Field cage studies with a genetic sexing strain in the Mediterranean fruit fly, Ceratitis capitata

Using a genetic sexing line based on pupal colour dimorphism in Ceratitis capitata Wied., field cage experiments were carried out with a wild population in Italy. The experiments were designed to monitor the competitiveness of males of this line in comparison with wild males and to assess the effect of removal of females prior to release on the proportion of wild females mated by the released males.The results showed that the genetic sexing strain competed well with the wild population but there was a large degree of intra-strain mating. Removal of females prior to release radically changed the mating pattern from intra to inter-strain mating. This change greatly increases the efficiency of the sterile insect method of control.It was concluded that as well as the obvious economic advantages of genetic sexing, significant biological advantages would also be associated with the integration of such a strain in a sterile insect control programme.

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Résumé Des expériences en cage en champ ont été réalisées en Italie avec des populations naturelles de Ceratitis capitata en utilisant une lignée dont le sexage était basé sur le dimorphisme de la couleur des pupes. Les expériences ont été conçues pour contrôler la compétitivité des mâles de cette lignée face aux mâles sauvages, et d'évaluer l'effet du retrait des femelles avant le lâcher sur le pourcentage de femelles sauvages accouplées avec des mâles lâchés.Les expériences montrent que la compétition ne défavorise pas la lignée génétiquement sexable mais révèlent une fréquence élevée d'accouplement intralignée.Le retrait des femelles avant le lâcher modifie radicalement l'orientation des accouplements d'intra vers interlignées. Ce changement accroît fortement l'efficacité de la technique d'utilisation des mâles stériles dans la lutte biologique. On en a conclu que non seulement des intérêts économiques évidents mais aussi des avantages biologiques significatifs seraient retirés de l'intégration de telles lignées sexables génétiquement dans les programmes de lutte avec des mâles stériles.

     

Genetic sexing strains in medfly,Ceratitis capitata,sterile insect technique programmes

The introduction of genetic sexing strains (GSS) into medfly, Ceratitis capitata(Wiedemann), sterile insect technique (SIT) programmes started in 1994 and it was accompanied by extensive evaluation of the strains both in field cages and in open field situations. Two male-linked translocation systems, one based on pupal colour, wp, and the other based on temperature sensitivity, tsl, have been used in medfly SIT programmes and they have quite different impacts on mass rearing strategy. In strains based on tsl, female zygotes are killed using high temperature and for wpstrains, female and male pupae are separated based on their colour. In all these systems the colony females are homozygous for the mutation requiring that the mutation is not too deleterious and the males are also semi-sterile due to the presence of a male-linked translocation. Managing strain stability during large-scale mass rearing has presented some problems that have been essentially solved by selecting particular translocations for GSS and by the introduction of a filter rearing system (FRS). The FRS operates by removing from the colony any recombinant individuals that threaten the integrity of the strain. The use of GSS opens up the possibility of using the SIT for suppression as opposed to eradication and different radiation strategies can be considered. Some of the many field trials of the strains that were carried out before the strains were introduced into operational programmes are reviewed and an overview is given of their current use.     

Molecular sexing in the Mediterranean fruit fly, Ceratitis capitata

Molecular methods have been devised for sexing Mediterranean fruit fly (medfly) individuals using minimal amounts of material from any stage of the life cycle. Molecular sexing methods are particularly valuable when material is obtained from pre-adult stages and sex identification based on morphological characters is not possible. These methods may also be useful for adult stage material in situations where only limited amounts or poorly preserved specimens are available. The sexing methods described here use the polymerase chain reaction (PCR) to amplify sequences known to originate from the sex chromosomes of this species. One method co-amplifies homologous regions of the ITS1 ribosomal DNA from both the X and Y chromosomes. Males and females are distinguished based on the restriction fragment pattern produced after digestion of the PCR products with the restriction enzyme ApoI. A second method identifies males based on the positive amplification of a repetitive DNA sequence originating from the Y chromosome. Both methods are shown to be capable of establishing the sex identity of individuals using only minimal amounts of material from any stage of the life cycle.     

