**Thermal Stress and Thermoregulation During Feeding in Mosquitoes**

Chloé Lahondère and Claudio R. Lazzari

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56288

## **1. Introduction**

[111] Vythilingam I, Foo LC, Chiang GL, Chan ST, Eng KL, Mahadevan S, et al. The impact of permethrin impregnated bednets on the malaria vector *Anopheles maculatus* (Dip‐ tera: Culicidae) in aboriginal villages of Pos Betau Pahang, Malaysia. Southeast Asian

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[114] Chareonviriyaphap T, Bangs MJ, Ratanatham S. Status of malaria in Thailand. South‐ east Asian Journal of Tropical Medicine and Public Health 2000;31:225-37.

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apmenorg/storage/newsmedia/Vietnam assessed 18 Sept 2012.

1985;79:677-80.

510 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Many arthropods have acquired the ability to use the blood of endothermic vertebrates as their main or even unique food. Among insects, haematophagy has evolved independently in different groups [1], which have converged to this way of life under strong selective pressures that modelled many morphological, physiological and behavioural traits.

Blood is a rich source of nutrients and, except for the possible presence of parasites, otherwise sterile. However, being haematophagous is a risky task, as the food circulates inside vessels hidden beneath the skin of mobile hosts, able to defend themselves from biting or even predate on blood-sucking species. Thus, in order to minimize the contact with the host, blood-sucking insects need to pierce the host-skin without being noticed and gather blood in relatively high amounts and as quick as possible. Large blood-meals produce a strong osmotic misbalance at its ingestion and toxic metabolites as by products of its digestion. In addition, the rapid ingestion of a fluid which temperature can exceed that of the insects by 20°C or more and account for many times the insect's own body weight also implies a rapid transfer of heat into the insect's body. Thus, the inner temperature of the insect could exceed the physiological limits of certain functions, causing deleterious effects [2]. Numerous studies report the impact of temperature on different behavioural [3] and physiological processes such as development [4-6], metabolism [7, 8], blood-feeding and reproduction [9] of mosquitoes and insects in general.

Thermal stress may not only affect the insect itself but also its symbiotic flora [10-12] and the parasites that it transmits with an important impact on vector infectivity [13-15]. Finally, heat constitutes a main cue to find a food source (*i.e.* a warm-blooded vertebrate). Consequently, a recently fed insect could be exposed to cannibalism if its body temperature is higher than that

© 2013 Lahondère and Lazzari; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Lahondère and Lazzari; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of the surrounding environment, facilitating the horizontal transmission of parasites between vectors [16-17].

Provided their ectothermic nature, as well as their ability to colonize all kind of habitats, insects must cope with highly variable temperatures. Therefore, many insect species have developed particular physiological and behavioural mechanisms and strategies to avoid the risk to be submitted to thermal stress [18, 19]. To avoid the effect of environmental heat, insects can seek for fresher environments or adjust their water loss to increase evaporation. In the case of haematophagous insects such as mosquitoes, they must in addition confront the exposition to thermal stress at each feeding event.

The problem of heat transfer between hosts and blood-sucking insects during blood feeding remained largely overlooked until recently, when unexpected physiological mechanisms against thermal stress were unravelled in mosquitoes. We present in this chapter a brief account of these findings and the perspectives that they open in both, fundamental and applied research.

## **2. Thermal stress and protective strategies in** *Anopheles*

The first evidences of thermal stress during feeding in haematophagous insects were obtained only recently [20]. The variation of the temperature of the body during the feeding process was measured in different species of blood-sucking insects, including two mosquitoes, *Aedes aegypti* and *Anopheles gambiae* using thermocouples. As soon as feeding begins, a steady increase of the body temperature occurs, reaching peak values of up to +10°C a few minutes later. After feeding, the temperature decreases gradually to come back similar to the environ‐ mental one. Depending on the values of environmental temperature, which is the initial temperature of the insect, and that of the blood, the amplitude and dynamics of heating and cooling vary.

Physiological responses of insects to heat include molecular changes, as is a rapid increase in the level of heat shock proteins (Hsps), which have a role as molecular chaperones that preserve the function of enzymes and other critical proteins [20]. More than a dozen Hsps are synthe‐ tized after exposure to high temperature, being the Hsp70 the most widely recognised as associated to thermal and other stresses. As in many other organisms, mosquito Hsp70s have been shown to increase during environmental stress [21, 22].

Benoit and co-workers [20] showed that, correlated with feeding and the associated elevation of the body temperature, a synthesis of heat-shock proteins occurs in *Aedes aegypti* in the few hours following a blood meal, in particular of Hsp70. In this species, the Hsp70 synthesis peaks 1 hour after feeding, reaching maximal expression in the mosquito midgut, where the relative amount of Hsp70 increases about 7 times after feeding. Similar increases in Hsp70 were showed immediately after blood feeding in *Culex pipiens* and in *Anopheles gambiae*, as well as in the bed bug *Cimex lectularius*. Nevertheless this increase, measured as the relative increase of mRNA by Northern blot, is not identical in the three mosquito species. Whereas in *Aedes aegypti* and *Culex pipiens* the relative level increases between nine and ten times, in *Anopheles gambiae* only three times. This last result is particularly interesting, since it is probable that the last named species would be less submitted to thermal stress, as we will discuss in the following sections.

## **3. Heterothermy during feeding in** *Anopheles*

of the surrounding environment, facilitating the horizontal transmission of parasites between

Provided their ectothermic nature, as well as their ability to colonize all kind of habitats, insects must cope with highly variable temperatures. Therefore, many insect species have developed particular physiological and behavioural mechanisms and strategies to avoid the risk to be submitted to thermal stress [18, 19]. To avoid the effect of environmental heat, insects can seek for fresher environments or adjust their water loss to increase evaporation. In the case of haematophagous insects such as mosquitoes, they must in addition confront the exposition to

The problem of heat transfer between hosts and blood-sucking insects during blood feeding remained largely overlooked until recently, when unexpected physiological mechanisms against thermal stress were unravelled in mosquitoes. We present in this chapter a brief account of these findings and the perspectives that they open in both, fundamental and applied

The first evidences of thermal stress during feeding in haematophagous insects were obtained only recently [20]. The variation of the temperature of the body during the feeding process was measured in different species of blood-sucking insects, including two mosquitoes, *Aedes aegypti* and *Anopheles gambiae* using thermocouples. As soon as feeding begins, a steady increase of the body temperature occurs, reaching peak values of up to +10°C a few minutes later. After feeding, the temperature decreases gradually to come back similar to the environ‐ mental one. Depending on the values of environmental temperature, which is the initial temperature of the insect, and that of the blood, the amplitude and dynamics of heating and

Physiological responses of insects to heat include molecular changes, as is a rapid increase in the level of heat shock proteins (Hsps), which have a role as molecular chaperones that preserve the function of enzymes and other critical proteins [20]. More than a dozen Hsps are synthe‐ tized after exposure to high temperature, being the Hsp70 the most widely recognised as associated to thermal and other stresses. As in many other organisms, mosquito Hsp70s have

Benoit and co-workers [20] showed that, correlated with feeding and the associated elevation of the body temperature, a synthesis of heat-shock proteins occurs in *Aedes aegypti* in the few hours following a blood meal, in particular of Hsp70. In this species, the Hsp70 synthesis peaks 1 hour after feeding, reaching maximal expression in the mosquito midgut, where the relative amount of Hsp70 increases about 7 times after feeding. Similar increases in Hsp70 were showed immediately after blood feeding in *Culex pipiens* and in *Anopheles gambiae*, as well as in the bed bug *Cimex lectularius*. Nevertheless this increase, measured as the relative increase of mRNA by Northern blot, is not identical in the three mosquito species. Whereas in *Aedes aegypti* and

**2. Thermal stress and protective strategies in** *Anopheles*

been shown to increase during environmental stress [21, 22].

vectors [16-17].

research.

cooling vary.

thermal stress at each feeding event.

512 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

To better understand to what extent mosquitoes are exposed to thermal stress during feeding, we recently conducted a real-time infrared thermographic analysis of the evolution of the body temperature of *Anopheles stephensi* during feeding on live hosts at different skin temperatures and using an artificial feeder [23].

Thermal imaging analysis has first revealed that during feeding, the different regions of the mosquito's body exhibited different temperatures. When *Anopheles stephensi* fed on mice or human volunteers, their head temperature remained close to that of the ingested blood while the abdomen temperature stayed closer to that of the ambient temperature (Figure 1). The thermal profile along a mosquito's body during feeding, notwithstanding the exact tempera‐ ture of the host skin, can be summarized as in this: *T°head > T°thorax > T°abdomen*. The fact of main‐ taining different temperatures in different regions of the body by an animal is named "regional heterothermy" and it is common in vertebrates living in cold aquatic or terrestrial environ‐ ments. When the body temperature changes with time, this condition is called "temporal heterothermy". A combination of both types of heterothemy is frequently found in insects that perform pre-heating of flight muscles before taking off. By means of simultaneous isometric contractions of antagonist muscles, insects like bumble-bees and moths heat their thorax up to reach the optimal temperature for muscular work [18].

In the case of *Anopheles stephensi*, an average difference of 3.3° C between *T°head* and *T°abdomen* was measured when the *T°host* was 34° C and 2.2° C when *Thost* was 28° C. At the end of feeding, when mouthparts are retracted from the skin, the mosquito temperature returns rapidly to environmental temperature (ectothermy).

Infrared thermography revealed a quite different pattern of body temperature in *Aedes aegypti*. In this species, the abdominal temperature during feeding remains close to that of the host, rather than to that of the environment as in *Anopheles stephensi* [23]. On the other hand, when the two species fed on sugar solution, despite the muscular activity of the ingestion pump, no heterothermy occurs: the temperature of the whole body remained that of the environment. As a consequence males, which don't feed on blood, exhibit a typically ecto‐ thermic thermal profile even when resting on a warm host, demonstrating that heating is only due to blood ingestion and not to the proximity of the host [23].

## **4. Prediuresis and drop-keeping**

During blood feeding, most haematophagous species excrete drops of fluid, a process referred in mosquitoes as "prediuresis". The physiological function of prediuresis has been related to

**Figure 1.** Thermographic image of an *Anopheles stephensi* female at the beginning of feeding on an anesthetized mouse (*T°host* = 28° C, *T°environment* = 22° C). The temperature of the head is very close to the mouse one and a tempera‐ ture gradient along the mosquito body can be observed (i.e., heterothermy).

erythrocytes concentration and elimination of water excess. The eliminated fluid is in most insects composed of urine, but in some blood-sucking species, such as mosquitoes and sandflies, it also contains fresh ingested blood that gives to the drop a bright red appearance. In mosquitoes, which feed not only on vertebrate blood, but also on nectar, prediuresis occurs during blood-feeding but it is rare or absent when they take a sugar meal.

