ENTOMOPHAGA 39 (3/4), 1994, 385-395

TAKE-OFF CAPACITY AS A CRITERION FOR QUALITY CONTROL IN MASS-PRODUCED PREDATORS, GRANDIS (COL. : RHIZOPHAGIDAE) FOR THE BIOCONTROL OF BARK , MICANS (COL. : SCOLYTIDAE)

D. COUILL1EN & J.-C. GRI~GOIRE

Section interfacultaire d'Agronomie, Laboratoire de Biologie animale et cellulaire, CP 160/12, Universit6 Libre de Bruxelles, 50 av. E D. Roosevelt, 1050 Bruxelles, Belgique

Time spent by adult beetles in cold storage at 3-7 ~ accounted for 81% of the loss of take-off capacity in Gyllenhal in windtunnel experi- ments. At the age of three weeks, the were at their highest take-off capacity at about 80 %. This was followed by a steady decrease, 7 % of the insects failing to take-off each month. Changes in the fat reserves during cold storage could explain at least partly this reduction of flight capacity although there was no significant difference in fresh weight between insects that were able/unable to take-off. Sex had a significant influence on take-off rates, with an 8.7 % higher take-off rate in females. Take-off capacity was further reduced when the insects were mass-produced in cultures using parent beetles submitted to a blend of synthetic oviposition stimulants instead of live prey larvae. Response to synthetic attractants by those insects which took-off, however, was not influenced by cold storage or by the use of synthetic stimulants in the culture medium.

KEY-WORDS : , Rhizophagtls grandis, Scolytidae, Rhizoph- agidae, biological control, mass-rearing, flight, take-off, windtunnel, quality con- trol.

The specific predator, Rhizophagus grandis Gyllenhal has been widely used in biological control programmes against the greater , Dendroctonus micans (Kugelann) (Coleoptera: Scolytidae) in the Republic of Georgia (Kobakhidze, 1965), in France (Gr6goire et al., 1984), in the United Kingdom (King & Evans, 1984) and in Turkey (N. Uslu, personnal communication). Different techniques of mass-production have been used for this (Kobakhidze et al., 1968; Gr6goire et al., 1985, 1986, 1992a; King & Evans, 1984). One common feature of these techniques is that they involve cold storage of the mass-produced beetles for a period of a few days to several months, between production and releases. Prey location is achieved by flight. It is therefore essential that the released beetles keep their fullest flight capacities. We demonstrate here that, under laboratory conditions, there is a reduction in take-off capacity which is correlated with age and that rearing techniques influence take-off rates. Response to kairomones, however, is independent of age and of rearing techniques. 386 D. COUILLIEN & J.-C. GRI~GOIRE

MATERIAL AND METHODS

WINDTUNNEL The windtunnel is similar in many respects to that described by Wyatt et al. (1993). It is 2 m long, 0.6 m wide and 1.2 m high. The lateral walls, floor and ceiling are made of 6 mm thick glass plates. Access is allowed by sliding lateral glass plates. The upwind and downwind walls are made of nylon gauze allowing free movement to the airflow but not to the insects. The windtunnel is lit from above by five fluorescent tubes (Philips TLD 58W/33) hanging 25 cm above the transparent ceiling. A gentle airflow (ca 0.05 m/s) is produced by a fan situated upwind. The airflow is made laminar by passing it through a honeycomb structure placed immediately upwind of the gauze screen.

INSECT PRODUCTIONAND CONDITIONING Insects were mass-reared using pieces of fresh spruce bark and live larvae of D. micans as described by Grrgoire et al. (1986). After metamorphosis, the adults were stored in moist bark powder in a refrigerator at 3-7 ~ Prior to the experiments, the insects were sorted by sex and kept for 12 h in a polystyrene Petri dish on moist filter paper at 20-23 ~ under the lights of the windtunnel. Six batches of 50-70 insects (3 sets of 2 batches of insects of different ages ; 1 batch of males, 1 of females for each age) were tested dally. Ages to be tested each day were selected at random. Each insect was only tested once. For assessing the effect of age on take-off, insects from several origins (Loz~re, France ; Tervueren, Belgium) and from different lab generations (F2, F3, F4) were used. Insects used for assessing a possible link between fresh weight and take-off capacity also came from Loz~re and Tervueren breeding stocks (F2 or F3).