Recombination between homologous autosomes in medfly (Ceratitis capitata) males: type-1 recombination and the implications for the stability of genetic sexing strains

The sterile insect technique (SIT) is an environmentally safe technology to control insect pests. To improve this technology, genetic sexing strains (GSS) have been developed for the Mediterranean fruit fly, Ceratitis capitata. Such strains are based on Y-autosome translocations linking a selectable marker to the male sex and their long-term stability, especially under large-scale mass rearing conditions, is threatened by genetic recombination in the heterozygous males. We have measured male recombination in order to be able to construct GSS that are more stable. Our results show that male recombination occurs at very low frequencies, that is, below 1% per generation. Furthermore, recombination in medfly males occurs premeiotically. By selecting strains where the Y-autosome translocation breakpoint and the selectable marker are closely linked, the deleterious effects of recombination on the stability of GSS can be minimized. In such strains recombination is reduced by ca. 80% as compared to previously studied GSS. Although recombinants still occur at very low frequencies they still pose a threat to the integrity of the sexing system if they possess a selective advantage. Under mass rearing condition such recombinants will accumulate according to their relative fitness and additional measures, such as improved mass rearing strategies, are required to preserve the accuracy of the sexing system. As a conclusion it is shown that current GSS are stable enough to allow mass rearing at levels exceeding 1000 million male medflies per week.     

Genetic sexing in the mediterranean fruit fly,Ceratitis capitata,using the alcohol dehydrogenase locus

Summary Using the alcohol dehydrogenase (ADH) locus a genetic sexing system is being developed in the Mediterranean fruit fly Ceratitis capitata based on the sensitivity of ADH null mutations to environmental ethanol. A series of null mutants have been induced at this locus, however, none proved viable as homozygotes. One of these null mutants was translocated to the male determining chromosome and this line can be used for genetic sexing. When larvae from this line were reared on larval medium containing various concentrations of allyl alcohol, 97% of the emerging adults were males; in the absence of the allyl alcohol the sex ratio in the line is distorted in favour of the females. It is proposed that the higher ADH activity of the females (homozygous positive) in comparison with the males (heterozygous null) is responsible for their lower survival in larval medium containing allyl alcohol. ADH converts the allyl alcohol to the lethal ketone. The possible use of this line to sex large populations of medflies for use in sterile insect release programmes is discussed.     

Assessment of quality control parameters during mass rearing of a pupal colour genetic sexing strain of the Mediterranean fruit fly

Quality control charts were employed to monitor the quality of several parameters during mass rearing of the T : Y(wp ⁺)30C strain of Ceratitis capitata (Wiedemann), in which male and female flies emerge from brown and white puparia, respectively. Egg hatch and pupal recovery in this genetic sexing strain were below that reported for a wild-type Sohag strain, whereas pupal size, adult emergence and flight ability were higher. The overall recovery from eggs to adult flies capable of flight was similar in the two strains but egg production in T:Y(wp ⁺)30C was reduced to about 56% of that of Sohag. The quality control charts showed an acceptable level of variation within sets of data but the level between data sets was occasionally out of control. This indicated the presence of uncontrolled conditions during the rearing process, possibly arising from variations in the quality of the larval diet. It is concluded that the quality of T : Y(wp ⁺)30C renders this strain suitable for mass rearing in sterile insect technique (SIT) programmes requiring the elimination of the female flies prior to the release of sterilized males. The next step should involve further quality assessments both under field cage and under field release conditions. The importance of optimal and standardized dietary conditions is stressed in relation to the quality of mass reared medflies.

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Résumé Des normes de qualité ont été utilisées pour relever la valuer de plusieurs paramètres lors de l'élevage industriel de la souche T : Y(wp ⁺)30C de C. capitata Wied. dont les mâles et les femelles émergent respectivement de pupes brunes et blanches. Les taux d'éclosion et d'obtention de pupes sont plus faibles avec cette souche qu'avec la souche souvage Sohag; par contre, la taille des pupes, le taux d'émergence des adultes et l'aptitude au vol étaient plus élevés. La fréquence totale des adultes capables de voler par rapport au nombre initial d'ufs a été de 0,297 contre environ 0,311 pour Sohag. Cependant, la production d'ufs a été réduite à environ 56% de celle de Sohag. Les tableaux de contrôle de la qualité ont révélé un niveau de variation acceptable à l'interieur rieur des lots de données, mais la variabilité entre les lots excédait les normes admissibles. Ceci implique des paramètres incontrôlés pendant l'élevage de masse, probablement liés à des variations de la qualité de l'aliment. On peut en conclure que la qualité de la souche T : Y(wp ⁺)30C la rend utilisable pour un élevage de masse dans des programmes d'utilisation d'insectes stériles (SIT) exigeant l'élimination des femelles avant le lâcher de mâles stériles. La prochaine étape impliquera de nouvelles épreuves de qualité dans des cages en champ et dans les conditions de libération dans la nature. L'importance des conditions optimales d'une alimentation standardisée est soulignée par la qualité des mouches obtenues en élevage de masse.

