciscana G2 Jingyu artemia G2 A. franciscana G4 Jingyu artemia G4

120

100

80

60

40 Concentrations (nmol/mg)

20

0 Asp Glu Ser His Gly Thr Arg Ala Tyr Val Met Phe Ile Leu Lys Hyp Pro Amino acids

Fig. 3 Total amino acids in carcasses of Macrolophus pygmaeus females reared on eggs of Ephestia kuehniella, cysts of Artemia franciscana or cysts of Artemia sp. (Jingyu Lake) during consecutive generations; G2: generation 2, G4: generation 4; mean values for each amino acid were not significantly different among treatments (P > 0.05; Duncan’s test).

Besides complete removal of the cyst shell, longer piercing the outer cyst shell with their rostrum. decapsulation times may also cause damage to the Likewise Arijs and De Clercq (2001) reported that hatching membrane, and as such make the embryo O. laevigatus also had difficulties penetrating the hard more easily available for feeding predators. When outer alveolar layer of dry non-decapsulated cysts, the cysts are hydrated, they may be easier to which led them to conclude that for this predator penetrate by the predator’s stylets; in that case, the hydration of the cysts was necessary for successful decapsulation process is probably of less importance. development. Thus, exposing non-hydrated cysts for a longer time Thompson (1999) pointed out that insects may to the decapsulation treatment makes them a more show a loss in fitness only after they have been suitable food for M. pygmaeus. Non-hydrated cysts reared on a diet for multiple generations, as nutri- should be preferred as they lead to fewer problems tional deficiencies may only then become with moulds in the rearing containers than fully expressed. Coudron et al. (2002) noted that insects hydrated cysts, which contain more than 90% water may also adapt to a new diet, resulting in a gradual (De Clercq et al. 2005). Preliminary studies indicated improvement of biological parameters. In this study, that in cultures where plant materials are supplied, even after four consecutive generations of rearing dry cysts (ca. 4% water) hydrate when they are on on brine shrimp cysts, there was no significant a leaf surface until they reach a water content of decrease in survival, adult weight and fecundity of 20% or more within 24 h, facilitating feeding by the the predator. However, on A. franciscana cysts, bugs. immature development took significantly longer in Hydrated cysts resulted in better nymphal devel- the fourth generation than in the second, whereas opment than non-hydrated cysts when they were this was not the case on Artemia sp. (Jingyu Lake) poorly decapsulated. Hydration of poorly or non-de- cysts, suggesting that latter cysts are nutritionally capsulated cysts may make them easier to penetrate superior to the former. This may be related to the by the predators. When the cysts are completely higher content in HUFA, or alternatively to the decapsulated, hydration is of little importance. The larger size of Artemia sp. (Jingyu Lake) cysts longer the predators had access to non-decapsulated (ca. 320 lm) compared to A. franciscana cysts from hydrated cysts in their nymphal life, the better their San Francisco Bay (ca. 227 lm) (Van Stappen et al. chances of survival were. The relative importance of 2003; Camargo et al. 2005). The predator may have offering hydrated A. franciscana cysts decreased with needed to spend less time and energy to handle nymphal age: access to hydrated cysts proved of such larger cysts and extract nutrients from them. greatest importance for the first two instars. Small Castan˜ e et al. (2006) did not find differences nymphs of M. pygmaeus have the most difficulty in any of the discussed biological parameters of

ª 2008 The Authors 8 Journal compilation ª 2008 Blackwell Verlag, Berlin B. Vandekerkhove et al. Artemia as a factitious food for Macrolophus pygmaeus

