What is known about modified insects for disease prevention?: a systematic review

The modification and release of insects to suppress or replace natural insect vectors constitutes a promising tool for vector control and disease prevention, facing the unprecedented global emergence of vector-borne diseases. Little is known regarding these innovative modification strategies and available evidence is not standardized turning it difficult to reflect on their actual efficacy and eventual effects. This work conducted a systematic review, gathering and analyzing research articles from PubMed and Biblioteca Virtual em Saúde databases whose results directly report efficacy and effects of the use of modified insects for disease prevention until 2016. Within more than 1500 publications that were screened a total of 349 where analyzed. A total of 12/3.4% reported field-based evidence, and 41/11.7% covered modification stretagies’ efficacy after insects’ release, their epidemiology impact or its long-term efficacy. Examples of successful results were the replacement of natural field populations by wolbachia-infected mosquitoes in 5 weeks, and the elimination of a population in laboratory cages after transgenic mosquitoes release over 10–20 weeks. Variability in the effective results were described (90/25.7%) questioning its reproducibility in different settings. We also found 38/10.9% publications reporting reversal outcomes, such as an increase of vector population after release. Ecological effects such as horizontal transfer events to non-target species (54/15.5%), and wolbachia-induced worsening pathogenesis on mammal filarial diseases (10/2.9%) were also reported. Present work revealed promising outcomes of both suppressing and replacing approaches. However, it also revealed a need of field-based evidence mainly regarding epidemiologic and long-term impact of insect modification strategies. It pointed out some eventual irreversible and important effects that must not be ignored when considering open-field releases, and that may constitute constraints to generate the missing field evidence. Moreover, the level of variability of existing evidence suggests the need of local/specific evidence in each setting of an eventual release. Author summary Innovative strategies are needed to arrest the unprecedented increase of vector-borne disease incidence, distribution and severity. Several modification techniques are being tried all over the world. However this is still an emergent topic with scarce available information and of complex understanding. Present work is the unique structured review regarding the use of modified insects for vector-borne disease prevention, bringing neutral and robust evidence that will contribute with critical insights regarding these approaches. Here we explored more than 1200 publications and analyzed 349 publications on this subject, describing the actual efficacy and reported effects of several modification strategies. More than 30 categories were reported such as, the type of modification, the year of the publication, the species were results were tested, the type of study, and also type of Efficacy Outcome (from modification to long-term) and/or the type of Effects Outcome (from physiologic to ecologic effects). Analysis revealed promising outcomes regarding vector-control and disease prevention. However insects’ modification strategies still lack field-based evidence mainly regarding epidemiological and long-term efficacy. Eventual reversal outcomes on disease transmission, or irreversible biological effects (including horizontal transfer to non-target species or worsening pathogenesis in particular diseases in mammals), were also described. These effects need to be explored, dispelled or resolved before field trials occur in human residential areas. Some of these questions could only have a robust answer if these strategies would be implemented, needing to take the risk to observe reversal outcomes and/or irreversible effects. These findings reflect the big dilemma that is under the use of modified insects to prevent vector-borne diseases. Findings could support health authorities in decision-making and regulatory committees during advisory processes, by evaluating the pros/cons of each modifying technique for a particular setting. Moreover they could also summarize what is crucial to inform to communities if planning open field releases in residential areas.