     

Improved stability of genetic sex-separation strains for the Mediterranean fruit fly, Ceratitis capitata.

In the existing genetic sexing strains for the medfly, Ceratitis capitata, male recombination leads to breakdown of the sexing mechanism under mass rearing conditions. The rate of breakdown depends on the recombination frequency and on the fitness of the recombinants. We have tested two different sexing genes, white pupa and a temperature sensitive lethal, in combination with the translocation T(Y;5)30C. Both sexing strains broke down, although at very different rates. In the case of the white pupa strain, 3.5% recombinants were observed after rearing the strain for 15 generations. The second strain, utilizing white pupa and the temperature sensitive lethal as selectable markers, already reached a comparable level after six generations and was broken down completely in the ninth generation. In these strains the frequency of recombination is high because the breakpoint of T(Y;5)30C and the sexing gene(s) are far apart. To remedy the situation, we have isolated four new translocations with breakpoints located closer to the sexing genes. Mass rearing was simulated for several generations with strains based on these translocations and no breakdown was observed under the conditions used.     

Sensitivity of wild and genetic sexing strain Ceratitis capitata to high-temperature disinfestation of Valencia oranges

Abstract:  The purpose of this research was to investigate post-harvest heat treatment of Valencia oranges as an effective disinfestation protocol (fast, no fruit damage) for the Mediterranean fruit fly Ceratitis capitata (Wiedemann). Forced high-temperature air was applied under the following conditions: (a) exposure to air temperature of 56°C, for fast increase of temperature in the interior of the fruit, (b) reduction in air temperature to 47°C when the fruit centre reached 47°C and (c) maintenance of fruits in the chamber for another 30 min. Relative humidity in the treatment chamber was kept between 50% and 65% during treatment. Forced air at 47°C applied for 30 min on eggs before hatch or late third instar larvae (the most heat-tolerant stages) resulted in complete kill. Egg and larval sensitivity to high temperature differed between a wild strain and a laboratory genetic-sexing strain based on white pupa mutation. In this strain males emerge from brown pupae and females from white pupae. In particular, mature eggs from the wild strain were significantly more temperature resistant than eggs from the laboratory strain. Exposure of Valencia oranges of a diameter of 7–7.5 cm to 56°C forced air for about 86 and 99 min was required to increase temperature to 47°C at 1.5 cm depth and the fruit centre respectively. Treated oranges showed no substantial peel or interior deterioration, or change in colour and taste when kept at 25°C and 50–60% RH for a period of up to 1 month following treatment. Treatment in 1% O₂ atmosphere, produced by flushing of CO₂ into the treatment chamber, resulted in about 1°C reduction in killing temperature and faster increase in temperature inside the fruit to a lethal level.     

Isolation and cytogenetic analyses of genetic sexing strains for the medfly,Ceratitis capitata

Over the last 10 years, several genetic sexing strains have been isolated for the Mediterranean fruit fly, Ceratitis capitata, with the aim of improving the Sterile Insect Technique. However, a major problem with the currently used genetic sexing system, which is based on translocations, is their potential genetic instability. Therefore, careful monitoring and chromosome analyses are necessary when new genetic sexing strains are developed. Instability of a genetic sexing strain can be the consequence of recombination or the survival of aneuploid individuals occurring as a consequence of adjacent-1 segregation in the meiosis of males with Y-autosome translocations. Recently, genetic sexing strains have been isolated that show only low levels of recombination. However, many aneuploid flies are produced by these strains. Therefore, we have made an attempt to isolate new genetic sexing strains that show a low percentage of recombination and no survival of aneuploid individuals. We report their genetic behaviour and the polytene chromosome structure of these new strains.     