In *Anopheles stephensi*, notwithstanding the nature of the host, blood-feeding almost always proceeds in a similar way: drops of fluid start being excreted during the first or second minute after the insect begins to feed. Frequently, a drop remains attached to the end of the abdomen for several minutes, increasing its size during feeding. Eventually the drop felt, and a new one is emitted and retained at the abdomen's end. The number of drops produced until complete gorging may vary.

Real-time thermography revealed that when *Anopheles stephensi* performs prediuresis and keeps a drop attached to its anus, a transient fall of 2° C or more of the abdominal tem‐ perature occurs and the characteristic heterothermy along its body becomes even more pronounced (Figure 2). The same phenomenon was observed in females of this species feeding in mice, human volunteers or using an artificial feeder [23]. Besides, when ingest‐ ing blood at the same temperature, the abdominal temperature of drop-keepers is signifi‐ cantly lower than that of mosquitoes that just perform prediuresis but that do not keep drops. These results demonstrate the existence of a physical cooling process in *Anopheles stephensi*. Conversely, drop-keeping was never observed to occur in *Aedes aegypti* among the individuals producing pre-urine while feeding, even if the frequency of prediuresis is the same in both mosquito species [23]*.*

**Figure 2.** Thermographic image of the same *Anopheles stephensi* female as Figure 1, but during prediuresis. The mos‐ quito performs evaporative cooling. The retention of the fluid drop attached to the abdomen end leads to a fall of the abdomen temperature causing a clear temperature gradient along the mosquito body. The colour of the droplet does not reflect the real temperature, because of the difference in the emissivity between the cuticle of the mosquito and the drop surface.

## **5. Thermoregulation in** *Anopheles*

erythrocytes concentration and elimination of water excess. The eliminated fluid is in most insects composed of urine, but in some blood-sucking species, such as mosquitoes and sandflies, it also contains fresh ingested blood that gives to the drop a bright red appearance. In mosquitoes, which feed not only on vertebrate blood, but also on nectar, prediuresis occurs

**Figure 1.** Thermographic image of an *Anopheles stephensi* female at the beginning of feeding on an anesthetized mouse (*T°host* = 28° C, *T°environment* = 22° C). The temperature of the head is very close to the mouse one and a tempera‐

In *Anopheles stephensi*, notwithstanding the nature of the host, blood-feeding almost always proceeds in a similar way: drops of fluid start being excreted during the first or second minute after the insect begins to feed. Frequently, a drop remains attached to the end of the abdomen for several minutes, increasing its size during feeding. Eventually the drop felt, and a new one is emitted and retained at the abdomen's end. The number of drops produced until complete

Real-time thermography revealed that when *Anopheles stephensi* performs prediuresis and keeps a drop attached to its anus, a transient fall of 2° C or more of the abdominal tem‐ perature occurs and the characteristic heterothermy along its body becomes even more pronounced (Figure 2). The same phenomenon was observed in females of this species feeding in mice, human volunteers or using an artificial feeder [23]. Besides, when ingest‐

during blood-feeding but it is rare or absent when they take a sugar meal.

ture gradient along the mosquito body can be observed (i.e., heterothermy).

514 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

gorging may vary.

Many insects, in particular those having easy access to water, produce and retain drops of fluid, such as nectar, honey-dew, water or urine, depending on species, which evaporates in contact with the air, causing heat loss by evaporative cooling and the consequent decrease of the temperature of the insect body. Evaporative cooling constitutes an adaptive and effective response to risks associated to high temperature and has been observed in different groups of insects [24, 25].

This decrease of temperature helps them to avoid the deleterious physiological consequences of thermal stress. Some insects such as honeybees and bumblebees produce heat with their thoracic muscles while flying (endothermy) and regurgitate a droplet of nectar through their mouthparts to cool down their head, thus keeping the brain safe from overheating [26, 27]. Moths emit fluid, which is retained on the proboscis to refresh their head whereas others, like aphids, excrete honey-dew through their anus that consequently refresh their abdomen. The recorded loss of temperature is between 2 and 8° C depending on species [28].

**Figure 3.** Evolution of the body temperatures of *Anopheles stephensi* during feeding on an anesthetized mouse. The arrow indicates the excretion of a droplet. (*T°host* = 35.5° C, *T°environment* = 28° C)

In *Anopheles* mosquitoes, the abdominal temperature of drop-keepers decreases of about 2° C during drop retention. For mosquitoes and in general for all haematophagous insects that need to manage an excess of water into their body during feeding and keep a well-adjusted water balance, evaporative cooling represents an efficient protective mechanism against overheating. To what extent prediuresis and drop-keeping occurs is variable and it is known that the rate of production and the size of the droplets excreted in mosquitoes during prediuresis differ not only between species but also within the same species, as also differs the amount of erythro‐ cytes from the ingested blood [29].

**Figure 4.** Sequence of thermographic images showing the production of a drop during feeding and the subsequent cooling of the abdomen in an *Anopheles stephensi* female. The insect fed on a human host (*T°host* = 36° C, *T°environment* = 23° C). Images were taken every 5 seconds.

## **6. A novel significance of prediuresis**

the temperature of the insect body. Evaporative cooling constitutes an adaptive and effective response to risks associated to high temperature and has been observed in different groups of

This decrease of temperature helps them to avoid the deleterious physiological consequences of thermal stress. Some insects such as honeybees and bumblebees produce heat with their thoracic muscles while flying (endothermy) and regurgitate a droplet of nectar through their mouthparts to cool down their head, thus keeping the brain safe from overheating [26, 27]. Moths emit fluid, which is retained on the proboscis to refresh their head whereas others, like aphids, excrete honey-dew through their anus that consequently refresh their abdomen. The

**Figure 3.** Evolution of the body temperatures of *Anopheles stephensi* during feeding on an anesthetized mouse. The

In *Anopheles* mosquitoes, the abdominal temperature of drop-keepers decreases of about 2° C during drop retention. For mosquitoes and in general for all haematophagous insects that need to manage an excess of water into their body during feeding and keep a well-adjusted water balance, evaporative cooling represents an efficient protective mechanism against overheating.

arrow indicates the excretion of a droplet. (*T°host* = 35.5° C, *T°environment* = 28° C)

recorded loss of temperature is between 2 and 8° C depending on species [28].

insects [24, 25].

516 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

Even though the occurrence of prediuresis and the elimination of fresh blood have been largely reported, it has been always considered just a way of concentrating erythrocytes and reducing the insect weight for take-off [30]. Nevertheless, two puzzling aspects of prediuresis in mosquitoes remained unsolved. The first one is the elimination during feeding of some of the just ingested blood containing erythrocytes [29]. It is widely accepted that strong selective pressures made blood-sucking insects minimize their contact time with a host in order to reduce the risk of being predated [1]. Thus, throwing away some of the food they ingest appears, at first glance, as a maladaptive strategy. From a point of view of thermoregulation, however, this "waste" makes sense, since it allows a quick increase in the volume (and evaporative surface) of the droplet and perhaps the surface properties of the drop, influencing its retention. Thus, the excretion of fresh blood during feeding in mosquitoes can be explained in terms of an adaptive response of evaporative cooling when exposed to thermal stress associated to feeding.

The second puzzling aspect of prediuresis is that not all mosquito species perform it. In fact, it has been shown that species that perform prediuresis need more time to reach repletion during a blood meal than species that do not produce pre-urine [31, 32]. Thus, the production of pre-urine could be seen, again, as a maladaptive strategy. However, an increase in feeding time could represent a trade-off between feeding quickly and avoiding overheating in species that are particularly sensitive to thermal stress. Others may be less sensitive or, as *Aedes* minimize the consequences of thermal stress by synthetizing more heat-shock proteins as, for example, *Anopheles* mosquitos.

Drop-keeping as evaporative cooling mechanism is in accordance with the particular position adopted by *Anopheles* species, which keep their abdomen away from the host surface. This causes the drop to be more exposed to the ambient air facilitating evaporation and cooling, and also avoiding the drop to be lost by contact with the host skin.

## **7. Thermoregulation and pathogens transmission**

When anopheline mosquitoes ingest a blood meal from an infected host, mature and functional *Plasmodium* gametocytes are present in the erythrocytes and undergo differentiation in the mosquito midgut, a process that is influenced by temperature. Indeed, high temperatures negatively affect early stages of the parasite life cycle and no exflagellation occurs above 30° C, holding parasites in an inactive state [14]. Later processes such as ookinete formation or migration of sporozoites towards the salivary glands are also influenced by temperature [15, 33, 34]. Furthermore, it has been well demonstrated that different species of *Plasmodium* are thermo-sensitive and that temperature has a direct impact on the incubation period of parasites in the mosquito [13]. On the other hand, the proliferation and dispersion of flaviviruses in *Aedes* mosquitoes is also under the influence of temperature but contrary to *Plasmodium*, this latter constitutes one of the most important factor positively influencing the extrinsic incuba‐ tion period (*EIP*). It has been shown that high temperatures are important for flaviviruses, acting on the rate of viral multiplication and consequently on the vector competence [35, 36, 37].

Moreover, *Plasmodium* parasites have to cope with the formation of the peritrophic matrix that follows each blood meal, which restrain their penetration through the gut wall [38, 39]. During the process of differentiation, *Plasmodium* ookinetes have to cross the peritrophic matrix and the midgut epithelium, before they turn into oocysts [40]. The time needed for the formation of the peritrophic matrix positively correlates with the vectorial capacity of mosquitoes, taking a longer time in *Anopheles* species than in species of *Aedes* or *Culex* [1, 41]. Thus, for *Plasmodi‐ um* parasites, insect's heterothermy could represent an important advantage, since when they enter into the mosquito's body, they are exposed to a rapid fall in temperature, which could immediately trigger exflagellation. Parasites could therefore penetrate the gut wall before the peritrophic matrix is fully formed.

pressures made blood-sucking insects minimize their contact time with a host in order to reduce the risk of being predated [1]. Thus, throwing away some of the food they ingest appears, at first glance, as a maladaptive strategy. From a point of view of thermoregulation, however, this "waste" makes sense, since it allows a quick increase in the volume (and evaporative surface) of the droplet and perhaps the surface properties of the drop, influencing its retention. Thus, the excretion of fresh blood during feeding in mosquitoes can be explained in terms of an adaptive response of evaporative cooling when exposed to thermal stress

The second puzzling aspect of prediuresis is that not all mosquito species perform it. In fact, it has been shown that species that perform prediuresis need more time to reach repletion during a blood meal than species that do not produce pre-urine [31, 32]. Thus, the production of pre-urine could be seen, again, as a maladaptive strategy. However, an increase in feeding time could represent a trade-off between feeding quickly and avoiding overheating in species that are particularly sensitive to thermal stress. Others may be less sensitive or, as *Aedes* minimize the consequences of thermal stress by synthetizing more heat-shock proteins as, for

Drop-keeping as evaporative cooling mechanism is in accordance with the particular position adopted by *Anopheles* species, which keep their abdomen away from the host surface. This causes the drop to be more exposed to the ambient air facilitating evaporation and cooling,

When anopheline mosquitoes ingest a blood meal from an infected host, mature and functional *Plasmodium* gametocytes are present in the erythrocytes and undergo differentiation in the mosquito midgut, a process that is influenced by temperature. Indeed, high temperatures negatively affect early stages of the parasite life cycle and no exflagellation occurs above 30° C, holding parasites in an inactive state [14]. Later processes such as ookinete formation or migration of sporozoites towards the salivary glands are also influenced by temperature [15, 33, 34]. Furthermore, it has been well demonstrated that different species of *Plasmodium* are thermo-sensitive and that temperature has a direct impact on the incubation period of parasites in the mosquito [13]. On the other hand, the proliferation and dispersion of flaviviruses in *Aedes* mosquitoes is also under the influence of temperature but contrary to *Plasmodium*, this latter constitutes one of the most important factor positively influencing the extrinsic incuba‐ tion period (*EIP*). It has been shown that high temperatures are important for flaviviruses, acting on the rate of viral multiplication and consequently on the vector competence [35, 36, 37]. Moreover, *Plasmodium* parasites have to cope with the formation of the peritrophic matrix that follows each blood meal, which restrain their penetration through the gut wall [38, 39]. During the process of differentiation, *Plasmodium* ookinetes have to cross the peritrophic matrix and the midgut epithelium, before they turn into oocysts [40]. The time needed for the formation of the peritrophic matrix positively correlates with the vectorial capacity of mosquitoes, taking

and also avoiding the drop to be lost by contact with the host skin.

