Insects are the most numerous of all animals and are found in almost every inhabitable place on earth. The order Diptera (true flies) contains many members that are notorious agricultural pests, nuisance or vectors of diseases. The list is long: mosquitoes, tsetse flies, screw worms, fruit flies, sand flies, blow flies, house flies, gall and biting midges, black flies, leaf miners, horse flies, and so on. Efforts to combat some of these pests and vectors have resulted in control measures such as the chemical, physical, and cultural control methods. These methods, though largely beneficial, have disadvantages and limitations, which sometimes seem to outweigh the problems initially sought to be controlled. The chemical method, for example, is not environment-friendly since it negatively affects many nontarget organisms and disrupts ecosystem balance. Development of insecticide resistance by pests/vectors is another concern. Molecular biotechnology has introduced vast arrays of novel ways to tackle pests and disease vectors, as well as improve the potency of existing control methods. This chapter looks at transgenic and paratransgenic biotechnologies and how they have been applied so far to develop and expand the arsenal against dipteran pests and disease vectors. Further, we discuss the advantages, disadvantages, and limitations of these technologies.
- crop pests
- disease vectors
Insects are highly abundant and are the most numerous classes of all described living animals. They account for about half or more of all living animals and are found in almost every inhabitable place on earth [1, 2]. Their success and abundance could be attributed to their ability to adapt and colonize diverse habitats. Among insects, the order Diptera is one of the largest orders with an estimated 120 families and 250,000 described species . They are generally regarded as the two-winged insects or true flies [2, 3]. The main characteristics for members of this order include larvae that lack legs (apodous maggots), pupae enclosed in a thick larval cuticle (puparium), and adults that possess a pair of membranous forewings, vestigial hindwing modified into halters, as well as a tubular sucking or sponging mouthparts . Dipterans are longtime foes and arguably considered the insect archenemy of man. This stems from the fact that many members of this order constitute pests of cultivated crops, are major causes of annoyance or are highly notorious as vectors of human diseases either in their larval or adult stages. Examples of pest, annoyance causing and vector dipterans are given in Table 1 [4–8].
|Family||Genus/species involved||Problematic or damage-causing stage; problem caused|
|Agromyzidae (leaf miners)||Larva; damage to leaves of crops|
|Anthomyiidae||Larva; damage to stems of crops like cauliflower and sorghum causing disease like “dead heart”|
|Calliphoridae (blow flies)||Larva; myiasis or flesh infesting damage to man and livestock|
|Cecidomyiidae (gall midges)||Larva; damage to leaves of crops such as rice, pear, sorghum, etc.|
|Ceratopogonidae (biting midges)||Adult; blood sucker from man and livestock|
|Chloropidae (chloropid flies)||Larva; damage to leaves of crops like rice and cereals|
|Culicidae (mosquitoes)||Adult; blood sucker from man and livestock transmitting parasites that cause various diseases like malaria, dengue fever, West Nile fever, yellow fever, encephalitis, O’nyong nyong fever, Bancroftian filariasis, chikungunya, Igbo-Ora, Zika, etc.; nuisance, major cause of disturbance and annoyance to man at night|
|Drosophilidae||Larva; damage to fruits|
|Glossinidae (tsetse flies)||Adult; blood sucker from man and livestock transmitting the causative agent of Trypanosomiasis (sleeping sickness)|
|Muscidae (house flies)||Adult; transmits microorganisms that cause cholera and amoebic dysentery; nuisance, major cause of annoyance to man during the day|
|Oestridae (warble or bot flies)||Larva; myiasis or flesh infesting damage to man and livestock|
|Psychodidae (sand flies and moth flies)||Adult; blood sucker from man transmitting the parasite causing disease leishmaniasis|
|Sarcophagidae (flesh flies or screw worms)||Larva; myiasis or flesh infesting damage to man and livestock|
|Simuliidae (black flies)||Adult; blood sucker from man transmitting the causative agent of the disease onchocerciasis (river blindness)|
|Tabanidae (horse flies)||Adult; blood sucker from man and livestock transmitting the causative agent of diseases like trypanosomiasis (sleeping sickness) and loasis|
|Tephritidae (fruit flies)||Larva; serious damage to fruits and vegetables|
Efforts by man in the fight against dipteran pest and vector insects have resulted in the generation of an arsenal with several weapons. These range from chemical method which involves the use of insecticides to cultural methods such as sanitation, physical interference or destruction of breeding sites, and cropping methods. However, many of these methods have major disadvantages and (or) limitations that sometime seem to outweigh their benefits. For example, the chemical method is very widely used, but has the disadvantages of environmental consequences such as pollution, health challenges on man and livestock, killing of nontarget insect species, as well as the challenge of the targeted insect species developing resistance to the insecticides applied [9, 10]. Most cultural methods applied against dipteran pest or vector control are labor-intensive and can only be most suitably applied on a small scale.