Susceptibility of the Mediterranean fruit fly (Ceratitis capitata) and the Natal fruit fly (Ceratitis rosa) to entomopathogenic nematodes

The potential of entomopathogenic nematodes, Heterorhabditis bacteriophora, Heterorhabditis zealandica and Steinernema khoisanae, to infect pupariating larvae, pupae and adults of Ceratitis capitata and Ceratitis rosa was investigated in laboratory bioassays. Pupariating larvae and adult flies were susceptible to nematode infection, with no infection recorded for the pupae. Pupariating larvae of C. capitata were generally more susceptible to infection than those of C. rosa. Significantly more larvae of C. capitata were infected by H. bacteriophora. For C. rosa, highest infectivity of larvae was obtained with H. zealandica. In contrast, adults of both species were highly infected by S. khoisanae.     

地中海实蝇地理种群遗传分化研究

以mtDNA的CO Ⅰ基因的部分序列作为分子标记,研究了来自安哥拉、黎巴嫩、刚果共和国、尼日利亚、贝宁、多哥、加纳、科特迪瓦、南非、埃及、法国、约旦、秘鲁、智利、美国佛罗里达和美国夏威夷等16个国家和地区共75个个体的地中海实蝇Ceratitis capitaza地理种群序列特征和单倍型特征,并分析了各种群间的遗传分化水平和进化关系,结果表明,在供试的样品中发现单倍型的数目是29,其中有5种为共享倍型.地中海实蝇各地理种群的遗传多态性水平差异较大,总体遗传距离D等于0.007.非洲南撒哈拉区(安哥拉、刚果、尼日利亚、贝宁、多哥、加纳、科特迪瓦)的种群的单倍型数、单倍型百分比和群内遗传距离等遗传多态性参数值都高于其它供试地区,多样性明显较其他地区丰富.     

Field releases of two genetic sexing strains of the Mediterranean fruit fly (Ceratitis capitata Wied.) in two isolated oases of Tozeur governorate, Tunisia

The recent development of medfly genetic sexing strains (GSS) enables only male adults to be produced and released for area-wide control using the sterile insect technique (SIT). Before these strains can be incorporated into large operational field programmes, information is required on all aspects of field performance.
In a pilot project in Tunisia, medfly pupae from GSS SEIB-40 [based on a pupal colour mutation ( wp ) and VIENNA-43/44 [based on a temperature sensitive lethal mutation ( tsl ) were shipped from the FAO/IAEA Agriculture and Biotechnology laboratory (Seibersdorf, Austria) and the emerged sterile males released weekly from the ground in oases of the Tozeur Governorate from mid-February until October 1994. During that period, flies emerging from an average of 5000 pupae per hactare were released weekly throughout 105 hectares. Transportation problems were encountered which resulted in damage to the pupae. Nevertheless, the monitoring of the wild population through a trapping network using Jackson and McPhail traps showed that both strains performed equally well inducing a three- to five-fold decrease of the wild population compared with the control oasis (10 wild flies per Jackson trap per day in October in Ain-El-Karma, five in Tamerza and 30 in Bir Kastilia control oasis). Considering the economical and technical benefits inherent in the use of these new strains in the field and as a result of their good field performance, similar strains have been introduced into mass-rearing facilities in Guatemala (1994), Argentina (1995) and Madeira (1996).     

Host-specific demographic studies of the Mediterranean fruit fly Ceratitis capitata

Abstract. 1. This study was conducted in Greece using a wild strain of medflies ( Ceratitis capitata [Weidemann]) to assess age-specific vital rates in adults, and development and survival in pre-adults when reared on different host fruit. The results were used to construct life tables.
2. The demographic analyses suggested that there are basically four aspects of the medfly's life history which are of major importance: (i) multiple, highlyoverlapping generations; (ii) high net reproduction while young; (iii) high larval fitness in certain hosts; (iv) lack of diapause.
3. Reasons why these characteristics are felt to be important are briefly discussed.     