**7. Thermoregulation and pathogens transmission**

associated to feeding.

518 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

example, *Anopheles* mosquitos.

From an evolutionary point of view, it makes sense that *Plasmodium* parasites take an advant‐ age to be associated with species that undergo evaporative cooling, protecting them from lethal temperatures. On the other hand, flaviviruses associated with non drop-keeper species would benefit from a necessary warmer environment.

Evaporative cooling could also protect from heat stress the symbiotic microorganisms as‐ sociated to mosquitoes and that can play an important role in haematophagous insects [10]. *Asaia* bacteria have been found in high density in the gut of *Anopheles stephensi* fe‐ males as well as in ovaries [11]. Recently many genera have been identified in the midgut of natural populations of *Anopheles gambiae* [42]. In particular, the abundance of *Enterobac‐ teriaceae* in the mosquito midgut has been found to correlate significantly with the *Plasmo‐ dium* infection status [42].

## **8. Thermoregulation and thermotolerance in mosquitoes**

Finally, it is possible to speculate on two further implications of our interpretation of the functionality of prediuresis as thermoregulatory mechanisms. The first one concerns how environmental temperature may affect the survival of less thermotolerant mosquitoes. If we consider that the species that perform evaporative cooling could be more sensitive to heat, any change in the environmental temperature, due to local or global warming, would have a higher impact on them than on species that do not perform it, as for example *Culex spp.* that feed quickly and do not perform prediuresis while feeding [43]. It can be predicted that such species have been selected to reduce the contact time with their host and consequently to be more thermotolerant to temperature increases. Indeed, *Aedes aegypti* and its ability to produce Hsps represent an example of this [20].

The second implication of our finding is related to the control of mosquito populations. Prediuresis has deeper physiological consequences than just diuresis. In addition to excre‐ tion, it implies blood concentration and thermoregulation. The exploitation of the knowl‐ edge about excretion physiology to control disease vector insects by interfering with the function of Malpighian tubules has been already proposed for other haematophagous in‐ sects [44], and the same can be expected for mosquitoes. In this case, blocking or delay‐ ing the production of urine would have a double impact on disease transmission by affecting microorganisms transmitted by prediuresis [45, 46] and/or affecting the survival of mosquitoes exposed to overheating.

## **9. Conclusion**

*Anopheles* mosquitoes are capable to perform thermoregulation by evaporative cooling during blood intake. This mechanism protects the insect itself, as well as the associated microorgan‐ isms (both symbionts and parasites) from thermal stress. Thus, prediuresis which plays such different roles in the mosquito physiology, appears one more time as an interesting possible target for the control of disease vectors.

## **Acknowledgements**

We are very grateful to Catherine Bourgouin and the CEPIA staff (Institut Pasteur, France) for providing us anopheline mosquitoes and rearing advices as well as Rogerio Amino (Institut Pasteur, France) for his valuable comments on the manuscript and helpful discussions. We also thank Fabrice Chandre and Marie-Noelle Lacroix (IRD Montpellier, France) for providing us *Aedes aegypti* eggs. This work received financial support from ANR (EcoEpi), CNRS and the University of Tours (France).

## **Author details**

Chloé Lahondère and Claudio R. Lazzari\*

\*Address all correspondence to: claudio.lazzari@univ-tours.fr

Institut de Recherche sur la Biologie de l'Insecte, UMR CNRS - Université François Rabelais, Tours, France

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**9. Conclusion**

target for the control of disease vectors.

520 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**Acknowledgements**

University of Tours (France).

Chloé Lahondère and Claudio R. Lazzari\*

New York. 2nd ed.; 2005.

\*Address all correspondence to: claudio.lazzari@univ-tours.fr

Bulletin of Entomological Research 2004; 94 441-448.

Bulletin of Entomological Research 1938; 125-140.

**Author details**

Tours, France

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*Anopheles* mosquitoes are capable to perform thermoregulation by evaporative cooling during blood intake. This mechanism protects the insect itself, as well as the associated microorgan‐ isms (both symbionts and parasites) from thermal stress. Thus, prediuresis which plays such different roles in the mosquito physiology, appears one more time as an interesting possible

We are very grateful to Catherine Bourgouin and the CEPIA staff (Institut Pasteur, France) for providing us anopheline mosquitoes and rearing advices as well as Rogerio Amino (Institut Pasteur, France) for his valuable comments on the manuscript and helpful discussions. We also thank Fabrice Chandre and Marie-Noelle Lacroix (IRD Montpellier, France) for providing us *Aedes aegypti* eggs. This work received financial support from ANR (EcoEpi), CNRS and the

Institut de Recherche sur la Biologie de l'Insecte, UMR CNRS - Université François Rabelais,

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[20] Benoit JB, Lopez-Martinez G, Patrick KR, Phillips ZP, Krause TB, Denlinger, DL. Drinking a hot blood meal elicits a protective heat shock response in mosquitoes. Proceeding of the National Academy of Science of the United States of America 2011;

[21] Gross TL, Myles KM, Adelman ZN. Identification and characterization of heat shock 70 genes in *Aedes aegypti* (Diptera: Culicidae). Journal of Medical Entomology 2009;

[22] Benoit JB, Lopez-Martinez G, Phillips ZP, Patrick KR, Denlinger DL. Heat shock pro‐ teins contribute to mosquito dehydration tolerance. Journal of Insect Physiology

[23] Lahondère C, Lazzari CR. Mosquitoes cool down during blood feeding to avoid

[24] Adams PA, Heath JE. An evaporative cooling mechanism in *Pholus achemon*. Journal

[25] Mittler TE. The excretion of honey-dew by *Tuberolachnus salignus* (Gmelin) (Homo‐ ptera: Aphididae). Proceeding of the Royal Entomological Society, Series A General

[26] Heinrich B. Heat-exchange in relation to blood-flow between thorax and abdomen in

[27] Heinrich B. Keeping a cool head - honeybee thermoregulation. Science 1979; 205

[28] Prange HD. Evaporative cooling in insects. Journal of Insect Physiology 1996; 42

[29] Chege GMM, Beier JC Blood acquisition and processing by three *Anopheles* (Diptera: Culicidae) species with different innate susceptibilities to *Plasmodium falciparum*.

[30] Briegel H, Rezzonico L. Concentration of host blood protein during feeding by Ano‐ pheline mosquitoes (Diptera, Culicidae). Journal of Medical Entomology 1985; 22

[31] Vaughan JA, Noden BH, Beier JC. Concentration of human erythrocytes by Anophe‐ line mosquitoes (Diptera, Culicidae) during feeding. Journal of Medical Entomology

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46 496–504.

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[46] Blow JA, Turell MJ, Walker ED, Silverman AL. Post-bloodmeal diuretic shedding of hepatitis B virus by mosquitoes (Diptera: Culicidae). Journal of Medical Entomology 2002; 39 605-612.

## **The** *Anopheles* **Mosquito Microbiota and Their Impact on Pathogen Transmission**

Mathilde Gendrin and George K. Christophides

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55107

## **1. Introduction**

[46] Blow JA, Turell MJ, Walker ED, Silverman AL. Post-bloodmeal diuretic shedding of hepatitis B virus by mosquitoes (Diptera: Culicidae). Journal of Medical Entomology

2002; 39 605-612.

524 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

An ecosystem is composed of a biological community and its physical environment. A unique ecosystem is the metazoan digestive tract, which contains and interacts with many microor‐ ganisms, e.g. a single human gut contains 1013-1014 bacteria belonging to hundreds of species [4, 5]. These microorganisms are important for the host physiology, particularly in shaping the mucosal immune system [6] and protecting the host against infections by colonization resistance [7].

The term microbiota defines the microbial communities that live in contact with the body epithelia. They are composed of bacteria, viruses, yeasts and protists. To date, the bacterial component of the microbiota is the most studied and best characterized. Studies from *Drosophila* to mice have revealed that the microbial flora is tightly regulated by the immune system and that failures in this can have detrimental effects on the host [8, 9]. The microbiota composition and numbers undergo significant changes during a host's lifetime, in particular upon changes of the environment and feeding habits.

*Anopheles* mosquitoes are of great importance to human health. They transmit pathogens including malaria parasites, filarial worms and arboviruses (arthropod-borne viruses). These pathogens infect the mosquito gut when ingested with a bloodmeal, disseminate through the hemolymph (insect blood) to other tissues and are transmitted to a new human host upon another mosquito bite some days later. The time pathogens spend in mosquitoes is known as extrinsic incubation period. The malaria parasite, *Plasmodium*, undergoes sexual reproduction in the midgut lumen and develops into a motile form that, approximately 24h after infection, traverses the gut epithelium establishing an infection on the basal side that is bathed in the hemolymph [10]. A week to 10 days later, parasites travel to the salivary glands where they become infectious to man. Similarly, after shedding their protective sheath in the mosquito midgut lumen, the elephantiasis nematodes *Wuchereria* and *Brugia* microfilariae migrate

© 2013 Gendrin and Christophides; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Gendrin and Christophides; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

through the midgut epithelium to the thoracic muscles where they embark on larval devel‐ opment [11]. Some 10-14 days later, infectious larvae emerge from the mosquito cuticle or the proboscis and infect the human host via a skin wound, such as that caused by the mosquito bite. The O'Nyong Nyong virus (ONNV), the only arbovirus known to be transmitted exclusively by *Anopheles*, mosquitoes infects the muscle bands of the midgut and other visceral tissues after dissemination from infected gut cells [12, 13]. The next steps of the virus migration through the mosquito are not well characterized but it is thought that, as shown for its cousin Chikungunya virus, it infects the salivary glands from where it can be transmitted to the human host. Thus, for all three types of pathogens, the *Anopheles* mosquito midgut is an obligatory gateway to infection and transmission.