TAKE-OFF RATES The preconditioned insects were placed in the windtunnel's centre, 1 m downwind of the screen, on a take-off platform (see Wyatt et al., 1993). A 20 x 32 cm tray was placed under the platform, to collect the insects which had not taken-off. After 20 rain, the insects outside the tray were counted to determine the take-off rates. In some batches the insects were weighed individually after the experiments using a Mettler AE 163 electronic balance with an accuracy of 0.1 mg. Take-off rates (x) were analysed using linear regression and analysis of variance on transformed data ( y = 2 arcsin V~x ).

INFLUENCEOF AGE AND REARINGTECHNIQUES ON RESPONSETO ATTRACTANTS The possibility that age and rearing techniques might also influence orientation to prey was investigated by analysing from this standpoint a set of results published previously (Gr6goire et al., 1992b), and bearing on response of R. grandis to attractants mimicking prey odour. Response to various mixtures of synthetic chemicals had been compared to response to prey Crass in a windtunnel, following an experimental procedure first described by Wyatt et al. (1993). Several mixtures were as attractive as Crass to flying beetles. These mixtures and Crass provided therefore a homogeneous set of attraction rates which could be subsequently matched against age of the beetles for the purposes of the present analysis. The results of 35 tests were used, involving insects l, 2 or 3 months old (respectively 8, 23 and 4 tests). As two rearing methods had been used for producing the TAKE-OFF CAPACITY IN RHIZOPHAGUS GRANDIS 387 insects tested in these experiments [oviposition boxes containing either live D. micans larvae (29 tests) or 6 doses of oviposition stimulants (6 tests) ; for details concerning rearing techniques with oviposition stimulants see Gr6goire et al. (1991, 1992a)], rates of response to attractants were also matched against rearing methods.

RESULTS

TEMPORALCHANGES IN TAKE-OFFCAPACITY Males and females displayed clear changes in take-off capacity with age (fig. 1). Both age and sex independently influenced take-off capacity (two-way analysis of variance, transformed data. Age : F = 53.83, p < 0.001 with 10 d. f. ; sex : F = 13.02, p < 0.001 with 1 d. f. ; interaction : F = 1.68, p = 0.095 with 10 d. f.).

0 Females 80 ,. (2r ...... ---

g 60- 3) ......

"~ ~12) ~ ...... 40-

0 I I I I I I I I I I I I ' 0 1 2 3 4 5 6 7 8 9 10 11 AGE (months)

Fig. 1. Mean proportions of beetles taking off ( % detransformed,+_ 1 SEM), according to their age. Figures between brackets indicate the numbers of tested batches used for computing each mean.

Overall, a higher proportion of females (8.7 %) than of males took-off. For both sexes, there was an increase in take-off capacity, starting at around 40 % just after metamorphosis and reaching a peak of around 80 % after 3 weeks. Take-off rates then steadily declined. These changes in time are satisfactorily accounted for by linear functions fitted to transformed data. Among the males, aging accounted for a large part of the total variation in take-off [from 0 to 3 weeks : r 2 = 0.83 (p < 0.01, 6 d. f.) ; from 3 weeks to 11 months : r 2 = 0.86 (p < 0.0005, 54 d. f.)]. The same influence of ageing was also observed among 388 D. COUILLIEN & J.-C. GRI~GOIRE females [from 0 to 3 weeks: r2=0.84 (p<0.01, 6d. f.); from 3 weeks to 11 months: r 2 = 0.80 (p < 0.0005, 55 d. f.)]. The combined take-off rates for both sexes could also be fitted to linear functions [from 0 to 3 weeks : r 2 -- 0.79 (p < 0.0005, 14 d. f.) ; from 3 weeks to II months : r2 = 0.81 (p < 0.0005, 111 d. f.) see fig. 2]. From 3 weeks onward, the beetles lost a monthly average of 7 % take-off capacity.

2.5 --

i 2.0 g .~ 1.5 r2=0-79 ~'~,..8 ~ u O"-,,~D I ? " II

< o.5 II 8 i-.- MALES & FEMALES

0.0 I'1'1'1 '1'1'1'1't'1'1 0 1 2 3 4 5 6 7 8 9 10 11 AGE (months)

Fig. 2. Temporal changes in take-off capacity. Each point represents a batch of 50-70 males or females.