Author summary 2
Innovative strategies are needed to arrest the unprecedented increase of vector-borne disease 3 incidence, distribution and severity. Several modification techniques are being tried all over the 4 world. However this is still an emergent topic with scarce available information and of complex 5 understanding. 6 Present work is the unique structured review regarding the use of modified insects for vector-7 borne disease prevention, bringing neutral and robust evidence that will contribute with critical 8 insights regarding these approaches. 9 Here we explored more than 1200 publications and analyzed 349 publications on this subject, 10 describing the actual efficacy and reported effects of several modification strategies. More than 11 30 categories were reported such as, the type of modification, the year of the publication, the 12 species were results were tested, the type of study, and also type of Efficacy Outcome (from 13 modification to long-term) and/or the type of Effects Outcome (from physiologic to ecologic 14 effects). Analysis revealed promising outcomes regarding vector-control and disease prevention. 15 However insects' modification strategies still lack field-based evidence mainly regarding 16 epidemiological and long-term efficacy. Eventual reversal outcomes on disease transmission, or 17 irreversible biological effects (including horizontal transfer to non-target species or worsening 18 pathogenesis in particular diseases in mammals), were also described. These effects need to be 19 explored, dispelled or resolved before field trials occur in human residential areas. Some of these 20 questions could only have a robust answer if these strategies would be implemented, needing to 21 take the risk to observe reversal outcomes and/or irreversible effects. These findings reflect the 22 big dilemma that is under the use of modified insects to prevent vector-borne diseases. Findings 23 could support health authorities in decision-making and regulatory committees during advisory 24 processes, by evaluating the pros/cons of each modifying technique for a particular setting. 25 Moreover they could also summarize what is crucial to inform to communities if planning open 26 field releases in residential areas. Vector-borne diseases have a wide impact on human health being an mandatory topic on global 2 health agendas (1)(2). Even with the significant reduction of the global burden of malaria since 3 the beginning of the century, in 2016, this infectious disease was still responsible for 445 000 4 deaths (3). Due to human population growth, globalization, and climate change, arboviral 5 diseases outbreaks have been increasing in frequency, expansion, diversity and severity (4). Only 6 dengue's incidence grew more than 30-fold in the last 50 years (5). Although arboviruses dispersal 7 is partially conditioned by the environmental constraints that limit the distribution of its main 8 vectors, outbreaks of diseases such as, yellow fever, chikungunya and Zika have been reported all 9 over the world (6)(7)(8)(9)(10). The severity of Zika fetal malformations during 2015/2016 epidemics turn 10 it a public health emergency of international concern according to World Health Organization 11 (11). The lack of effective approved vaccines for some of these infections and the increase of 12 insecticide resistance in its most competent vectors, impose an urgent need for innovative 13 effective strategies to minimize these diseases (12)(13). 14 The release of modified insects is considered a promising approach for prevention and control of 15 vector-borne diseases. Innumerous techniques and insects' modification strategies had been 16 laboratorial tested, all of them fitting one of the two broad approaches: (i) modification and 17 release of sterile insects aiming the reduction/eradication of natural vector populations 18 (suppression approach / vector control approach) or (ii) modification and release of insects 19 refractory to pathogen transmission aiming the replacement of natural vector populations 20 (replacement approach / transmission prevention approach). Open releases of modified insects 21 have been occurring all over the world in an attempt to cope to the unprecedented vector-borne 22 diseases burden (14-21). However, none of these modifying technologies has yet been approved 23 by the WHO's Vector Control Advisory Group(22). 24 Few studies reported the effectiveness of insects modification strategies, and even less their 25 eventual effects exploring them only barely and theoretically(23-25). Important reviews on this 26 topic were recently published, but corresponding to the perspective of the author regarding the 27 subject or a summary of the authors' selection of publications (23)(26-29). This work presents a 28 unique structured review on the use of modified insects to control and prevent vector-borne 29 A two-stage inclusion process was applied. All references were initially screened by title and 1 abstract and included in the study if they met the selection criteria. In the second stage, the full 2 text reading of each publication was undertaken. To establish consensus in criteria application, 3 part of the publications (5% of the 1 st screening, and 50% of the 2 nd screening) were screened by 4 two reviewers (inter-reviewer check). Disagreements were resolved by discussion. In the end of 5 the 2 nd screening and after criteria had been discussed, the full-text screening was repeated by 6 one reviewer to ensure homogeneity of the criteria during the process (temporal check). All 7 documents considered relevant went to the next phase of extracting data and analysis. 8 9

Data synthesis and analysis 10
Data was extracted from the included publications into a digital data-extraction form. Two 11 investigators performed data extraction and analysis of 50 % of the included publications (inter-12 reviewer check). All extracted data was structured into two major themes, efficacy and effects of 13 the modification strategies, and each of them divided into several topics and sub-topics (see more 14 detailed information in Results section). These hierarchical categories were defined by the two 15 reviewers through a consensual process. Disagreements were resolved by discussion. When 16 consensus was attained, categorization and analysis of all included articles were re-checked in 17 order to ensure a homogeneous analysis (temporal check). According to the evidence reported, 18 publications were classified into as many categories as possible, in order to reduce the likelihood 19 of missing key points in the data. It was also extracted information regarding the year, type of 20 study (laboratory, semi-field, field and computational modelling), species involved in the 21 experiments, and modification strategy (Wolbachia, anti-pathogen Transgenesis, lethal 22 Transgenesis, etc.). As to Wolbachia-based studies, publications were classified according to the 23 endosymbiont origin: natural occurrence, artificially introduced or removed from natural or 24 artificially infected hosts. The classification regarding the type of study was used as a proxy of the 25 publication robustness, considering semi-field and field studies the most robust ones, and 26 computational modelling and laboratorial the less robust. Apart from qualitative analysis, 27 descriptive statistics analysis was performed. The softwares Excel (Microsoft Office, Windows 10) 28 and NVivo 10 (QSR International Pty Ldt, Doncaster, Victoria, Australia) were used during the 29 analysis. Results are presented by theme, modification strategy and species, publications are also 30 referred according to their chronological order on the manuscript' sections, tables and 31 supplementary information. This literature review followed the proceeding of a PRISMA 32 methodology (S1 Checklist).