Biologically-based approaches are generally friendlier to the environment, more sustainable and cost-effective than many other methods used for dipteran control. Here, control methods such as the use of natural enemies like predators and parasitoids are environment-friendly with varying levels of success, but the major limitation is the fact that it is unpredictable as chances are usually low on finding a suitable parasitoid or predator that can survive the weather and conditions wherever the pest or vector dipteran is and continue to effectively eat or parasitize the host . The time it takes to find a good parasitoid may be so long that farmers or entomologists concerned may opt for other control methods, in addition to the fact that the process of actual control by a parasitoid or predator itself is slow. The biological method of using pathogens (microbial or biopesticides) has been quite promising, but recently there has been concerns of insect resistance as is the case with
Another biologically-oriented and environment-friendly method for controlling dipterans is the use of pheromones or suitable attractants. However, the scale of its application and area that it covers is also limited, while the potency of the attractants does reduce gradually with time or could easily be influenced by environmental factors such as rainfall or masked by other chemicals within the vicinity.
Genetic methods such as the sterile insect technique (SIT) majorly use radiation to sterilize male insects and thus reduce the fertility of females that mate with them [11, 12]. This method works well with sexually reproducing insects and has so far has great success among many dipterans . Its major problem is the fact that the gamma radiation used for sterilization also reduces the fitness of the males and makes them less competitive than the wild males.
Obviously, no present method of pest control is devoid of disadvantages or limitations. As such, an integration of different control methods that are compatible is the recent paradigm. Integrated pest management (IPM) has offered a way to augment control methods to achieve a more efficient and sustainable management of pests and vectors.
The new millennium has witnessed advanced progress in genetic biotechnology which in turn has greatly influenced insect control. Biotechnology approaches have been used and are continually been pursued as a means to develop novel ways or improve some of the methods used to fight pest and vector dipterans. For example, new strains of reproductively sterile insects or strains exhibiting other desired traits could be engineered to control a population or designed to fit into control methods like SIT, entomopathogens or biopesticides that are adversely affected by weather conditions where a pest or vector is located or have environmental concerns regarding nontarget insects could be encapsulated in materials that will release the pathogens only in a desired condition, while nonharmful microorganisms could be engineered to deliver therapeutic or antiparasitic molecules to pathogens in their environment. Many of these new biotechnology approaches could also be used as a part of IPM programs which is suitable for other methods. In this chapter, we focus on how transgenic and paratransgenic biotechnologies have been applied to expand the array of weapons in man’s arsenal against dipteran crop pests and vectors of diseases.
2. Transgenic biotechnology
Transgenesis aims at the transformation of an organism by altering its genetic composition and the final outcome is the generation of a transgenic or a genetically modified organism (GMO). Basically, desired genes or genes-of-interest from a different organism(s) are inserted into the genome of a wild type organism majorly with the aid of “jumping genes” called transposable elements or transposons and the transgenic organism generated carry these desired genes (transgenes), while exhibiting characters or traits encoded by the transgenes as well (Figure 1). For insects, germline transformation is sought and microinjections are performed to achieve it, allowing the genome modification to be passed on from generation to generation . To enable detection of successfully transformed organisms, fluorescent proteins such as the green fluorescent protein (GFP), the red fluorescent protein (RFP), and fluorescent proteins of other colors are used as markers [15–18]. Consequently, GM dipterans harboring a transgene that incorporates a fluorescent protein gene cassette as marker would express the fluorescent protein and can be visualized under a fluorescent microscope (Figure 2).
2.1. Transgenic strategies against agricultural crop pest dipterans
The family Drosophilidae consists of many members including the well-known model fly
A proof-of-principle transgene-based, embryo-specific lethality system for insect control was developed by Horn and Wimmer . The system used embryo stage-specific promoters such as serendipity alpha (sryα) to regulate the expression of a hidAla5 lethal effector placed under the control of a tetracycline-response element . Such a strain would effectively achieve reproductive sterility in insect populations because the offspring die during the embryo stage and could replace radiation sterilization of insects as is the case for conventional SIT.