FLIGHT MUSCLES AND FAT BODIES In several cases, flight capacity in beetles has been related to fat body size (Atkins 1969, 1975 ; Botterweg 1982 ; Jactel 1991), or to flight muscle size (Vouland et al., 1984). A global assessment of these factors has been undertaken in an experiment where the beetles were weighed after take-off tests. Fresh weight was independent of take-off success, but not of sex ; females were lighter than males (two-way ANOVA ; take-off success : F = 0.46, p=0.499, 1 d. f. ; sex : F=69.12, p<0.0005, 1 d. f. ; interaction : F=0.54, p=0.443, 1 d. f.). Histological preparations (along the frontal plane) suggested that, whatever the sex, them is no obvious difference between the flight muscles and fat bodies of "flyers" and "non flyers". The mrgostemal, dorsal, and medial-longitudinal muscles could be clearly distin- guished ; light pink in colour, they had a remarkably smooth and regular aspect. They filled the thoracic space throughout the beetles' life. The fat bodies, however, seemed to vary in aspect and volume with time. At 7 months, they were well developed close to the alar muscles. Their aspect was that of a clustering of swollen globular structures. From TAKE-OFF CAPACITY IN RHIZOPHAGUS GRANDIS 389

9 months onward, the fat bodies were no longer closely attached to the thoracic muscles ; but had an alveolar aspect with less swollen, less abundant and denser globular structures.

ADDITIONAL EFFECTS OF MASS-REARING METHODS ON TAKE-OFF CAPACITY Until mid-1991, mass-rearings were based on the use of live larvae of D. micans for providing oviposition stimulants for the parent predators (Grtgoire et al., 1986). All the tests previously described were performed with insects produced with this technique. Later, natural prey was replaced in some instances by dead Calliphora larvae and a mixture of synthetic stimulants (GrEgoire et al., 1992a). A possible effect of this change in rearing method on offspring take-off capacity was investigated. Three rearing methods were compared : i) D. micans larvae ; ii) 3 doses of 50 lal of synthetic stimulants in the rearing boxes; iii)6 doses of 50 ~tl of synthetic stimulants in the rearing boxes. A three-way ANOVA showed a significant effect of the rearing methods (F = 142.77; p < 0.0005; 2 d. f. : see fig. 3), an effect of age (thus confirming all earlier results : F = 9.31 ; p = 0.004 ; 1 d. f.) but no effect of sex (f = 1.24 ; p = 0.271 ; 1 d. f.). Also, there were no significant 2- or 3-way interactions.

RESPONSE TO ATTRACTANTS Response to attractants was not influenced by the age of the beetles (r2 = 0.05 ; p > 0.05 ; 33 d. f. ; see fig. 4), take-off rate (r2 = 0.02 ; p > 0.05 ; 33 d. f.), or rearing conditiong (D. micans larvae or 6 doses of oviposition stimulants : one-way ANOVA, F = 0.005 ; p = 0.945 with i d.f.).

DISCUSSION

Our results show the influence that aging and rearing methods exert on take-off capacity in R. grandis. These factors should therefore be carefully taken into account when releases of these predators are planned. We also provide a fast and convenient technique for assessing take-off in mass-produced beetles. In practice, we have used this technique as a quality control test since 1993. Other quality control tests currently in use concern survival and reproduction. Quality control has been a growing concern in biological control for the last 20 years. To date, it has been mostly applied to species used for inundative release methods (e.g. Trichogramma spp.) or for seasonal inoculative release methods (e.g. Encarsia formosa Gahan, Phytoseiulus persimilis Athias-Henriot) (Bigler, 1989 ; Bigler et aL, 1991 ; Noldus, 1989; van Lenteren & Steinberg, 1991; van Lenteren, 1991, 1993). Beneficial organisms used for inoculative releases have so far not been much studied from this standpoint, even though the individual value of such organisms (each as the founder of a long-term colony) might exceed that of organisms used in inundative releases. Quality control is each time a difficult compromise between what we would find "nice to know" and what we "need to know" (Leppla & Fisher 1989), and choices depend upon each species' characteristics. Rhizophagus grandis is released in small numbers in forest stands infested by Dendroctonus micans and is expected to disperse over several hundred metres and find its prey, even if the latter is at very low densities (Grtgoire et al., 1985 ; Fielding et al., 1991). Optimal dispersal and prey location capacities are therefore needed, and such features must be controlled in mass-produced insects, aside from the more usual controls on longevity and fecundity. An additional characteristic of R. grandis is the fact 390 D. COUILLIEN & J.-C. GRI~GOIRE