1
Databases searches resulted in a total of 1567 publications (Fig 1). Following the removal of 2 duplicates, 1205 references were selected. After the two-stage selection process 377 articles 3 were included in the study, and 349 publications were analyzed. References from analyzed 4 publications were ordered from 1 up to 349 and cited in italic for differentiation from 5 manuscript's references (see full list of analyzed publications and summary of analysis in S1 6 Appendix).  The majority constituted laboratory studies, i.e. performed in a controlled experimental 11 environment (310/88.6%) and referred to Wolbachia or other symbiont-based modification 12 strategy (307/88.0%). Out of those 307, 2/0.7% publications referred to other symbiont 13 (Rickettsia and Sodalis) (1),(2). Several organisms' species integrated in the experiments of the 14 analyzed publications: five genera of insects vectors (Aedes, Anopheles, Culex, Mansonia, 15 Phlebotomos and Glossina) 54 genera of non-vector insects, and four mammals genera (see all 16 data regarding quantitative analysis in S1 Figure). 17 In what concerns the content of the publications, two major themes emerged from the analyzed 18 research articles: (i) efficacy of the modification strategies, and; (ii) effects induced by the 19 modifications. Both themes (efficacy and effects) were divided into several topics (see Fig 2).

Results' section 23
Efficacy outcomes were also divided into effective outcomes -reporting the success of the 24 modification strategy -and ineffective outcomes -reporting the failure of the modification 25 strategy. Ineffective outcomes includes outcomes that: (i) achieved no results, (ii) described 26 indirect results that call into question the efficacy of the modification strategy and/or (iii) 27 reported reversal results, i.e. that lead to the reverse of the aim of the modification strategy. 28 There were more publications contributing to efficacy (237/67.7%) than publications with results 29 regarding effects (156/44.6%) (Fig 3). Regarding themes and topics' analysis, each publication In what concerns publications covering efficacy outcomes, there were more publications 3 reporting ineffective outcomes (164/69.2%) than publications reporting effective ones 4 (150/63.3%). Out of the latter, only 41/27.3% constitute main effective outcomes (regarding its 5 release, epidemiologic and long-term efficacy), and out of the former, 38/23.2% constitute 6 reversal outcomes (Fig 4).  However, release of Wolbachia-insects also led to no/low invasion rates (98), (99). Several studies 6 suggested the need to release prohibitively large number of insects (100-102). To overcome that, 7 two solutions were reported: releases in a ratio of 95% male mosquitoes (requiring a mass rear 8 capacity) (11) or the introduction of insecticide resistance genes along with Wolbachia in the host 9 insect, combined with a pre-release intervention to reduce (adult) insect vector numbers (29), 10 Finally, also affecting Wolbachia efficacy is its variability. It was reported that a considerable 2 degree of variability may evolve in short evolutionary periods (126). Several articles described 3 Wolbachia evolution (127) Table 1 and its reversal outcomes in insect vectors is described in Table 2, see the 16 complete data (also (146)(147)(148)(149)(150)(151)(152)(153) and (154-201)) in S1 Transgenesis and other non-symbiont-based modification strategies (effective and ineffective 1

outcomes) 2
A total of 37 research articles (15.6% out of the 237 with efficacy outcomes) have results 3 regarding the efficacy of transgenesis or other non-transgenic and non-symbiont-based insect 4 modification (37/100% reported effective outcomes, 15/40.5% reported ineffective outcomes).  In some cases, the anti-pathogen transgene led to no or low fitness cost in the modified insects 2 (208),(222), thus allowing the modified insect to be reproductively competitive against their 3 natural counterparts. However, anti-pathogen gene insertion also caused fitness benefits in 4 Anopheles gambiae (206), and Anopheles stephensi that fed Plasmodium-infected blood 5 (207),(221). Since these outcomes increase vectors abundances and vectorial capacity, they 6 constitute reversal outcomes. Computational modelling studies reported that elimination of vector insects might be an 2 unrealistic objective. However, substantial suppression can nonetheless be achieved in certain 3 conditions, such as an uniform spatial pattern and multiple lethal elements (227), or a certain 4 release ratio and population size (229),(224). Elimination of a natural population after the release 5 of insects with a dominant lethal was though reported in semi field studies (228),(226). 