Research Article: Cryopreservation of the Mediterranean fruit fly (Diptera: Tephritidae) VIENNA 8 genetic sexing strain: No effect on large scale production of high quality sterile males for SIT applications

Date Published: January 25, 2019

Publisher: Public Library of Science

Author(s): Ihsan ul Haq, Adly Abd-Alla, Ulysses Sto Tomas, Jose Salvador Meza, Kostas Bourtzis, Carlos Cáceres, David A. Lightfoot.


The sterile insect technique (SIT) integrated in area-wide integrated pest management (AW-IPM) programmes is being used for the successful management of the Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) which is a horticultural pest of economic importance in tropical and subtropical countries. All programmes with an SIT component are using the VIENNA genetic sexing strains (GSS), mainly the VIENNA 8 GSS, which have been developed by applying classical genetic approaches. The VIENNA 8 GSS carries two selectable markers, the white pupae and the temperature sensitive lethal genes, which allows the production and release of only males thus increasing the biological efficiency and cost effectiveness of SIT applications. However, mass rearing may affect quality traits of the GSS, in which case replenishment of the colony with wild flies is recommended, a process which is tedious and time consuming. We previously reported the development of a cryopreservation protocol for the VIENNA 8D53+ strain. In the present study, we report on the evaluation of the cryopreserved strain VIENNA 8D53+/Cryo-228L, reared under semi mass rearing conditions, for production parameters, quality control indices and mating competitiveness of males, in a comparative way with the non-cryopreserved VIENNA 8D53+ strain, against wild type males. The VIENNA 8D53+ and VIENNA 8D53+/Cryo-228L strains were similar for production parameters viz. egg production, pupal production, pupal recovery, and quality control indices like fly emergence, sex ratio and flight ability. Males from both strains were equally competitive with males of the wild type strain in achieving mating with wild type females under field cage conditions. Results are discussed in the context of cryopreservation as a potential backup strategy for refreshing the mass rearing colony with biological material from a cryopreserved stock.

Partial Text

The Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is one of the most destructive pests in the world, causing extensive direct damages to horticultural produces and indirect losses by impeding the international trade [1,2]. It is extremely polyphagous, and attacks more than 350 species of fruits and vegetables [3,4]. Application of synthetic insecticides in addition to causing damages to the environment remained insufficient to suppress this pest population to a level that can meet the quarantine requirements. Therefore, management of this pest necessitates the application of strategies which are environmentally friendly and effective to suppress the population to a desired level [5–7]. The sterile insect technique (SIT) is an eco-benign technique which relies on the mass-rearing of insects of the target population, sterilizing them by ionizing radiation and releasing them in the target areas where released sterile mass-reared males mate with virgin wild females and transfer their sterile sperm which results in no off-springs [8]. Successive and sustained releases of sterile males can gradually reduce the density of the target population to a very low, economically acceptable level and, in some cases, eradication can also be achieved [8]. The SIT has been proven very effective for the suppression, containment, prevention or eradication of populations of the Mediterranean fruit fly [9]. Two main factors have made the SIT for the Mediterranean fruit fly more cost-effective: first, the technological developments that enabled the production of flies on a large scale i.e. more than 4000 million sterile pupae per week are produced in different facilities world-wide [10] and second, the development of genetic sexing strains (GSS) [9] which allowed the separation of sexes early in development. The Mediterranean fruit fly ‘first generation’ GSS was based upon a pupal color mutation that allowed the separation of male brown pupae (wild type) from female white pupae (due to a mutation in the wp gene) [11]. The ‘second generation’ GSS carried an additional mutation in the temperature sensitive lethal (tsl) gene [12,13]. Female embryos homozygous for the tsl mutation are sensitive to temperature and die upon exposure to 34 ºC for 24 h. Therefore, the GSS carrying the mutations in the wp and tsl genes allows the production and release of males only. This improves the cost effectiveness and the biological efficiency of the SIT application due to the following reasons: (i) male-only releases eliminated the assortative mating between sterile males and sterile females, making the SIT component much more effective [14], (ii) dispersal of the sterile males to find wild females for mating is higher which enabled them to transfer their sterile sperms to more wild females and (iii) male-only releases reduced the cost of releasing sterile males [15,16]. In view of these advantages, all operational programs applying the SIT for management of this pest are rearing the GSS of this species [17,18]. Despite the fact that GSS are performing very well in small scale rearing environments, they may be vulnerable to break down, due to recombination phenomena, under large scale mass rearing conditions [19]. To overcome this potential problem, the colony management practice i.e. filter rearing system (FRS) was introduced. The FRS relies on maintaining a small standby colony of GSS under less crowded conditions, with no recombinants, which can regularly refresh the mainstream of production with new material. The key factor of this practice is that no flies from the mainstream colony are returned to the filter colony [20]. An additional step for preventing recombination-related problems was the integration of an inversion, known as D53, on some versions of the VIENNA 8 GSS [13,18].

The VIENNA 8D53+ and VIENNA 8D53+/Cryo-228L strains were similar in egg production, pupal production, pupal recovery as well as in quality control indices such as fly emergence, sex ratio, and flight ability. Males from both strains were equally competitive with males of the wildish strain for achieving mating with wildish females under field cage conditions. These data are in accordance with the findings of previous studies on other strains of Ceratitis capitata as well as on strains of Anastrepha suspensa and Anastrepha ludens [26], indicating that cryopreservation does not adversely affect the quality of the medfly GSS and they clearly suggested that cryopreservation can be used as a tool to preserve valuable strains such as the medfly VIENNA genetic sexing strains used for SIT applications worldwide.




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