Two proofs-of-principle for transgenic sex-specific lethality systems for insect population control were developed: (i) using female-specific enhancers of yolk protein 1 (yp1) gene to drive expression of a hid effector under control of tetracycline-responsive element . (ii) using a female-specific yolk protein and fat-body enhancer Yp3 to regulate the expression of a Ras64BV12 effector under control of tetracycline-responsive element, as well as using a Hsp26 promoter to regulate expression of a dose-compensation gene, mutant male-specific lethal 2 (msl-2NOPU), under the control of a tetracycline-response element . These kinds of systems limit lethality or death of offspring to only female individuals and could be used for efficient sex separation of dipterans prior to field release during area-wide dipteran pest control programs such as SIT.
Besides these afore-mentioned transgenic lethality systems which were all based on the tetracycline-repressible binary expression system (Figure 3) , a gene-driven system capable of driving population replacement was also developed in Drosophila . Basically, a gene-driven system such as a maternal-effect dominant embryonic arrest (Medea) system use a combination of two genes that encode for a toxin and its antidote, respectively, to create a condition whereby a heterozygote female would express only the maternal toxin in half of her oocytes without the antidote resulting in death of those offspring. The Medea strain which was developed by Chen et al. in Drosophila used microRNA-mediated silencing of a maternally expressed embryonic development gene, my88, as its toxin and early zygotic expression of a rescuing transgene as the antidote. A more complex Medea system employing additional mechanisms such as targeting signaling pathways like the Notch pathway has since been also demonstrated in
For the crop pest Drosophilid, the spotted wing drosophila
2.1.2. Tephritid fruit flies
Tephritids are very important pests of fruits and vegetables and majority of transgenic strategies for crop pests have been developed against members of this group. Lethality systems that their proofs-of-principle have earlier been developed in Drosophila have also been successfully transferred to many Tephritids. Among these are the conditional embryonic lethality strains transferred from
For sex separation of Tephritids, transgenic sexing strains were developed for different fruit fly genera: (i) an RNA interference (RNAi) system developed for
Since area-wide dipteran pest control strategies like SIT involve release of sterile males, a way to monitor the released males is also as important as the sterilization and sex separation of the males. Scolari et al. developed a transgenic strain that would facilitate such monitoring in C. capitata by using the promoter of a sperm-specifically expressed gene β2-tubulin (β2t) to regulate the expression of RFP and GFP to only male testis. Males of this sperm-marked fly strain were shown to still have brightly glowing fluorescent testis for several months after they had died . As such, the released males could easily be monitored if caught in traps or found dead in the field in the case they were used in any SIT control program.
2.2. Transgenic strategies against dipterans of medical and veterinary importance
The battle against any other dipteran insects has perhaps never been as intense as it is for mosquitoes due to the wide range of diseases they vector and transmit. Almost every kind of approach that is imaginable is under development or has been developed in the effort to win the battle against mosquitoes. Ever since the first germline transformation of an Anopheles mosquito , several transgenic strategies have been constructed including gene drive systems, lethality, flightless, sperm-monitoring, as well as spermless systems, and mosquito strains that have been impaired in their ability to transmit a parasite.
Among the gene-driven systems include a maternally-regulated transposition system in the yellow fever mosquito,
Strains exhibiting dominant lethality or major incapacitation in the form of a flightless phenotype have also been generated. A transgenic strain based on the expression of dominant lethality in
For transgenic sexing and sperm monitoring, Catteruccia et al. established a strain that exhibited fluorescent sperms in the Asian malaria mosquito
Another strategy that has also been pursued is to transgenically impair the ability of mosquitoes to transmit malaria Plasmodium parasites. To this end, transgenic Anophelines were developed that were unable to vector Plasmodium parasites as they express an antiparasitic peptide, the salivary gland and midgut peptide 1 (SM1) in their midgut epithelia under regulation by a carboxypeptidase promoter . In the wake of insecticide and drug resistance by both vector and parasite, respectively, this approach offered an avenue to curtail transmission while not removing the vector and could easily be spread to wild mosquito population using some of the gene drive systems developed. Several other researchers have followed this strategy and developed transgenic mosquitoes that cannot transmit their parasites. Bee venom phospholipases, synthetic antimalaria proteins like vida3, single chain antibodies (scFv) targeting malaria parasites, as well as an antimicrobial peptide cecropin A have been used as effectors and mosquitoes engineered to express them lack the ability to effectively transmit parasites [52–56]. RNAi-based resistance to dengue virus has also been engineered in
2.2.2. Blow flies
Veterinary pests such as Blow flies that inflict enormous damage to sheep and other livestock have also received attention lately. Transgenic sexing strains that allow male-only production for control of the Australian sheep blow fly Lucilia cuprina were developed using both the single and binary component tetracycline-repressible expression system. An initial single component female-specific lethality system showing lethality in pupa used a heat shock promoter Hsp70 and the transformer (tra) intron from
Though the very successful strategy of SIT had originally been developed against the New World Screwworm
3. Paratransgenic biotechnology
Similar to transgenesis, paratransgenesis also involves the genetic transformation of organisms. However, paratransgenesis targets to achieve the genetic transformation or transgenesis of the symbionts that live inside an insect instead of the insect itself and cause the symbionts to express or secrete substances that act against parasites and pathogens that are transmitted by the insect (Figure 4). Consequently, paratransgenesis is suitably applied against disease vectors. Originally developed against the triatomine bug vector of Chagas disease, Rhodinus prolixus using its symbiont Rhodococcus rhodnii and the antimicrobial peptide cecropin A as an effector , this strategy has been adopted for many dipterans that vector diseases of humans and livestock.