6  Long-term (lethal transgenesis) 7 One computational modelling study suggested lethal transgenesis long-term efficacy to be 8 compromised by invasion of wild type insects (224). 9 From all publications in this section, only one publication reported a field study, describing the 10 ability to mate and copulate of a radiated insect, modified by a sterilizing technique (SIT) (231). 11 Also only three publications reported reversal outcomes, related to fitness benefits (as above 12 mentioned). Main effective outcomes of transgenic and other non-symbiont-based modified 13 insect vectors are presented in Table 3 (the complete data is presented in S2 Table). 14 15 Five articles (2.1% out of the 237 with efficacy outcomes) contributed to the efficacy of insect 1 modification as a vector control approach, regardless of the modification strategy used (see S8 2 Table). immune response or microbiome of the modified insect; (ii) at the insect species level, concerning 14 its evolution and/or behavior; and (iii) at the ecosystem level, affecting any other organism of the 15 modified insect ecosystem (Fig 2 and Fig 7). 16 Wolbachia also induced effects on the immunity of the modified insect mainly through the up-8 regulation of effector genes. Report of Wolbachia-mediated induction of immune system was 9  Table.  19 20

Populational effects (at insect population level) 21
According to 40/11.5% analyzed articles (nTotal=349), Wolbachia also affected its host 22 population in several ways such as, altering its mitochondrial DNA (mtDNA) pattern, interfering 23 in speciation process, on its behavior ecology or others. Changes in mtDNA patterns were 24 reported in several non-vector insects (272-283), and in the vector mosquito Culex pipiens (7). 25

Ecological effects (at insect ecosystem level) 5
Finally, 64/18.3% publications reported that Wolbachia is also able to induce alterations in other 6 organism rather than its host, interfering thus, with host ecosystem. The majority of the 7 publications covering this type of effect described the report or the estimation of horizontal 8 transfer events (i.e. transfer between neighboring contemporary species) of genetic material, 9 such as a gene or a symbiont. Horizontal transfers (HT) can occur through bacteriophages,  To our knowledge, the present work constitutes a unique review on modified insects for vector-2 borne disease prevention, for several reasons. Rather than being the perspective of an author or 3 a summary of the authors' selection of publications, this review followed a structured 4 methodological procedure. Furthermore, authors from present review are not involved in 5 scientific projects to modify insects having, thus, no conflict of interest in the outcomes of this 6 reflection. Moreover, present review enclosed several types of modifications in any vector or 7 non-vector insect species, covering an uncommon comprehensiveness, and thus, offering an 8 exceptional opportunity to observe trends and to outline the big picture of the modified insects. 9 In that sense, this review constitutes a baseline of knowledge that not only can be fed with 10 forthcoming publications to follow up trends, but also point out to questions that may need to 11 be further explored. Additionally, this review developed a framework of themes, topics and 12 outcomes, that organizes the extensive information available. Also, it goes beyond modifications' 13 efficacy, most commonly covered in current reviews (26)(23) successful releases of modified insects, its positive epidemiological impact and/or its long-term 28 efficacy (the remaining cover ineffective or primary effective outcomes). Out of the three main 29 efficacy topics, the epidemiological impact was the less covered, reported in only five 30 publications, none of them based on the most robust field studies, but all based on computational 31 modelling studies instead. Main effective outcomes were obtained for several insect 32 modifications, namely, Wolbachia-based strategies (replacement and suppression approaches), and paratransgenesis (replacement approach). Seven publications (out of the 41) reported main 1 effective outcomes based on semi-field or field studies, (corresponding to outcomes obtained 2 with Wolbachia or RIDL strategies). Even though not many, these publications achieved critical 3 outcomes: (i) field-released wMel-aegypti mosquitoes not only reached near fixation (despite a 4 persistent low frequency of uninfected mosquitoes), but also maintained their effective traits 5 such as CI, fixation and pathogen protection for at least two years after the release (27)(28) (from 6 S1 Appendix) (ii) weekly introduction of Aedes albopictus males with a dominant lethal (RIDL) led 7 to the eradication of a laboratory cages population in 10-20 weeks (228) (from S1 Appendix). 