The mosquito gut microbiota has recently emerged as an important factor of resistance against pathogens. In particular, midgut bacteria have been shown to have a substantial negative impact on malaria parasite burden through colonization mechanisms involving either direct *Plasmodium*-microbiota interactions or bacteria-mediated induction of the mosquito immune response [1, 2, 14]. Equivalent effects of the microbiota on infection with the Dengue virus and *Brugia* microfilariae are shown in the mosquito *Aedes aegypti* [15-17]. Therefore, the research field of mosquito microbiota has received great attention in the last years and new concepts of microbiota-mediated transmission blocking are currently investigated. These studies face an important challenge: the microbiota of a female mosquito changes considerably as the mosquito shift environments during metamorphosis, from the aqueous developing larva to an air-living adult, and yet during adulthood as its feeding behaviour alternates between flower-nectar feeding and blood feeding [18, 19]. The diversity of the bacterial community is shown to decrease during mosquito development and after the first bloodmeal, whereas bacteria massively proliferate, with a 10 to 900-fold increase registered 24h to 30h after a bloodmeal [18, 20, 21].

In this chapter, we provide an overview on the current knowledge of the composition of the *Anopheles* mosquito microbiota, including important findings from recent high-throughput sequencing studies. We then review studies about the impact of the microbiota on mosquito physiology and infection, focusing in particular on resistance to infection by human pathogens. Finally, we discuss the potential use of this knowledge toward reducing the mosquito vectorial capacity and transmission blocking.

## **2. The diversity of the** *Anopheles* **microbiota**

The microbiota composition has been studied in several anophelines mainly by culturing or sequencing of the 16S rRNA [14, 18, 20, 22-41]. Together, studies on field-collected or labora‐ tory-reared mosquitoes identified as many as 98 bacterial genera excluding genera of low abundance identified by high-throughput sequencing analyses (Table 1). Of these, 41 genera were found in more than one *Anopheles* species while 9 were reported in at least 7 of these 23 studies and thus appear to be frequently associated with *Anopheles*. *Pseudomonas* was the most frequent of those genera, detected in 16 studies, followed by *Aeromonas*, *Asaia*, *Comamonas, Elizabethkingia*, *Enterobacter, Klebsiella*, *Pantoea* and *Serratia*, detected in 7-10 studies. No single bacterial genus was found in all the studies, even if culture-dependent studies are not consid‐ ered – as culturing techniques might be an issue. Thus, there is presumably no obligate symbiont in the *Anopheles* genus, as is the case of some other blood-sucking insects such as the Tsetse fly that hosts *Wigglesworthia spp.*, an obligatory bacterial symbiont important for fly fecundity [42] or the head louse that hosts *Riesia pediculicola* [43]. As the most frequent genera are present in both laboratory and field-collected mosquitoes, it is suggestive that laboratory colonies retain bacterial communities established prior to laboratory colonisation (Table 1 and [18]). There are, however, substantial differences between field-collected and laboratoryreared mosquitoes, as reflected by the loss of microbiota species richness in laboratory-reared mosquitoes [18, 22]. **Table1. List of the genera of bacteria associated to** *Anopheles* **mosquitoes reported in the following studies [11, 17, 19, 21-‐40].** For high-‐throughput sequencing studies, only genera found to represent at least 1% of the total population in at least one study/condition are listed. Genera are classified by phyla, which are indicated in bold. In column "Conditions", F, F\* and L indicate field, semi-‐natural and laboratory conditions, respectively. In column "Stage", L, P and A indicate larvae, pupae and adults, respectively. Column "Example" shows NCBI accession number of a sequence example for each genus (first hit after BLAST). Columns "Deep seq", "Culture", "Non culture" list studies based on 16S rRNA gene deep

sequencing, culture-‐dependent methods, conventional sequencing (including 16S rRNA gene libraries and DGGE) and gas chromatography, respectively. In the line "*Pantoea*", \* refers to what was identified in [19]

as *Enterobacter 
 agglomerans*, 
 since 
 then 
 renamed 
 *Pantoea 
 agglomerans*.

through the midgut epithelium to the thoracic muscles where they embark on larval devel‐ opment [11]. Some 10-14 days later, infectious larvae emerge from the mosquito cuticle or the proboscis and infect the human host via a skin wound, such as that caused by the mosquito bite. The O'Nyong Nyong virus (ONNV), the only arbovirus known to be transmitted exclusively by *Anopheles*, mosquitoes infects the muscle bands of the midgut and other visceral tissues after dissemination from infected gut cells [12, 13]. The next steps of the virus migration through the mosquito are not well characterized but it is thought that, as shown for its cousin Chikungunya virus, it infects the salivary glands from where it can be transmitted to the human host. Thus, for all three types of pathogens, the *Anopheles* mosquito midgut is an obligatory

The mosquito gut microbiota has recently emerged as an important factor of resistance against pathogens. In particular, midgut bacteria have been shown to have a substantial negative impact on malaria parasite burden through colonization mechanisms involving either direct *Plasmodium*-microbiota interactions or bacteria-mediated induction of the mosquito immune response [1, 2, 14]. Equivalent effects of the microbiota on infection with the Dengue virus and *Brugia* microfilariae are shown in the mosquito *Aedes aegypti* [15-17]. Therefore, the research field of mosquito microbiota has received great attention in the last years and new concepts of microbiota-mediated transmission blocking are currently investigated. These studies face an important challenge: the microbiota of a female mosquito changes considerably as the mosquito shift environments during metamorphosis, from the aqueous developing larva to an air-living adult, and yet during adulthood as its feeding behaviour alternates between flower-nectar feeding and blood feeding [18, 19]. The diversity of the bacterial community is shown to decrease during mosquito development and after the first bloodmeal, whereas bacteria massively proliferate, with a 10 to 900-fold increase registered 24h to 30h after a

In this chapter, we provide an overview on the current knowledge of the composition of the *Anopheles* mosquito microbiota, including important findings from recent high-throughput sequencing studies. We then review studies about the impact of the microbiota on mosquito physiology and infection, focusing in particular on resistance to infection by human pathogens. Finally, we discuss the potential use of this knowledge toward reducing the mosquito vectorial

The microbiota composition has been studied in several anophelines mainly by culturing or sequencing of the 16S rRNA [14, 18, 20, 22-41]. Together, studies on field-collected or labora‐ tory-reared mosquitoes identified as many as 98 bacterial genera excluding genera of low abundance identified by high-throughput sequencing analyses (Table 1). Of these, 41 genera were found in more than one *Anopheles* species while 9 were reported in at least 7 of these 23 studies and thus appear to be frequently associated with *Anopheles*. *Pseudomonas* was the most frequent of those genera, detected in 16 studies, followed by *Aeromonas*, *Asaia*, *Comamonas, Elizabethkingia*, *Enterobacter, Klebsiella*, *Pantoea* and *Serratia*, detected in 7-10 studies. No single bacterial genus was found in all the studies, even if culture-dependent studies are not consid‐

gateway to infection and transmission.

526 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

bloodmeal [18, 20, 21].

capacity and transmission blocking.

**2. The diversity of the** *Anopheles* **microbiota**



#### *Proteobacteria*



*Lactobacillus Lactobacillaceae Bacilli* FJ608053 F, F\* L, A *gambiae,* 

*Lysinibacillus Bacillaceae Bacilli* GU204964 <sup>F</sup> <sup>L</sup> *maculipennis,* 

*Paenibacillus Paenibacillaceae Bacilli* EF426449 <sup>F</sup> <sup>A</sup> *arabiensis,* 

*Staphylococcus Staphylococcaceae Bacilli* FJ608067 F, F\*, L L, A

*Streptococcus Streptococcaceae Bacilli* FJ608047 F, F\* L, A

*Proteobacteria*

*Aquabacterium*

*Acetobacter Acetobacteraceae Alpha-*

528 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*Achromobacter Alcaligenaceae Beta-*

*Acidovorax Comamonadaceae Beta-*

*Acinetobacter Moraxellaceae Gamma-*

*Aeromonas Aeromonadaceae Gamma-*

*Agrobacterium Comamonadaceae Beta-*

*Alcaligenes Alcaligenaceae Beta-*

*Anaplasma Anaplasmataceae Alpha-*

*Asaia Acetobacteraceae Alpha-*

*Azoarcus Rhodocyclaceae Beta-*

*Bordetella Alcaligenaceae Beta-*

*Bradyrhizobium Bradyrhizobiaceae Alpha-*

*Brevundimonas Caulobacteraceae Alpha-*

*Burkholderia Burkholderiaceae Beta-*

*Buttiauxella Enterobacteriaceae Gamma-*

*Cedecea Enterobacteriaceae Gamma-*

*Citrobacter Enterobacteriaceae Gamma-*

*Comamonas Comamonadaceae Beta-*

*Burkholderiales Genera incertae sedis*

*Beta-*

*stephensi* [17] [38]

*stephensi* [38, 22]

[17, 21,

*stephensi* [23] [30]

*stephensi* [24] [30]

*stephensi* [38] [33]

*stephensi* [21] [26, 27]

[21] [19, 29] [30]

[17, 21] [38, 35, 39, 40] [30]

[21, 36] [25, 38,

[21, 36] [38]

36] [19, 38] [38, 26]

23, 24] [22, 33]

28, 37] [26, 28]

[17, 36] [19, 38,

[21, 36] [11, 26-

40] [38, 26]

*stephensi* [24]

*funestus, gambiae, maculipennis, quadrimaculatus, stephensi*

*funestus, gambiae, stephensi*

*albimanus, funestus, gambiae, stephensi*

*coustani, darlingi, funestus, gambiae, maculipennis, stephensi*

*proteobacteria* <sup>L</sup> <sup>A</sup> *stephensi* [26]

*proteobacteria* FJ608301 <sup>F</sup> <sup>A</sup> *stephensi* [38]

*proteobacteria* AY837725 <sup>F</sup> <sup>A</sup> *arabiensis* [22]

*proteobacteria* FJ607997 <sup>L</sup> <sup>A</sup> *stephensi* [38] [38]

*proteobacteria* AY837739 <sup>F</sup> <sup>A</sup> *arabiensis* [22]

*proteobacteria* <sup>F</sup> <sup>A</sup> *gambiae* [26]

*proteobacteria* FJ608071 <sup>F</sup> <sup>L</sup> *stephensi* [38]

*proteobacteria* <sup>F</sup> <sup>A</sup> *darlingi* [33]

*funestus, gambiae (ss, sl), stephensi*

*dureni, funestus, gambiae, quadrimaculatus, stephensi*

*proteobacteria* HQ832874 <sup>F</sup> <sup>A</sup> *stephensi* [23]

*proteobacteria* AB740924 F\* <sup>A</sup> *gambiae* [21]

*proteobacteria* GU204962 <sup>F</sup> L, A *funestus,* 

*proteobacteria* AY391283 F, F\*, L <sup>A</sup> *gambiae,* 

*proteobacteria* FJ608234 <sup>F</sup> <sup>A</sup> *darlingi,* 

*proteobacteria* DQ068869 F, F\*, L <sup>A</sup>

*proteobacteria* EF426440 F, F\* P, A

*coustani, funestus, gambiae, maculipennis, stephensi*

*proteobacteria* FJ608267 F, F\*, L L, A

*proteobacteria* FJ608130 F, F\*, L L, A

*proteobacteria* FN821398 F, F\*, L L, A

*proteobacteria* HQ832875 <sup>F</sup> <sup>A</sup> *funestus,* 


**Table 1.** List of bacterial genera associated with *Anopheles* mosquitoes reported in the following studies: [11, 17, 19, and 21-40]. For high-throughput sequencing studies; only genera found to represent at least 1% of the total population in at least one study/condition are listed. Genera are classified by phyla, which are indicated in bold. In column "Conditions", F, F\* and L indicate field, semi-natural and laboratory conditions, respectively. In column "Stage", L, P and A indicate larvae, pupae and adults, respectively. Column "Example" shows NCBI accession number of a sequence example for each genus (first hit after BLAST). Columns "Deep seq", "Culture", "Non culture" list studies based on 16S rRNA gene deep sequencing, culture-dependent methods, conventional sequencing (including 16S rRNA gene libraries and DGGE) and gas chromatography, respectively. In the line "*Pantoea*", \* refers to what was identified in [19] as *Enterobacter agglomerans*, since then renamed *Pantoea agglomerans*.