3.1. Paratransgenic strategies against medical and veterinary important dipterans
Since mosquitoes transmit several disease-causing pathogens, many paratransgenic studies have been conducted on it. Inhibition of vectorial competence in mosquitoes via bacterial symbiont paratransgenesis were demonstrated in the malaria mosquito
Besides bacteria, fungi and viruses have also been utilized in mosquito paratransgenesis. The entomopathogenic fungi Metarhizium anisopliae was engineered by Fang et al. to express the anti-Plasmodium molecules SM1, a sporozoite-agglutinating scFv, as well as an antimicrobial toxin scorpine in An. gambiae . Using the densonucleosis virus (DNV) in a proof-of-concept viral paratransgenesis work in
Efforts on control of sandfly vectors that transmit Leishmania parasites which cause the disease Leishmaniasis has been done mainly with chemical insecticides. To develop a more environment-friendly strategy, Hurwitz et al. recently demonstrated the feasibility of paratransgenesis for sandflies in a proof-of-principle work in
3.1.3. Tsetse flies
Although several control strategies including SIT have been applied against tsetse flies, continual effort is made to develop other methods that would not have limitations of the existing methods, be more sustainable, more cost-effective or suitable for use in IPM control. To investigate the possibility of paratransgenesis in tsetse flies, transgenic
4. Advantages, disadvantages and limitations of transgenic and paratransgenic technologies
4.1. Transgenic technology
4.1.1. Advantages of transgenic technology
The main advantage of transgenic biotechnology is its ability to generate strains that possess traits that are unique and special, and accurately designed or tailored to be specific as desired. Also, the flexibility of transgenic technology allows generation of such desired strains in many species which would have been very difficult or impossible to achieve by other means. Transgenic strains are usually generated after one generation (Figure 1), and hence take less time to generate compared to other methods like classical genetics. Moreover, generation of strains possessing desired traits in one species can almost always be reproduced and transferred to related species with relative ease . Quite unlike earlier genetic methods such as SIT where the use of radiation generates unknown and uncharacterized genetic mutations, transgenic technologies generate known and characterizable genetic modifications. Transgenes could easily be thoroughly characterized, and same goes for genomic positions in the dipteran insect where desired transgenes had got inserted. Also, most transgenic strategies are environment-friendly, sustainable and target-specific. For example, while chemicals developed against fruit flies may kill pollinator insects, transgenic strains developed for control of fruit flies are not likely to have any negative effect on pollinators that their wild counterparts do not already exhibit. Also, the development of resistance against control agents is less likely to occur when using transgenics.
In terms of costs, transgenic technologies as well as many other control strategies are not so cheap to develop. But it is difficult to say with all certainty whether transgenic (and paratransgenic technology) is cheaper than many other methods as there have not been any such economic studies to the best of our knowledge. Nevertheless, transgenic (and paratransgenic) approaches are considered less expensive with regards to the farmer or public beneficiaries as they are usually area-wide-oriented and implemented by big organizations at overall little or no cost to the individual farmers or the public.
4.1.2. Disadvantages of transgenic technology
Probably due to the fact that transgenic technology is just beginning to move from laboratory to the field [79, 80], there are yet no scientifically proven disadvantages. Despite this, many public negative concerns already exist on the use of transgenics , mostly environmental and social, as well as safety and ethical issues. These are mainly due to speculations and the uncertainty as to what might happen in nature following field use of transgenics, and whether unintentional and unforeseen mutations could lead to harmful consequences (though these can potentially occur also in nontransgenics). There are also thoughts on how field use of transgenics could interfere with diversity and evolution due to possible loss of genetic material of original insects and the associated future downstream events. Potential horizontal transfer of transgenes could also be a potential disadvantage that could be associated with the use of transgenic technology. However, a ”self-limiting” transgenic approach such as use of transgenic lethality dipteran strains should not present some of the afore-mentioned environmental problems since they are most likely to remove themselves from the environment with time unlike “self-sustaining” strains . Though not always the case, there may be fitness costs that might arise in dipterans and other insects due to the various loads of transgenes they carry [82, 83]. When well assessed, the fitness costs could be determined and measures taken to eliminate them if necessary or avoid using strains that suffer such lack of fitness. New transgenic strains that may not have the observed fitness cost could also be developed and utilized instead. Proper assessment should be done to determine the associated risks and benefits before any GMO can be utilized .