70 I1 Age 1 month J'~ Age == 2 months

uJ (/3

-H

Q ',' riO r 0 h U3 Z < 40 n~ p- Ld 0 Z < 30 taJ

I,I

a: 20 h 0 U.II Y 10

0 PREY 3 DOSES 6 DOSES REARING METHOD

Fig. 3. Mean take-off capacity ( % detransformed, + I SEM) of beetles produced by three different rearing proces- ses : i) rearing boxes containing live prey larvae ; ii) rearing boxes containing 3 doses of synthetic oviposition stimulants ; iii) rearing boxes containing 6 doses of synthetic oviposition stimulants. Results for males and females are pooled. that adults may be subjected to cold storage for several months before they are released. This is unusual in mass-production of for biological control. In Aphidoletes aphidimyza (Rondani), storage (at 25 ~ does not exceed 12-15 days, in Aphidius matri- cariae Haliday, 6-7 days, in Encarsia formosa, 4-5 days (Enkegaard & Reitzel, 1991). Stinner (1977 and references therein) reports that it is possible to store Chrysopa carnea Stephens adults at 5 ~ for 150 days, and pupae ofAphidius smithi Sharma & Rao for more than 2 months between 1 and 4 ~ in mummified aphids. Changes in flight propensity in mass-reared insects have rarely been analysed. Adverse effects of gamma ray irradiation have been observed in Ceratitis capitata (Wiedemann) and Rhagoletis cerasi (Linn6) when mass-produced for sterile-fly releases (Boller, 1987); rearing conditions have been shown to affect flight capacity and behaviour in Cochlyomiya TAKE-OFF CAPACITY IN RH1ZOPHAGUS GRANDIS 391

1.2 --

I ~ 1.0- .,I,1f1,.

-~" 0.8- W

W Z 0 ~ 0.6- W

0.4 m

I I I 1 2 3 AGE (months) Fig. 4. Relationshipbetween response rates to attractants (transformed)and age of the beetles. hominivorax (Coquerel) when reared for similar purposes (Bush et al., 1976). Bigler et al. (1991) have started to use flight propensity as a quality control criterion for Trichogramma evanescens Westwood and T. minutum Riley. For this last species, the quality of mass- produced insects in China is also controlled by testing flight propensity (Noldus, 1989). Simple techniques for assessing flight propensity have been developed by Enkegaard & Reitzel (1991) for Encarsia formosa, Aphidius spp. and Aphidoletes aphidimyza. In Rhizophagus grandis, changes in flight propensity were related to ageing. Although a crude approach, the two linear functions used to describe these relationships fitted ex- tremely well to the data. After an optimum take-off capacity at three weeks, this dropped by 7 % each following month. Release strategies would need to be adapted by the addition of 7 % more insects for each month spent in cold storage. A more detailed analysis of take-off rates shortly before and after three weeks is still needed to obtain a clearer picture of the changes occuring around this turning point. 392 D. COUILLIEN & J.-C. GRI~GOIRE