Three metagenomics studies were recently carried out using 16S RNA from bacteria found in the *Anopheles* gut [18, 22, 37]. Wang and co-workers examined the microbiota composition throughout the mosquito life cycle, using a laboratory colony of *A. gambiae* mosquitoes (the main vector of malaria in sub-Saharan Africa) reared in semi-natural microcosms in Kenya [18]. The microcosms contained local rainwater and topsoil and were kept outside to allow microbial colonization. Boissière and co-workers investigated the microbiota of adult *A. gambiae* mosquitoes in Cameroon and how these microbiota may be related to *Plasmodium* infection [22]. They collected larvae from the field, reared them to adulthood in the laboratory and monitored the microbiota composition of individual mosquitoes 8 days after infection with *Plasmodium falciparum* sampled directly from gametocytemic patients. Finally, Osei-Poku and co-workers collected adult mosquitoes in Kenya and analysed the microbiota of individual mosquitoes of 8 different species, including 3 species of *Anopheles* (*A. coustani*, *A. funestus* and *A. gambiae*) [37].

*Pseudomonas Pseudomonadaceae*

530 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*Rahnella Enterobacteriaceae Gamma-*

*Raoultella Enterobacteriaceae Gamma-*

*Salmonella Enterobacteriaceae Gamma-*

*Serratia Enterobacteriaceae Gamma-*

*Shewanella Shewanellaceae Gamma-*

*Thorsellia Enterobacteriaceae Gamma-*

*Vibrio Vibrio Gamma-*

*Xenorhabdus Enterobacteriaceae Gamma-*

*Yersinia Enterobacteriaceae Gamma-*

*Zymobacter Halomonadaceae Gamma-*

*Sphingobium Sphingomonadaceae*

*Sphingomonas Sphingomonadaceae*

*Stenotrophomonas Xanthomonadaceae*

*Others Bacillariophyta* 

*Chlorophyta (green* 

*Rhizobium Rhizobiaceae Alpha-*

*Schlegelella Comamonadaceae Beta-*

*Ralstonia Burkholderiaceae Beta-*

*Gamma-*

*Alpha-*

*Alpha-*

*Gamma-*

*(Eukaryota: Diatom)* JQ727029 F\* <sup>L</sup> *gambiae* [17]

*algae)* EF114678 F\* <sup>L</sup> *gambiae* [17]

*Cyanobacteria-GpI* HM573452 F\* P *gambiae* [17] *Cyanobacteria-GpIIa* JQ305084 F\* L *gambiae* [17] *Cyanobacteria-GpV* AB245143 F\* L *gambiae* [17] *Fusobacterium Fusobacteriaceae Fusobacteriia* JX548360 F\* A *gambiae* [17, 21]

identified in [19] as *Enterobacter agglomerans*, since then renamed *Pantoea agglomerans*.

*Calothrix Rivulariaceae* (no data) FJ608095 F L *stephensi* [38] *Deinococcus Deinococcaceae Deinococci* FJ608089 F L *stephensi* [38] [38] *Mycoplasma Mycoplasmataceae Mollicutes* AY837724 F A *arabiensis* [22] *Spiroplasma Spiroplasmataceae Mollicutes* AY837733 F A *funestus* [22]

**Table 1.** List of bacterial genera associated with *Anopheles* mosquitoes reported in the following studies: [11, 17, 19, and 21-40]. For high-throughput sequencing studies; only genera found to represent at least 1% of the total population in at least one study/condition are listed. Genera are classified by phyla, which are indicated in bold. In column "Conditions", F, F\* and L indicate field, semi-natural and laboratory conditions, respectively. In column "Stage", L, P and A indicate larvae, pupae and adults, respectively. Column "Example" shows NCBI accession number of a sequence example for each genus (first hit after BLAST). Columns "Deep seq", "Culture", "Non culture" list studies based on 16S rRNA gene deep sequencing, culture-dependent methods, conventional sequencing (including 16S rRNA gene libraries and DGGE) and gas chromatography, respectively. In the line "*Pantoea*", \* refers to what was

*proteobacteria* EF426444 F, F\*, L L, P,

A

*proteobacteria* GU204974 <sup>F</sup> <sup>L</sup> *stephensi* [24]

*proteobacteria* AY191852 F\* <sup>A</sup> *gambiae* [21]

*proteobacteria* HQ811336 F\* <sup>A</sup> *gambiae* [17]

*proteobacteria* DQ814410 F\* <sup>L</sup> *gambiae* [17]

*proteobacteria* FR774570 F\* <sup>A</sup> *gambiae* [21]

*proteobacteria* GU940735 F\* <sup>A</sup> *gambiae* [17]

*proteobacteria* HQ591421 <sup>F</sup> <sup>L</sup> *stephensi* [23]

*proteobacteria* FJ608116 <sup>F</sup> L, A *arabiensis* [38, 22]

*proteobacteria* FJ608329 <sup>F</sup> <sup>A</sup> *stephensi* [38]

*proteobacteria* <sup>F</sup> <sup>A</sup> *darlingi* [33]

*proteobacteria* <sup>F</sup> *funestus,* 

*proteobacteria* FJ608101 F, F\*, L L, A

*proteobacteria* GU204960 F, F\*, L L, A

*proteobacteria* EF426435 F, F\* <sup>A</sup>

*proteobacteria* NR\_043217 F, F\* L, A *gambiae,* 

*proteobacteria* FR851711 <sup>F</sup> <sup>A</sup> *funestus,* 

*albimanus, darlingi, dureni, funestus, gambiae (ss, sl), maculipennis, quadrimaculatus stephensi*

*albimanus, dureni, gambiae, maculipennis,q uadrimaculatus, stephensi*

*funestus, gambiae, stephensi*

*arabiensis, funestus, gambiae*

*gambiae* [36]

[17, 21, 36]

*gambiae sl* [30]

[17, 21]

*stephensi* [17] [38, 22] [38, 34]

[11, 19, 25, 31, 37-40]

[21, 36] [11, 24] [26]

[17, 21] [35] [22, 30]

[38]

[11, 19, 22-24, 29, 35, 38, 39, 40]

[38, 26, 30, 33]

> These studies led to 5 main observations. First, the microbiota diversity is high: when defining species as OTU97%, V1-V31 , Wang et al. detected more than 2,000 species in a pool of 30 adult *A. gambiae* [18]. The highest diversity was registered in larvae and pupae, with an estimate of 4,000-8,000 species in a pool of 30 individuals of each stage. Diversity decreased during adulthood to 2,000-4,000 species upon emergence and dropped further to 600-900 species after a bloodmeal. As all of these high-throughput sequencing studies used bacterial DNA, which is a very stable molecule, an important question is whether these results genuinely reflect the *Anopheles* gut communities or include environmental contaminants. By direct sampling of the larval aquatic environment, Wang et al. indeed showed that the microbial communities differed from those in the larvae, suggesting that – at least in this study – bacteria were able to persist in, if not colonise, the mosquito host (Figure 1A).

> Second, this diversity is partially explained by significant diversity within a single mosquito [22, 37], varying from 5 to 71 OTUs97%, V3 per individual (median: 42 OTUs97%, V3) [37]. Diversity is higher than what observed by metagenomics studies in other insects such as the honeybee which hosts 8 dominant species (OTU97%, V6-V8), the estimated species richness within a colony being 9-10 [44], and *Drosophila* where 31 OTUs97%,V2 were observed in a pool of 50 females [45]. Nevertheless, a single OTU97%, V3 represents on average 67% of a mosquito bacterial community and the median mosquito gut species richness is only 17% to that of humans, where an individual hosts 150-300 OTUs99%, whole 16S [4, 37].

> Third, another component of the observed biodiversity lies within the high variability in microbial communities between individuals. This is quantified by calculating the UniFrac distance between mosquitoes. UniFrac varies from 0 when two mosquitoes have exactly the same microbiota to 1 when there is no phylogenetic overlap between the microbiota of two mosquitoes. The mean UniFrac distance between individuals is high, 0.72 and 0.74 in *A. funestus* and *A. gambiae*, respectively [37]. This variability is almost as high between *Anopheles* individuals of the same species as between mosquitoes of different species and/or genera [37].

<sup>1</sup> As not all the studies were based on the same region of 16S or the same threshold of differences, we refer here to OTU97%, V1-V3 as the operational taxonomic unit with more than 97% identity in the V1-V3 regions of 16S rRNA gene sequences.

in larval habitat and in larvae found in [17]. **B, C:** Natural habitat of *A. gambiae*. Permanent habitats such as rice fields (**B**) are colonized with M molecular form of *A. gambiae* and temporary water ponds (**C**) with S plus M forms (mostly S). **D, E:** Mosquitoes feeding on *Senna siamea* flowers (**D**) and papaya fruit - *Carica papaya* (**E**). **Figure 1.** Anopheles microbiota and environment. A: Abundance of bacterial genera in larval habitat and in larvae found in [17]. B, C: Natural habitat of *A. gambiae*. Permanent habitats such as rice fields (B) are colonized with M mo‐ lecular form of *A. gambiae* and temporary water ponds (C) with S plus M forms (mostly S). D, E: Mosquitoes feeding on *Senna siamea* flowers (D) and papaya fruit-*Carica papaya* (E).