不同饲养温度对橘小实蝇蛹色伴性遗传品系的影响

分别在19℃,22℃,25℃,28＇t2,31℃下饲养橘小实蝇蛹色伴性遗传品系,统计各温度下卵到蛹的的获得率、蛹重、羽化率、存活率及飞出率。结果表明卵到蛹的获得率28℃最大,为62．12％,19℃最小,为43．12％;蛹重25℃最重为1．3854g／100粒,19℃最轻,为1．2610g／100粒;羽化率25℃最高,为93％,31℃最低,仅82．67％;存活率31℃最高,为66％,19℃最低,仅为4．67％;飞出率25℃最高,为65％,31℃最低,为16％。综合不同温度下的卵到蛹的的获得率、蛹重、羽化率、存活率及飞出率得出25℃-28℃是大量饲养橘小实蝇蛹色伴性遗传品系的最佳温度。     

Viability and behaviour of translocations that suppress female recombination in the Mediterranean fruit fly,Ceratitis capitata (Wied.)

Summary The viability of a series of recombination suppressor (RS) strains in Ceratitis capitata, all previously found to contain a reciprocal autosomal translocation, was assessed for egg hatchability and adult emergence in both the homozygous and heterozygous state. Except in T 30C, which contains a Y-autosome translocation in addition to the A-A translocation, egg hatch was significantly reduced in all heterozygous translocation strains, and ranged from 42.4% to 58.5% in seeded eggs compared to a control value of 82.8%. Adult emergence from hatched eggs was affected to a lesser extent, but with a range of 59.5% to 84.2%, compared to the control value of 83.1%, remained significantly reduced in 4 of the 6 translocation strains, as well as in the male line of T 30C. In the homozygous configuration all strains, except T 19 and T 109, showed a significant reduction in egg hatchability, whereas adult emergence was not adversely affected. A significant reduction in the egg hatchability of the translocation heterozygotes compared to that of the homozygotes was observed in 5 of the 7 strains, the observed reduction in T 55/109 being non-significant while that of T 30C was significantly increased. The behaviour of translocations as recombination suppressors and their suitability for inclusion in breeding schemes for the isolation of induced recessive mutations is discussed.This work forms part of a Joint FAO/IAEA research programme on the development of genetic sexing mechanisms for the Mediterranean fruit fly, Ceratitis capitata (Wied.)     

Hosts of Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) from Broome and the Broome Peninsula, Western Australia

Abstract  In the Kimberley region of Western Australia, Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is only established at Broome. From 1998 to 2001 extensive fruit sampling was carried out at Broome during a trial investigating the eradication of medfly from the Kimberley using the sterile insect technique. Fruit was collected from 82 native and introduced plants growing in and surrounding the town of Broome and held for emergence of medfly. Eighteen plants, including the native tree Terminalia petiolaris , were found to be medfly hosts . On the basis of abundance, fruiting phenology and host suitability, eight species (kumquat Fortunella japonica , mango Magnifera indica , Barbados cherry Malpighia glabra , orange jessamine Murraya paniculata , guava Psidium guajava , Pacific almond Terminalia catapa , blackberry tree Terminalia petiolaris and yellow oleander Thevetia peruviana ) were the most important hosts ensuring medfly survival and population growth. Despite medfly being reared from T. petiolaris , it is likely that medfly can only maintain populations in areas close to human habitation, and eradication from these areas would lead to eradication from the whole Kimberley region.     

Cytogenetic analysis of three genetic sexing strains of Ceratitits capitata

Summary Polytene chromosomes of three genetic sexing strains of Ceratitis capitata were analyzed. The genetic sexing mechanism is based on a pupal color dimorphism (white-brown) and is the result of a reciprocal translocation between the Y chromosome and the autosome bearing the w locus (white pupal case). The analyzed polytene chromosomes were derived from two different pupal tissues, the orbital bristle and fat body cells. The Y chromosome is visible in both tissues, while the autosomes present a different banding pattern. Based on these features, the autosome breakpoints in the three Y; autosome translocations were mapped, and the homology of the translocated autosome in both tissues was established. In addition, the location of the break-points was compared to the stability of these three strains.     