M. caliginosus offered different strains of Artemia sp. Acknowledgements (species was not identified in the study), neither in The authors wish to thank Christ Mahieu for his the first, nor in the sixth generation; their experi- excellent technical assistance and two anonymous ment included a strain with low HUFA content and reviewers for their helpful comments. one with high HUFA content. Besides data on developmental and reproductive performance, biochemical analysis may allow a fur- References ther evaluation of the quality of insects reared on Arijs Y, De Clercq P, 2001. Rearing Orius laevigatus on alternative foods (Grenier and De Clercq 2003). cysts of the brine shrimp Artemia franciscana. Biol. Con- Amino acid contents of carcasses of M. pygmaeus trol 21, 79–83. females did not differ between those fed E. kuehniella Callebaut B, Van Baal E, Vandekerkhove B, Bolckmans eggs and those fed Artemia cysts, even after four gen- K, De Clercq P, 2004. A fecundity test for assessing the erations of rearing. Fatty acid contents of M. pygma- quality of Macrolophus caliginosus reared on artificial eus females reared on Artemia cysts showed diets. Parasitica 60, 9–14. deficiencies in palmitic, oleic and linoleic acid as Camargo WN, Duran GC, Rada OC, Hernandez LC, Lin- compared to those reared on flour moth eggs, indi- ero JC, Muelle IM, Sorgeloos P, 2005. Determination cating that these fatty acids were either unavailable of biological and physicochemical parameters of Art- for the predators or present at insufficient levels in emia franciscana strains in hypersaline environments for the cysts. Most of the fatty acids consumed by aquaculture in the Colombian Caribbean. Saline Syst. insects are stored as energy sources in neutral lipids, 1, 1–11. and some others have a key role as structural Canavoso LE, Jouni ZE, Karnas KJ, Pennington JE, Wells elements incorporated in membrane phospholipids MA, 2001. Fat metabolism in insects. Annu. Rev. Nutr. or as signal molecules (Downer 1985; Stanley 2000). 21, 23–46. It is well accepted that specific lipids are necessary Castan˜ e C, Quero R, Riudavets J, 2006. The brine dietary requirements for all insects, particularly the shrimp Artemia sp. as alternative prey for rearing the immature stages (Reinecke 1985). Many insects predatory bug Macrolophus caliginosus. Biol. Control require dietary polyunsaturated fatty acids to sup- 38, 405–412. port growth and reproduction, and either linoleic Coudron TA, Wittmayer J, Kim Y, 2002. Life history and or linolenic acid usually satisfies their nutritional cost analysis for continuous rearing of Podisus maculiv- entris (Say) (Heteroptera: ) on a zoo- need (Canavoso et al. 2001). Development on phytophagous artificial diet. J. Econ. Entomol. 95, A. franciscana cysts is slower in G4 as compared to 1159–1168. G2, but this is not reflected in the biochemical carcass De Clercq P, 2004. Culture of natural enemies on facti- analysis of the insects. This ‘generation effect’ only tious foods and artificial diets. In: Encyclopedia of occurred for insects fed A. franciscana cysts but not entomology. Ed. by Capinera JL, Kluwer Academic for those fed Artemia sp. (Jingyu Lake) cysts. Interest- Publishers, Dordrecht, 650–652. ingly, the amino acid and fatty acid patterns of De Clercq P, Arijs Y, Van Meir T, Van Stappen G, M. pygmaeus adults correspond well to those reported Sorgeloos P, Dewettinck K, Rey M, Grenier S, for eggs of E. kuehniella (De Clercq et al. 2005). Febvay G, 2005. Nutritional value of brine shrimp In conclusion, non-hydrated and fully decapsulat- cysts as a factitious food for Orius laevigatus ed cysts of Artemia sp. proved to be a viable alterna- (Heteroptera: Anthocoridae). Biocontrol Sci. Technol. tive food source for rearing M. pygmaeus, provided 15, 467–479. that plant materials were offered. Although hydrated Downer RGH, 1985. Lipid metabolism. In: Comprehen- cysts also constitute a good food, their use is not rec- sive insect physiology biochemistry and pharmacology. ommended as they are more prone to microbial con- Vol 10: Biochemistry. Ed. by Kerkut GA, Gilbert GI, tamination, especially when fully decapsulated. Pergamon Press, Oxford, 77–113. However, some loss of fitness was observed after Ferran A, Rortais A, Malausa JC, Gambier J, 1996. several generations of exclusive rearing on certain Ovipositional behaviour of Macrolophus caliginosus types of Artemia materials. Artemia cysts may none- (Heteroptera: ) on tobacco leaves. Bull. theless constitute an economically viable alternative Entomol. Res. 86, 123–128. food for the production of M. pygmaeus, as it may Grenier S, De Clercq P, 2003. Comparison of artificially reduce inputs of expensive lepidopteran eggs in parts vs. naturally reared natural enemies and their potential of the production process of this important biocon- for use in biological control. In: Quality control and production of biological control agents – theory and trol agent.

ª 2008 The Authors Journal compilation ª 2008 Blackwell Verlag, Berlin 9 Artemia as a factitious food for Macrolophus pygmaeus B. Vandekerkhove et al.

testing procedures. Ed. by van Lenteren JC, CABI Pub- Riudavets J, Arno J, Castan˜ e C, 2006. Rearing predatory lishing, Cambridge, 115–131. bugs with the brine shrimp Artemia sp. as alternative Lavens P, Sorgeloos P, 1996. Manual on the production prey food. IOBC/WPRS Bull. 29, 235–240. and use of live food for aquaculture. FAO Fisheries SPSS Inc., 1989–2007. User’s guide, version 15.0. SPSS Technical Paper 361, Rome. Inc., Chicago, IL. Mahdian K, Kerckhove J, Tirry L, De Clercq P, 2006. Stanley DW, 2000. Eicosanoids in invertebrate signal Effects of diet on development and reproduction of the transduction systems. Princeton University Press, predatory pentatomids Picromerus bidens and Podisus Princeton, NJ. maculiventris. Biocontrol 51, 725–739. Thompson SN, 1999. Nutrition and culture of entomoph- Martinez-Cascales JI, Cenis JL, Cassis G, Sanchez JA, agous insects. Annu. Rev. Entomol. 44, 561–592. 2006. Species identity of Macrolophus melanotoma (Costa Van Stappen G, Sui L, Xin N, Sorgeloos P, 2003. Charac- 1853) and Macrolophus pygmaeus (Rambur 1839) teristation of high-altitude Artemia populations from (Insecta : Heteroptera : Miridae) based on the Qinghai-Tibet Plateau, PR China. Hydrobiologia morphological and molecular data and bionomic 500, 179–192. implications. Insect Syst. Evol. 37, 385–404. Vandekerkhove B, Van Baal E, Bolckmans K, De Clercq Perdikis D, Lykouressis D, 2000. Effects of various items, P, 2006. Effect of diet and mating status on ovarian host plants, and temperature on the development and development and oviposition in the polyphagous pred- survival of Macrolophus pygmaeus Rambur (: ator Macrolophus caliginosus (Heteroptera: Miridae). Biol. Miridae). Biol. Control 17, 55–60. Control 39, 532–538. Reinecke JP, 1985. Nutrition: artificial diets. In: Compre- Zapata R, Specty O, Grenier S, Febvay G, Pageax J-F, hensive insect physiology, biochemistry and pharma- Delobel B, Castan˜ e C, 2005. Carcass analysis to cology. Vol. 4: Regulation: digestion, nutrition, improve a meat-based diet for the artificial rearing of excretion. Ed. by Kerkut GA, Gilbert GI, Pergamon the predatory mirid bug Dicyphus tamaninii. Arch. Press, Oxford, 391–420. Insect Biochem. Physiol. 60, 84–92.

ª 2008 The Authors 10 Journal compilation ª 2008 Blackwell Verlag, Berlin