In our histological observations, flight muscles were not altered through time, but fat bodies were. This last phenomenon might have been responsible for this gradual loss in flight propensity in R. grandis, as fat reserves are directly used for flight in several other insects such as the bark beetles Dendroctonus ponderosae (Atkins, 1969, 1975) and Ips calligraphus (Siansky & Haack, 1986). However, reduction of the fat bodies was observed in R. grandis only after 9 months at 3-7 ~ and may not be directly related to early changes in take-off capacities. Such an independence between the state of the fat bodies and flight capacity has been recorded in the bark beetles Ips typographus (Botterweg, 1982), and lps sexdentatus (Jactel, 199I). In Dendroctonus pseudotsugae, fat reserves are used only after sustained flight. Initially, the insects use glucids (glycogen, trehalose) stored in their flight muscles (Thompson & Bennett, 1971), as do other insects such as locusts and aphids (Chapman, 1971). Measuring glucids in the flight muscles of R. grandis might provide a clue to the causes of age-related losses in take-off capacity in this species. Furthermore, the possibility of using a diet rich in glucids for restoring take-off capacity to older beetles should be explored. Morphological changes at cellular level could constitute other possible causes for age-related changes in take-off capacity. For example, in Musca domestica, Rockstein & Miquel (1974) report an increase in number and size of the mitochondria present in alar muscles during the first two weeks of imaginai life. After this, the mitochondria clearly decreased in numbers and size. These changes were correlated respectively with an increase in take-off rates during the first 15 days, then with a decrease in take-off rates. In our experiments, the use of oviposition stimulants had an influence on flight propen- sity in R. grcmdis. The mechanisms for this are unknown, but it should be noticed that the insects underwent only short exposure to these chemicals (i.e. 8 days for egg incubation plus 4-5 days as larvae) before the larvae were transferred to stimulant-free containers. As take-off capacity is reduced by approximately 20 % when 3 doses of oviposition stimulants have been used for producing the insects, an economic appraisal must be made where the costs of using live prey (D. micans) in the oviposition boxes must be put in balance with the losses due to artificial stimulants. Since the cost of using live prey varies with their scarcity in the field, such an economic appraisal would have to be run regularly. As a basis, the cost of producing D. micans should not exceed 20 % of the total cost if artificial stimulants are to be avoided: Although concerned with a limited age range, the analysis of previously published results suggests that orientation to prey (in the present case response to prey frass or to a synthetic mixture of chemicals mimicking prey odour) was not age-dependent nor correlated in any other way with take-off capacity. Rearing methods had no influence either. In other mass-produced arthropods though, dietary effects on response to kairomones have been observed (Dicke et al., 1989).

ACKNOWLEDGEMENTS

Thanks are due to Dr. Malcolm Gillham for his critical reading of the manuscript, and to L&icia Chrrtien and Alain Drumont for rearing the predators. Part of this work was financed by a grant from Rrgion Languedoc-Roussillon/FEOGA Special Programme E2.89, Project 5.6.1.1.d.J.-C. G. acknowledges the Belgian Funds for Scientific Research. TAKE-OFF CAPACITY IN RHIZOPHAGUS GRANDIS 393

P,~StrME

La capacit6 d'envol comme crit~re de contr61e de qualit6 pour la production en masse du pr~xlateur, Rhizophagus grandis (Col. : Rhizophagidae) en vue de la lutte biologique contre le seolyte, Dendroctonus micans (Col. : Scolytidae).

Le temps pass6 en conservation h basse temp6rature (3-7 ~ depuis la m6tamorphose est intervenu pour 81% dans la perte de la capacit6 d'envol ehez le col~opt~re pr6dateur Rhizophagus grandis Gyll. lot's d'exl~dences en tunnel de vol. A l'lge de trois semaines, les insectes sont h leur plus haut niveau d'envol (envoi d'environ 80 % des insectes). Par la suite, il y a un d~clin constant de la capacit6 d'envol, ~t raison de 7 % des insectes ehaque mois. Des changements dans les r6serves lipidiques peuvent partiellement expliquer eette r6duction, bien qu'il n'y ait pas eu de diff6rence entre le poids frais d'insectes capables de s'envoler et celui d'individus qui en 6taient incapables. Le sexe a une influence sur le taux d'envol, avec un taux d'envol significativement plus ~lev6 de 8.7 % chez les femelles. La capacit6 d'envol est encore r6duite chez des insectes qui ont 6t6 produits darts des 61evages de masse oi~ les parents 6taient soumis ~t un m61ange de stimuli de ponte de synth~se au lieu d'Stre mis en pr6sence de larves de D. micans vivantes. Chez les insectes qui prennent leur vol, cependant, la r6ponse aux attractifs de synth~se est ind6pendante de l'fige ainsi que des conditions d'61evage. Received : 11 July 1994 ; Accepted : 30 March 1995.

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