Electroantennogram responses of the Mediterranean fruit fly, Ceratitis capitata to identified volatile constituents from calling males

Fifty-six compounds from the odor of calling, sexually mature, laboratory reared males of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) were isolated by headspace trapping on Tenax columns and identified using GC/MS techniques (69 total compounds were detected). Electroantennogram responses (EAGs) to 54 of the 56 identified compounds as well as 5 analogs were tested on both sexes. Significant differences between the sexes in their responsiveness were found in 9 of the 54 identified compounds tested. There was no correlation between the amplitude of the EAG response and the relative abundance of compound identified from headspace analysis. Of the five major identified components, three elicited relatively small EAG responses, while two elicited large EAGs compared to the hexan-1-ol standard. The relative ranking of EAG responses were: methyl and ethyl hexenoates and hexanoates > C₄–C₆ esters and/or acetates > ethyl and methyl octenoates > monoterpenes > sesquiterpenes > C₂–C₅ acetates, alcohols and ketones. Behavioral bioassays on each of the five major identified components as well as a blend of six of the compounds showed some degree of attractancy to virgin females which in some cases approached the response to a pheromonal standard (male odors absorbed onto filter paper). These results are discussed in relationship to the insect's antennal sensitivity to putative pheromone components and/or allomonal components and to other reported C. capitata pheromone studies.

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Résumé Cinquante-six composés de l'odeur de mâles de C. capitata Weidemann, élevés en laboratoire, sexuellement mûrs et en appel, ont été isolés par piégeage sur colonnes tenax et identifiés par la technique GC/MS (69 composés avaient été détectés en tout). Les électroantennogrammes (EAGs) ont été examinés chez les deux sexes pour 54 des 56 composés identifiés et 5 de leurs analogues. Des différences significatives entre les sexes ont été observées pour 9 des 54 composés identifiés. Il n'y avait pas de corrélation entre l'ampleur de l'EAG et l'abondance relative du composé lors de son isolement. Pour les 5 principaux composés identifiés, 3 ont induit des EAGs relativement faibles, tandis que 2 étaient importants, par comparaison avec l'Hexane-1-ol utilisé comme témoin. Le classement relatif des EAG a été: hexénoates et hexanoates d'éthyl et de méthyl C₄–C₆ esters et/ou acétates octénoates d'éthyl ou de méthyl monoterpènes sesquiterpènes C₂–C₅ acétates, alcools et kétones. Les expériences de comportement avec chacun des 5 composés principaux identifiés, comme avec des mélanges de 6 composés ont mis en évidence une attraction des femelles vierges qui dans quelques cas avoisine la réponse à la phéromone témoin (odeur du mâle absorbée sur papier filtre). Ces résultats sont discutés en fonction de la sensibilité de l'antenne d'insexte aux composés supposés de la phéromone et aux composés allomonaux, et en fonction des autres études connues sur les phéromones de C. capitata.

     

Effects of larval rearing temperature on the development of the mediterranean fruit fly, Ceratitis capitata

As the temperature at which larvae of C. capitata were reared decreased from 30° to 25° to 20° the yield of pupae from eggs increased from 15% to 25% to 30% and the mean weight of pupae decreased. Within each temperature regime the weight of pupae produced on successive days declined and the fat content of those pupae varied.

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Résumé On a étudié l'effet de température d'élevage larvaire de 20°, 25° et 30°. La durée du développement s'accroît de 7 à 13 jours pour des températures respectives de 30° et 20°. Lorsque la température d'élevage baisse, le nombre de pupes produites par rapport aux oeufs s'accroîts de 15% à 20% et 30% et le poids moyen des pupes diminue.Pour chaque régime de température il y a une nette réduction du poids des pupes produites au cours des jours successifs et la teneur en lipides de celles-ci varie également. L'émergence moyenne des adultes à partir de pupes produites dans un élevage à 30° est plus élevée que celle obtenue dans des élevages à 25° et 20°.Tandis que la vitesse du développement, le contrôle du métabolisme des graisses et la production de pupes peuvent être des facteurs primaires intervenant dans le choix de la température d'élevage pour la production de masse de la mouche des fruits, il faudrait aussi considérer les effets de cette température d'élevage sur d'autres aspects de la biologie de l'insecte.