4.1.3. Limitations of transgenic technology
Transposable elements or “jumping genes” have been the main tool relied on to achieve germline transformation and generate transgenic dipterans. However, most of the transposons used in dipterans (and other insects too) are insect-derived  and a major concern is that a transposon could potentially be remobilized from its integrated genomic position in the insect if transposases required for its activity is encountered in the field. The consequence of such transgene-transposase exposure could be the remobilization of a transgene to another genomic location or total loss of a transgene from an insect’s genome. Measures to avoid potential transgene remobilization in engineered dipterans such as postintegrational transgene modification to alter the transposon and achieve nonmobilization or stability has been demonstrated in
Transgenesis is not yet possible in all dipterans as not all members are amenable to it. Since the development of a transgenic insect strain involves germline transformation (Figure 1), it is therefore important that the biology of a target insect must be in such a way that allows the necessary manipulations to achieve genetic transformation. Tsetse flies are yet to be genetically transformed due to their viviparity which makes it difficult to obtain embryos needed for microinjections and subsequent germline transformation .
4.2. Paratransgenic technology
4.2.1. Advantages of paratransgenic technology
While similar to transgenic approach in terms of its ability to generate within a short time strains that possess unique and special traits designed specifically as desired, paratransgenesis also has an additional advantage of leaving the insect itself genetically unmodified and rather targets the parasites transmitted. This gives paratransgenic approaches a major plus in the sense that it has a more positive public perception than transgenic approaches as many of the disadvantages with use of transgenics would not be present [78, 89]. In addition, this technology has a high potential to be transferred between different species . Moreover, paratransgenic biotechnology mostly employs microorganisms that live within the target dipterans (symbionts) and as a result also has a high likelihood of field success. Another advantage of paratransgenics is the absence of fitness cost of genetic manipulation compared to transgenics or other control strategies .
4.2.2. Disadvantages of paratransgenic technology
Field application of paratransgenic strategies is yet to be actualized and any potential disadvantage of this technology is still to be proven scientifically. Nevertheless, safety concerns and risk assessments have become necessary requirements that need to be addressed to ensure that the benefits outweigh the risks of utilized genetically modified organisms . One concern for paratransgenics is the potential exposure of engineered symbionts to the environment and likely consequences such as horizontal gene transfer. Measures such as symbiont encapsulation to ensure regulated release are being taken to address some of these regulatory concerns .
4.2.3. Limitations of paratransgenic technology
Despite the known advantages of the paratransgenic approach, a major limitation is that it is not suitable for most dipteran crop pests and has been developed mostly for those dipterans (and other insects) that transmit disease pathogens. Symbiont choice and utilization in a paratransgenic expression approach depend not only on availability of symbionts that can be isolated, cultured, reintroduced, and survive well in the targeted host, but also on the ability of the symbiont to be genetically transformed and to possibly express antiparasitic molecules . The lack of some of these requirements would render several good symbionts unusable for paratransgenic control. The bacteria symbiont Wolbachia is one such microorganism that is promising for paratransgenic application, but the lack of success in genetically transforming it has hindered its further utilization for expression of antiparasitic molecules.
5. Future of transgenic and paratransgenic technologies
In the near future, transgenic and paratransgenic pest/vector control strategies may become common place and more widely applied than it is now. Some of the novel approaches of these technologies are promising and offer great hopes for control of several human diseases and could be implemented in the near future if regulatory and ethical issues are satisfied [92, 93]. This could usher in a new era where cases major dipteran-vectored diseases of man such as malaria and dengue, as well as agricultural pest like Tephritid fruit flies become much reduced or even eradicated.
The arrays of weapons in man’s arsenal against his dipteran enemies are also expected to continue to expand. Continuous improvement will be made to existing control strategies, while new and better strategies are expected to be developed in the future as more advances are made in genetics and molecular biology. The RNA-guided genome editing tool, the CRISPR/Cas9 endonuclease recently developed from bacteria such as
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