**Figure 1.** *Anopheles* **microbiota and environment. A:** Abundance of bacterial genera

Fourth, the microbiota composition partly reflects the larval origin but bacteria acquired during adulthood may affect the microbiota composition to the extent that the geographic origin cannot be traced. Osei-Poku and co-workers did not observe any correlation between geographic location and microbiota composition in their Kenyan adult collections [37]. This is in sharp contrast to the Boissière et al. observations that microbiota were more similar between adults derived from larvae breading in the same pond than between adults derived from larvae of different geographic origins [22]. These results are, however, not contradictory if we consider differences in experimental designs of these studies. The latter study focused almost exclusively on bacteria transmitted from larvae to adults since larvae from the field were sampled and adults where fed with sterile sugar upon emergence, while the former study additionally sampled bacteria acquired during adulthood, and related to presumably diverse adult life histories. Together, these studies suggest that the acquisition of new strains of bacteria during adulthood can potentially increase the inter-individual diversity and mask similarities linked to the larval origin. However, this hypothesis requires further investigation, as mos‐ quitoes from the two geographical origins reported in the Boissière et al. study belonged to the M and S molecular forms of *A. gambiae*, respectively, which are thought to be emerging species breading in different types of aquatic environments, i.e. permanent and temporary (rain-dependent) water pools, respectively (see Figures 1B, C) [22]. These environments are likely to contain different microbiota that largely determine the mosquito enterotype. Addi‐ tionally, genetic differences between the two molecular forms may also partly account for the observed differences in microbiota composition.

Fifth, when considering the *Plasmodium* infection status, Boissière and co-workers found that the abundance of bacteria of the *Enterobacteriaceae* family was higher in *P. falciparum*-infected mosquitoes than in non-infected mosquitoes fed with the same infectious bloodmeal. This observation may indicate that *Enterobacteriaceae* favour *P. falciparum* infection or, conversely, that *P. falciparum* infection influences the composition of microbiota to the benefit of *Entero‐ bacteriaceae* [22].

## **3. Bacterial colonization of mosquitoes**

Fourth, the microbiota composition partly reflects the larval origin but bacteria acquired during adulthood may affect the microbiota composition to the extent that the geographic origin cannot be traced. Osei-Poku and co-workers did not observe any correlation between geographic location and microbiota composition in their Kenyan adult collections [37]. This is in sharp contrast to the Boissière et al. observations that microbiota were more similar between adults derived from larvae breading in the same pond than between adults derived from larvae of different geographic origins [22]. These results are, however, not contradictory if we consider differences in experimental designs of these studies. The latter study focused almost exclusively on bacteria transmitted from larvae to adults since larvae from the field were sampled and adults where fed with sterile sugar upon emergence, while the former study additionally sampled bacteria acquired during adulthood, and related to presumably diverse adult life histories. Together, these studies suggest that the acquisition of new strains of bacteria

**Figure 1.** *Anopheles* **microbiota and environment. A:** Abundance of bacterial genera in larval habitat and in larvae found in [17]. **B, C:** Natural habitat of *A. gambiae*. Permanent habitats such as rice fields (**B**) are colonized with M molecular form of *A. gambiae* and temporary water ponds (**C**) with S plus M forms (mostly S). **D, E:** Mosquitoes feeding on *Senna siamea* flowers (**D**) and papaya fruit - *Carica papaya*

**Figure 1.** Anopheles microbiota and environment. A: Abundance of bacterial genera in larval habitat and in larvae found in [17]. B, C: Natural habitat of *A. gambiae*. Permanent habitats such as rice fields (B) are colonized with M mo‐ lecular form of *A. gambiae* and temporary water ponds (C) with S plus M forms (mostly S). D, E: Mosquitoes feeding on

Genus abundance (%)

(**E**).

*Senna siamea* flowers (D) and papaya fruit-*Carica papaya* (E).

A

532 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*Klebsiella* 

*Raoultella* 

*Serratia* 

*Enterobacter* 

*Aeromonas* 

*Pseudomonas* 

*Elizabethkingia* 

*Acinetobacter* 

B C

D E

*Comamonas* 

*Propionibacterium* 

*Stenotrophomonas* 

*Bacillariophyta* 

*Thorsellia* 

*Finegoldia* 

*Chlorophyta* 

*Methylocystis* 

*Cyanobacteria-GpIIa* 

*Roseomonas* 

*Novosphingobium* 

*Aerococcus* 

*Corynebacterium* 

*Lactobacillus* 

*Cloacibacterium* 

*Rhizobium* 

*Porphyrobacter* 

*Agromyces* 

*Cyanobacteria-GpV* 

*Clostridium* 

*Hydrogenophaga* 

*Cyanobacteria-GpI* 

*Methylophilus* 

*Fusobacterium* 

*Chryseobacterium* 

Habitat Larva

*Pelagibacter* 

*Sphingobium* 

In addition to metagenomics studies, factors determining the composition of the adult mosquito microbiota were also investigated by conventional methods. Evidence that mosqui‐ toes are colonized by bacteria both found in the environment and transmitted between individuals or developmental stages was revealed, but the relative contribution of these transmission routes to the microbiota diversity remains largely unknown. Laboratory studies investigated the vertical (from parent to progeny), transstadial (between developmental stages) and horizontal (between individuals of the same stage) transmission of specific bacterial strains. In particular, horizontal transfer of *Asaia sp*. is found to occur both by feeding and by mating (from male to female), but it is yet unclear whether vertical transmission occurs via egg spreading or by contamination of the environment during egg-laying [27]. Transstadial transmission of *Pantoea stewartii* is shown to occur from larvae to pupae but not from pupae to adults [36]. This is likely due to gut sterilization during metamorphosis; bacterial counts are high in the gut of fourth instar larvae, decrease after final larval defecation, increase again during pupal development and are very low or null in newly emerged adults [46].

Two mechanisms are thought to be involved in gut sterilization during adult emergence [46]. Firstly, bacteria are enclosed in the degenerated larval midgut, the meconium, enveloped by 2 meconial peritrophic matrixes and egested during molting. Secondly, during emergence, adults ingest exuvial liquid that has bactericidal properties. Nevertheless, sterilisation is thought to be incomplete, thus allowing some direct transmission from pupae to adults [46] and being responsible for the contribution of the larval/pupal breading sites to the adult microbiota, as mentioned earlier [22]. Moreover, emerging adults have been reported to ingest water and uptake bacteria during or shortly after emergence, with colonization efficiencies depending on the bacterial strains, e.g. *Elizabethkingia anophelis* (previously thought to be *E.* *meningoseptica)* is more successful than *Pantoea stewartii*[33, 36]. During adulthood, mosquitoes take sugar-meals of floral and extra-floral nectar, sap, ripe fruit and honeydew (Figure 1D, E) [47-49]. These meals potentially provide new bacterial species and are likely to affect the relative growth of existing species or strains depending on their properties, such as the concentration of each sugar type, typically glucose, fructose or gulose [50]. This might well be the case for *Asaia* and *Gluconacetobacter*, two genera usually found in flowers, and which have been identified as part of the adult *Anopheles* microbiota [22, 27].

The *Anopheles* tissue specificity of *Asaia sp.* was studied using a bacterial strain expressing GFP (green fluorescent protein) [27]. *Asaia* was found in the female gut and salivary glands, two tissues of particular interest to vector biology, but also in the male reproductive tract and the larval gut, which are potentially important tissues for the bacterial spread [27]. The microbiome of *Anopheles* other tissues than the gut has not yet been characterized. Interestingly, *Wolbachia sp.*, a maternally transmitted intracellular bacterium able to colonize multiple tissues in other insects, has not yet been found in any *Anopheles* species. This is of particular interest, as this endosymbiont colonizes around half of the insect species including several *Culex* and *Aedes* mosquito species [51]. Reasons for the apparent incompatibility between *Anopheles* and *Wolbachia* are unknown, but the generation of *Wolbachia*-infected *Anopheles* colonies is currently being pursued. Laboratory infection has been achieved for *Ae. aegypti* [52, 53], where *Wolba‐ chia* is a promising candidate for reducing the vector competence (see below). To our knowl‐ edge, no endosymbiont has been described in *Anopheles* to date.

Non-bacterial members of the *Anopheles* microbiota are poorly understood. Such studies are of special interest, as these microorganisms can potentially interact directly with the bacterial microbiota as well as the human pathogens and are likely to affect the mosquito physiology. An initial study, based on sequencing a 18S-library, identified 6 fungal clones related to *Candida sp*., *Hanseniaspora uvarum, Pichia sp*., *Wallemia sebi, Wickerhamomyces anomalus* and uncultured fungi in laboratory-reared *A. stephensi* [54]. *W. anomalus* is also found in wild and laboratory-reared *A. gambiae* [55]. TEM observation of mosquito tissues revealed the presence of yeasts in the female midgut and of actively dividing yeasts in the male gonoduct of *A. stephensi* [54, 55].

## **4. Impact of microbiota on** *Anopheles* **physiology and pathogen transmission**

The studies reviewed above suggest that *Anopheles* mosquitoes do not host any particular obligate symbiont. However, bacteria as a whole appear to be essential for mosquito physiol‐ ogy. In particular, it has not been possible to date to maintain *Anopheles* colonies on conven‐ tional laboratory diet in axenic conditions. In addition, *A. stephensi* larval development is slowed down in the presence of antibiotics and putatively blocked at the 3rd or 4th instar, but an antibiotic-resistant strain of *Asaia* is sufficient to revert this effect [56]. Although the mechanism involved in this dependence is unknown, several lines of experimental evidence point to the important nutritional role of gut commensals. First, the development of aseptic *A. stephensi* mosquitoes was achieved from sterilized eggs to adults in a custom aseptic medium [57], although no mention is made about adult fertility under these conditions. Second, a delay in the development was also observed in *Drosophila melanogaster* raised in axenic conditions under protein deprivation, which was rescued by the addition of live *Lactobacillus plantarum* in the fly medium [58]. *L. plantarum* was shown to promote larval growth under poor dietary conditions by enhancing nutrient sensing in a TOR-dependent manner, thus acting on ecdysone and insulin-like-peptide pathways [58]. Third, larval mortality was reported in the clothing louse deprived of its bacterial symbionts and can be avoided by supplementing the blood with B-vitamins (ß-biotin, pantothenate and nicotinic acid) [59]. The *Anopheles* micro‐ biota may also participate in metabolism, as adult mosquitoes fed with radiolabelled-Glycine *Pseudomonas* displayed radioactive signal throughout their body [40]. Interestingly, *Plasmodi‐ um* oocysts and sporozoites developing in these mosquitoes also contained radioactive compounds, suggesting that bacteria also participate in parasite nutrition [40].

