Malaria is one of the deadliest infectious diseases worldwide. Plasmodium parasites, the causative agents of malaria, are transmitted to humans through the bite of infected female Anopheles mosquitoes. Control of malaria is based primarily on reducing vector populations with insecticides and using antimalarial drugs [1]. These tools have been ineffective due to the development of mosquito insecticide resistance and parasite drug resistance [1]. The development of new tools to combat this disease is of high priority.
Paratransgenesis, the genetic manipulation of insect symbiotic microorganisms to block pathogen transmission, is a promising strategy for controlling insect-borne diseases. Its effectiveness is enhanced by the fact that bacteria share the same compartment, the midgut, with the pathogens transmitted by the insects and because bacterial numbers increase dramatically following a blood meal [2, 3]. The facultative aerobic and gram-negative rod-shaped bacteria of the genus Serratia (Enterobacteriaceae) are common components of the midgut microbiota. This genus is a symbiont of many arthropods, such as mosquitoes, bees, sandflies, ticks, and aphids [4–9].
The potential of Serratia eGFP AS1 (Serratia marscescens, AS1 strain) for paratransgenesis has been demonstrated [10–12]. The gene encoding green fluorescent protein (eGFP) has been integrated into the Serratia eGFP AS1 chromosome. This bacterium contains a plasmid with five anti-Plasmodium effector genes [(MP2) 2 - Scorpine - (EPIP) 4 - Shiva1 - (SM1) 2] under the control of a single promoter, which inhibits the development of Plasmodium falciparum in female Anopheles gambiae and Anopheles stephensi [10]. Both SM1 and MP2 (midgut peptides 1 and 2, respectively) bind to the mosquito midgut surface and inhibit Plasmodium invasion [13, 14]. Scorpine is an antimicrobial peptide found in the venom of the scorpion Pandinus imperator and prevents the formation of gametes and ookinetes of Plasmodium berghei [15]. EPIP (enolase-plasminogen interaction peptide) inhibits mosquito midgut invasion by preventing plasminogen binding to the ookinete surface [13]. The Shiva1 or cecropin-like synthetic antimicrobial lytic peptide kills P. falciparum [16]. All these effector proteins strongly inhibited this pathogen, reducing the oocyst load by up to 93% [10].
The use of any genetically modified organism (GMO) for biological control should impose minimal fitness cost to its insect carrier [2]. Moreover, a thorough risk assessment to the environment is required prior to introduction in the field [17]. These assessments include investigating the transfer routes of GMOs, which can be vertical (from mother to offspring), transstadial (between developmental stages), and horizontal (from one individual to another, without being parental), as well as from the effects of the GMO on behavior, survival, and reproduction of potential hosts [10, 11].
The horizontal transfer of the GMO between organisms can occur through the sharing of common resources [10, 18, 19], for example, by water contaminated with the GMO [12]. Normative institutions, such as CTNBio (National Technical Commission of Biosafety, Ministry of Science, Technology and Innovation, Brazil), also require the assessment of the interaction of paratransgenic individuals with the environment, which includes a particular concern regarding the possible harmful effects of GMO on non-target organisms [17], including pollinators [20]. Since bees, as pollinators, play a significant role in maintaining biodiversity [21, 22], these organisms are widely recognized for integrating risk assessment protocols [23–26].
Forager bees perform out-colony tasks, including the search for food resources (i.e., water, fiber, resin, nectar, and pollen), which are in direct contact with the external environment [27]. Therefore, risk assessments are preferably carried out on foragers and include assessments of lethality and, to a lesser extent, sublethal effects [28, 29]. Such assessment studies are mostly carried out with the honey bee Apis mellifera as a model organism, but this species is exotic in Neotropical environments such as South America [29, 30]. In this sense, stingless bees (Meliponini) are more representative of Neotropical ecosystems [22, 29] as pollinators of native and cultivated plants [31]. Therefore, stingless bees should be considered in studies to measure the potential risks of GMOs.
The ability of bees to withstand environmental stressors is linked to the gut microbiota [32, 33]. In addition, the gut microbiota can influence the behavior, metabolism, growth, and development of hosts [34, 35]. The microbiota is highly conserved among several species of stingless bees [36]. The stingless bee Partamona helleri (Meliponini) has a wide range of dominant bacterial genera (approximately 33), and the genus Serratia has also been found in this species [8]. Certain Serratia species can also be pathogenic to bees, as was observed for the S. marcescens sicaria strain (Ss1) in honeybee adults [37].
This work evaluated the risk to adult P. helleri workers of ingestion of the genetically modified Serratia eGFP AS1, carrying anti-Plasmodium effectors. We investigated survival, food ingestion (i.e., feeding rate), and walking and flight activities of adults as it relates to the evaluation of safety to the environment.