8 Present review also described reversal outcomes obtained after the release of transgenic insects 9 or insects modified with Wolbachia (reported in 39 publications). Out of those, only one 10 publication correspond to a field study, but in this case the release of modified insects may lead 11 to an increase in the vector insect population particularly if occurring when its natural abundance 12 is at its maximum (100) (from S1 Appendix).  In what concerns suppressing strategies, such as RIDL (lethal transgenesis), female killing (lethal 21 transgenesis), SIT or RNAi-mediated sterilization, the elimination of a species leads to profound 22 changes in its ecosystem, such as eventually putting some non-target species in risk or giving 23 opportunity to not-targeting species to expand (35)(23). Moreover, some authors have been 24 arguing that biodiversity loss may even be associated to emergence of vector-borne diseases (36) 25 (37). Regarding replacement approaches, such as anti-pathogen transgenesis, the effects of the 26 transgene in the ecosystem are unknown. When associated with Wolbachia as gene drive, there 27 are emerging questions regarding the lateral transfer of the inserted transgene to Wolbachia 28 itself or via Wolbachia to other organisms. 29 Since the inclusion criteria were restricted to studies on insects and mammals, results regarding 30 ecological effects may be limited to these taxonomic groups. Horizontal transfer events of strategy, other studies regarding Wolbachia were included (mainly regarding natural Wolbachia) 1 whenever their results contributed to the efficacy or the effects of Wolbachia as a vector control 2 strategy. Furthermore, Wolbachia is a unique term while for other strategies several diverse 3 terms may be used (such as, the name of the particular transgene), and may occur that not all 4 were covered within the research expressions . 5 In what concerns the year of the publications it is clear how recent this topic is, being almost the 6 totality been published in the last 20 years. This can, at least partially, explain why in several topics 7 we found a gap of knowledge such as the long-term efficacy or the epidemiological impact of the 8 modifications. 9 In conclusion, insects modifications strategies appear as a promising innovative alternative to 10 overcome an unprecedented increase of vector-borne, mainly arboviral, diseases. Nevertheless, 11 these modification tools still lack evidence on field-based efficacy mainly regarding 12 epidemiological and long-term impact. Field releases in endemic areas could provide that kind of 13 missing evidence. However, relevant questions remain without a solid understanding. Eventual 14 reversal outcomes on disease transmission, or irreversible biological effects (including effects on 15 mammals) need to be explored, dispelled or resolved. This leads for demand of studies before 16 testing their definite effectiveness in the field. However, some of these questions could only have 17 a robust answer if these strategies would be implemented, needing to take the risk to observe 18 reversal outcomes and/or irreversible effects, in order to confirm the efficacy of these strategies. 19 This reflect the current dilemma that is under the use of modified insects to prevent vector-borne 20 diseases. The level of variability of existing evidence suggests the need to generate local/specific 21 evidence in each setting of an eventual release. Importantly, available preventive strategies 22 should not remain on hold while modified insects do not offer an effective and safe solution. A 23 comprehensive cost-effectiveness analysis could be an important tool when deciding to proceed 24 or not with these innovative strategies, and/or to improve other available strategies. Therefore, 25 an adequate decision could be made for each particular setting, evaluating the pros and cons of 26 these approaches and of each modifying technique.