*meningoseptica)* is more successful than *Pantoea stewartii*[33, 36]. During adulthood, mosquitoes take sugar-meals of floral and extra-floral nectar, sap, ripe fruit and honeydew (Figure 1D, E) [47-49]. These meals potentially provide new bacterial species and are likely to affect the relative growth of existing species or strains depending on their properties, such as the concentration of each sugar type, typically glucose, fructose or gulose [50]. This might well be the case for *Asaia* and *Gluconacetobacter*, two genera usually found in flowers, and which have

The *Anopheles* tissue specificity of *Asaia sp.* was studied using a bacterial strain expressing GFP (green fluorescent protein) [27]. *Asaia* was found in the female gut and salivary glands, two tissues of particular interest to vector biology, but also in the male reproductive tract and the larval gut, which are potentially important tissues for the bacterial spread [27]. The microbiome of *Anopheles* other tissues than the gut has not yet been characterized. Interestingly, *Wolbachia sp.*, a maternally transmitted intracellular bacterium able to colonize multiple tissues in other insects, has not yet been found in any *Anopheles* species. This is of particular interest, as this endosymbiont colonizes around half of the insect species including several *Culex* and *Aedes* mosquito species [51]. Reasons for the apparent incompatibility between *Anopheles* and *Wolbachia* are unknown, but the generation of *Wolbachia*-infected *Anopheles* colonies is currently being pursued. Laboratory infection has been achieved for *Ae. aegypti* [52, 53], where *Wolba‐ chia* is a promising candidate for reducing the vector competence (see below). To our knowl‐

Non-bacterial members of the *Anopheles* microbiota are poorly understood. Such studies are of special interest, as these microorganisms can potentially interact directly with the bacterial microbiota as well as the human pathogens and are likely to affect the mosquito physiology. An initial study, based on sequencing a 18S-library, identified 6 fungal clones related to *Candida sp*., *Hanseniaspora uvarum, Pichia sp*., *Wallemia sebi, Wickerhamomyces anomalus* and uncultured fungi in laboratory-reared *A. stephensi* [54]. *W. anomalus* is also found in wild and laboratory-reared *A. gambiae* [55]. TEM observation of mosquito tissues revealed the presence of yeasts in the female midgut and of actively dividing yeasts in the

**4. Impact of microbiota on** *Anopheles* **physiology and pathogen**

The studies reviewed above suggest that *Anopheles* mosquitoes do not host any particular obligate symbiont. However, bacteria as a whole appear to be essential for mosquito physiol‐ ogy. In particular, it has not been possible to date to maintain *Anopheles* colonies on conven‐ tional laboratory diet in axenic conditions. In addition, *A. stephensi* larval development is slowed down in the presence of antibiotics and putatively blocked at the 3rd or 4th instar, but an antibiotic-resistant strain of *Asaia* is sufficient to revert this effect [56]. Although the mechanism involved in this dependence is unknown, several lines of experimental evidence point to the important nutritional role of gut commensals. First, the development of aseptic

been identified as part of the adult *Anopheles* microbiota [22, 27].

534 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

edge, no endosymbiont has been described in *Anopheles* to date.

male gonoduct of *A. stephensi* [54, 55].

**transmission**

*Anopheles* females appear to also sense bacterial presence in the water, which influences oviposition in a bacterial strain dependent manner [60]. The underlying stimuli are not known but they are likely semiochemicals, i.e. messenger molecules produced by bacteria [60]. A principal component analysis of volatiles emitted by 17 bacterial strains, including 6 oviposi‐ tion-inducing strains, failed to identify compounds shared between all oviposition-inducing bacterial strains, suggesting that such semiochemicals are acting as cocktails [60].

An aspect of the *Anopheles* microbiota that received great interest recently is the colonisation resistance effect towards *Plasmodium* infection, as depicted in Figure 2. First, bacterial growth after a bloodmeal is reported to trigger an immune response via the Immune-deficiency (Imd) pathway, which causes synthesis of antimicrobial peptides and other immune effectors [2]. These effectors target bacterial populations in the mosquito midgut and exert antiparasitic effects. Second, an *Enterobacter* strain (*EspZ*) isolated from wild *A. arabiensis* mosquitoes is shown to directly affect *Plasmodium* development in the mosquito gut via elevated synthesis of ROS (reactive oxygen species) [1]. Third, microbiota-dependent immune priming is reported upon *Plasmodium* infection. This effect protects mosquitoes from subsequent *Plasmodium* infections and is likely to be mediated by hemocyte differentiation [3].

As mentioned above, *Anopheles* mosquitoes are also vectors of filarial worms and ONNV (anophelines are also secondary vectors of West Nile virus). The effect of gut microbiota on infection with these pathogens has not been thoroughly investigated to date, but feeding *A. quadriannulatus* with an antibiotic/antimycotic mixture is shown to increase *Brugia malayi* infection [61]. In *Ae. aegypti*, antibiotic treatment increases the susceptibility of mosquitoes to Dengue virus via a decrease in antimicrobial gene transcription [53]. This can be reverted by addition of bacterial strains such as *Proteus sp.* and *Paenibacillus sp.* [62]. The role of *Anopheles* microbiota upon viral infections is still unclear, but our unpublished observations suggest that antibiotic treatment of *A. gambiae* increases significantly the prevalence of infection with ONNV.

Vertically-transmitted *Wolbachia* endosymbionts are under special focus as promising candi‐ dates to stop pathogen transmission. Research in this field has advanced in *Ae. aegypti*, where stable infections of *Wolbachia* strains have been established in laboratory colonies [52, 53]. The

**Figure 2. Mechanisms of colonization resistance conferred by** *Anopheles* **microbiota against** *Plasmodium* **infec‐ tion.** 1 — Direct effect via synthesis of ROS by the *Enterobacter EspZ* strain [1]. 2 — Indirect effect via induction of NFκB antibacterial responses that have antiparasitic effects [2]. This is likely to be the most general mechanism. 3 — Induction of hemocyte differentiation by unknown soluble hemolymph factors during *Plasmodium* infection, which has a priming effect against asubsequent *Plasmodium* infection [3].

fast growing wMelPop strain of *Wolbachia* halves the mosquito lifespan, thus potentially affecting the capacity of mosquitoes to transmit pathogens with long extrinsic incubation periods [52]. It also induces a constitutively elevated immune response that negatively impacts on the infection prevalence and intensity of *Brugia pahangi* microfilariae, Chikungunya and Dengue viruses and the avian parasite *Plasmodium gallinaceum* [15, 17]. wAlbB and wMel, which naturally infect the Asian tiger mosquito *Aedes albopictus* and *D. melanogaster*, respec‐ tively, also render *Aedes* mosquitoes resistant to Dengue virus when introduced into laboratory populations [16, 63, 64]. Moreover, wMel is shown to successfully spread into wild *Ae. aegypti* populations in North-Eastern Australia [65] and is a strong candidate for Dengue biocontrol. When injected into *Anopheles* mosquitoes, *Wolbachia* seems to positively or nega‐ tively impact on *Plasmodium* infection depending on the *Wolbachia*/*Plasmodium* strain/species combination [66-68].

The immune system of *Anopheles* is known to control the microbiota population, by both resistance and tolerance mechanisms. On the one hand, the Imd pathway is shown to control the midgut bacterial numbers, especially after a bloodmeal [2], together with the production of ROS [21]. The melanization reaction might also contribute to limiting the bacterial numbers, as shown in the hindgut of the silkworm *Bombyx mori* [69]. On the other hand, induction of the Duox-IMPer (Dual oxidase - Immunomodulatory peroxidase) pathway after a bloodmeal leads to the formation of a dityrosine-linked mucus layer in the space between the peritrophic membrane and the midgut epithelium that reduces the permeability to immune elicitors. This tolerance mechanism leads to increased bacterial and *Plasmodium*loads [21]. Interestingly, such protection from oxidative stress is also identified in *Ae. aegypti*, where blood heme induces a protein kinase C-dependent mechanism leading to decreased ROS production and bacterial proliferation [70]. In *Drosophila*, several negative regulators of the Imd pathway are involved in tolerance to gut bacteria, but equivalent tolerance mechanisms have not yet been described in *Anopheles*. In particular, PGRP-LB and PGRP-SC1A/B degrade peptidoglycan into nonimmunogenic fragments and Pirk downregulates the activity of the PGRP-LC and PGRP-LE receptors [71-76]. Orthologs of these regulators PGRPs, but not of Pirk, are present in *Anoph‐ eles* [77, 78].

In several insect species, microbiota are shown to also impact on host behavior. Notably, *Drosophila* mating preference is influenced by the microbiota composition [79]. *Klebsiella oxytoca* is proposed as a probiotic able to rescue the loss of copulatory performance that follows male sterilization by irradiation in medfly (*Ceratitis capitata*), by restoring the *Klebsiella*/ *Pseudomonas* ratio to its normal levels [80]. In termites, a Rifampicin treatment is shown to reduce the queen oviposition rate and to decrease longevity and fecundity of termite repro‐ ductives [81]. As *Anopheles* mosquitoes are able to sense the presence of bacteria in water as well as on human skin and modulate their oviposition rate and feeding behavior accordingly [60, 82], the microbiota composition could also influence the mosquito social and/or repro‐ ductive behavior and feeding preference. This may prove to be of particular importance to vector control.

## **5. Potential exploitations to reduce** *Anopheles* **vector competence**

fast growing wMelPop strain of *Wolbachia* halves the mosquito lifespan, thus potentially affecting the capacity of mosquitoes to transmit pathogens with long extrinsic incubation periods [52]. It also induces a constitutively elevated immune response that negatively impacts on the infection prevalence and intensity of *Brugia pahangi* microfilariae, Chikungunya and Dengue viruses and the avian parasite *Plasmodium gallinaceum* [15, 17]. wAlbB and wMel, which naturally infect the Asian tiger mosquito *Aedes albopictus* and *D. melanogaster*, respec‐ tively, also render *Aedes* mosquitoes resistant to Dengue virus when introduced into laboratory

has a priming effect against asubsequent *Plasmodium* infection [3].

536 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**Figure 2. Mechanisms of colonization resistance conferred by** *Anopheles* **microbiota against** *Plasmodium* **infec‐ tion.** 1 — Direct effect via synthesis of ROS by the *Enterobacter EspZ* strain [1]. 2 — Indirect effect via induction of NFκB antibacterial responses that have antiparasitic effects [2]. This is likely to be the most general mechanism. 3 — Induction of hemocyte differentiation by unknown soluble hemolymph factors during *Plasmodium* infection, which

> Reduction of the *Anopheles* competence to transmit human pathogens, especially malaria, will have great implications on public health. Any perspective of reducing vector competence should affect at least one of the parameters of the Ross-McDonald model of disease transmis‐ sion [83]. These parameters include the mosquito-to-man ratio, the mosquito biting rate, the probability of successful man-to-mosquito and mosquito-to-man transmission, the mosquito

daily survival probability, the days needed for the parasite in the mosquito to become infective and the daily rate at which humans become non-infectious to mosquitoes. From studies carried out to date and reviewed in preceding sections, it is evident that the mosquito microbiota can potentially affect most of these parameters except those referring only to disease progression in the vertebrate host. The most important of these parameters are mosquito longevity, feeding behavior and capacity to support pathogen development and/or replication.

A direct way to reduce vector competence using our current knowledge of the *Anopheles* microbiota would be to use bacterial strains that are naturally incompatible with pathogen development and/or replication. Potential candidates are either natural microbiota such as the EspZ strain of *Enterobacter* that causes resistance to *Plasmodium* [1] or artificially introduced bacteria such as *Wolbachia*, which apparently induce a wide spectrum of resistance to human pathogens [15]. The great advantage of the latter is its ability to spread into populations by manipulating insect reproduction in several ways. In particular, *Wolbachia* induces death of young embryos laid by *Wolbachia*-free females mated with infected males; *Wolbachia-*infected females are always fertile independently of the male infection status [84]. This so-called cytoplasmic incompatibility confers a reproductive benefit to *Wolbachia*-infected females and leads to propagation of *Wolbachia* even if it bears small fitness cost to the host, including reduced fecundity (discussed in [85, 86]). The challenge of this approach is the fact that *Wolbachia* and *Anopheles* seem to be incompatible in nature and introduction of the endosym‐ biont in laboratory colonies of *Anopheles* has not yet been achieved. Screening of *Wolbachia* strains able to infect the *Anopheles* reproductive tissues, when cultured *ex vivo*, has been reported [87]. Alternatively, preadaptation of *Wolbachia* strains by long-term culturing in mosquito cell lines has been suggested as a strategy to infect new hosts, as shown successfully for *Aedes* [52, 88]. As previously reported in *Aedes* [15-17], *Wolbachia* might impact both on mosquito longevity and successful development and/or replication of all three taxa of *Anopheles*-borne pathogens, i.e. *Plasmodium*, viruses and nematodes.

An alternative approach is paratransgenesis, the introduction of genetically modified bacteria into the vector, which would confer resistance to pathogens. *Pantoea agglomerans*, a natural *Anopheles* symbiont, is a candidate for this approach and has been successfully engineered to express and secrete proteins that either inhibit midgut invasion by *Plasmodi‐ um*, such as [EPIP]4 (*Plasmodium* enolase-plasminogen interaction peptide) that competes with *Plasmodium* EPIP for plasminogen binding, or by directly targeting the parasite, such as the scorpion-derived antiplasmodial scorpine [89, 90]. Green fluorescent protein (GFP) tagged *P. agglomerans* persists and grows in the *Anopheles* gut, while transgenic *P. agglomer‐ ans* confers resistance against *P. falciparum* infection in both *A. stephensi* and *A. gambiae* without affecting the mosquito lifespan [90]. Applicability to more than one mosquito species is particularly advantageous for a transmission blocking approach. *Asaia* has also been proposed as a candidate for paratransgenesis, as it is quite frequent in *Anopheles* microbiota and can be successfully transformed [27]. Interestingly, this genus has been found in all of the 30 individuals assessed in the metagenomics study of Boissière et al. suggesting that it can easily spread into field populations [22]. *Asaia* can be transmitted both horizontally and vertically presenting an additional advantage for the spread of a transgenic strain into mosquito populations [27]. The introduction of such microbiota into mosquito populations could be achieved by using baiting stations, i.e. clay jars containing cotton balls soaked with sugar and bacteria, around malaria endemic villages, but this approach requires further investigation [90].

Finally, transmission-blocking interventions could involve drugs or other interventions that would impact on the microbiota, thus affecting mosquito homeostasis and efficiency of pathogen development. For example, the effects of antibiotics in the human blood could significantly impact the mosquito microbiota upon blood feeding, indirectly influencing mosquito physiology and infection with pathogens. Depending on its spectrum, an antibiotic could influence the microbiota composition and thus have a positive or negative impact on pathogen development and/or replication.

## **6. Conclusion**

daily survival probability, the days needed for the parasite in the mosquito to become infective and the daily rate at which humans become non-infectious to mosquitoes. From studies carried out to date and reviewed in preceding sections, it is evident that the mosquito microbiota can potentially affect most of these parameters except those referring only to disease progression in the vertebrate host. The most important of these parameters are mosquito longevity, feeding

A direct way to reduce vector competence using our current knowledge of the *Anopheles* microbiota would be to use bacterial strains that are naturally incompatible with pathogen development and/or replication. Potential candidates are either natural microbiota such as the EspZ strain of *Enterobacter* that causes resistance to *Plasmodium* [1] or artificially introduced bacteria such as *Wolbachia*, which apparently induce a wide spectrum of resistance to human pathogens [15]. The great advantage of the latter is its ability to spread into populations by manipulating insect reproduction in several ways. In particular, *Wolbachia* induces death of young embryos laid by *Wolbachia*-free females mated with infected males; *Wolbachia-*infected females are always fertile independently of the male infection status [84]. This so-called cytoplasmic incompatibility confers a reproductive benefit to *Wolbachia*-infected females and leads to propagation of *Wolbachia* even if it bears small fitness cost to the host, including reduced fecundity (discussed in [85, 86]). The challenge of this approach is the fact that *Wolbachia* and *Anopheles* seem to be incompatible in nature and introduction of the endosym‐ biont in laboratory colonies of *Anopheles* has not yet been achieved. Screening of *Wolbachia* strains able to infect the *Anopheles* reproductive tissues, when cultured *ex vivo*, has been reported [87]. Alternatively, preadaptation of *Wolbachia* strains by long-term culturing in mosquito cell lines has been suggested as a strategy to infect new hosts, as shown successfully for *Aedes* [52, 88]. As previously reported in *Aedes* [15-17], *Wolbachia* might impact both on mosquito longevity and successful development and/or replication of all three taxa of

An alternative approach is paratransgenesis, the introduction of genetically modified bacteria into the vector, which would confer resistance to pathogens. *Pantoea agglomerans*, a natural *Anopheles* symbiont, is a candidate for this approach and has been successfully engineered to express and secrete proteins that either inhibit midgut invasion by *Plasmodi‐ um*, such as [EPIP]4 (*Plasmodium* enolase-plasminogen interaction peptide) that competes with *Plasmodium* EPIP for plasminogen binding, or by directly targeting the parasite, such as the scorpion-derived antiplasmodial scorpine [89, 90]. Green fluorescent protein (GFP) tagged *P. agglomerans* persists and grows in the *Anopheles* gut, while transgenic *P. agglomer‐ ans* confers resistance against *P. falciparum* infection in both *A. stephensi* and *A. gambiae* without affecting the mosquito lifespan [90]. Applicability to more than one mosquito species is particularly advantageous for a transmission blocking approach. *Asaia* has also been proposed as a candidate for paratransgenesis, as it is quite frequent in *Anopheles* microbiota and can be successfully transformed [27]. Interestingly, this genus has been found in all of the 30 individuals assessed in the metagenomics study of Boissière et al. suggesting that it can easily spread into field populations [22]. *Asaia* can be transmitted both horizontally and vertically presenting an additional advantage for the spread of a

behavior and capacity to support pathogen development and/or replication.

538 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*Anopheles*-borne pathogens, i.e. *Plasmodium*, viruses and nematodes.

Recent high-throughput sequencing studies of the *Anopheles* microbiota have revealed the extent of the microbiota diversity, mostly in field or semi-natural conditions. A diverse range of bacteria is able to colonize the *Anopheles* gut, and there is a vast diversity of microbiota between mosquitoes. To some extent, this diversity needs to be considered at the bacterial strain level, as different strains of one species may have diverse effects on the mosquito physiology and other microbes of the gut ecosystem. Although bacteria may be the most abundant and important members of the gut microbiota, characterization of the viral, fungal and protist communities could prove insightful into the understanding of the homeostasis of this complex biological system (e.g. phage predation is thought to regulate bacterial popula‐ tions [91]) and its effects on pathogen transmission. An important question that may arise from further studies is whether variability and/or discrepancies in experimental findings about the interactions between mosquitoes and pathogens could be attributed to differences in the microbiota between laboratories. Toward exploiting the knowledge on *Anopheles* microbiota to reduce vector competence, research is currently at its infancy, but some bacteria such as *Pantoea* and *Asaia* already emerge as promising candidates of paratransgenesis. The use of *Wolbachia* to reduce *Aedes* vectorial capacity and fitness may be of particular importance, if this technology can be effectively transferred to *Anopheles*. Finally, the possibility to use drugs such as antibiotics to target specific mosquito microbiota and affect vector competence or fitness is a new concept that merits further investigation.

## **Acknowledgements**

We thank Jiannong Xu, Jewelna Osei-Poku, Anne Boissière and Isabelle Morlais for providing example sequences of some of the bacterial genera shown in Table 1 and Thierry Lefèvre for helping with mosquito pictures presented in Figure 1.

## **Author details**

Mathilde Gendrin and George K. Christophides\*

\*Address all correspondence to: g.christophides@imperial.ac.uk

Department of Life Sciences, Imperial College London, UK

## **References**


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**Author details**

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Mathilde Gendrin and George K. Christophides\*

540 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

\*Address all correspondence to: g.christophides@imperial.ac.uk

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## **Bacterial Biodiversity in Midguts of** *Anopheles* **Mosquitoes, Malaria Vectors in Southeast Asia**

Sylvie Manguin, Chung Thuy Ngo, Krajana Tainchum, Waraporn Juntarajumnong, Theeraphap Chareonviriyaphap, Anne-Laure Michon and Estelle Jumas-Bilak

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55610

**1. Introduction**

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Factors allowing the development of a pathogen to reach the infecting stage in a mosquito are poorly known. On the 528 species of mosquitoes recorded within the *Anopheles* genus [1], only 70 to 60 are able to transmit parasites responsible for malaria and filariasis [2, 3]. In vectorparasite interactions, the mosquito gut represents the first point of contact between parasites ingested and the vector epithelial surfaces. In the midgut, the parasites will have the oppor‐ tunity to undergo their life cycle, but of the tens of thousands of *Plasmodium* gametocytes ingested by mosquitoes, less than five oocysts might be produced [4]. The factors responsible for this drastic reduction are still poorly understood. Recent studies showed that one of these factors concerns the primordial role played by the bacteria naturally present in mosquito midgut. Then, there is a growing interest on bacterial biodiversity in *Anopheles* mosquitoes and particularly those based on the identification of bacteria to be used for malaria transmission blocking based on bacterial genetic changes to deliver antiparasite molecules or paratransgenic approach [5-13]. Recent studies reported the presence of symbiotic bacteria, such as *Pantoea agglomerans* or *Asaia* in midgut lumen with anti-*Plasmodium* effector proteins that render host mosquitoes refractory to malaria infection [6, 10, 13]. Engineered *P. agglomerans* strains were able to inhibit *Plasmodium falciparum* development by 98% [13]. Other studies showed that insects with an important microbiota seem more resistant to infections and certain bacteria, such as *Enterobacter* sp. (Esp Z) inhibit partially or totally ookinete, oocyst and sporozoite formation [14-16]. In *Anopheles albimanus*, co-infections with the bacteria, *Serratia marcescens*,

© 2013 Manguin et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Manguin et al.; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and *Plasmodium vivax* resulted in only 1% of mosquitoes being infected with oocysts, compared with 71% infection for control mosquitoes without bacteria [17]. A recent meta-taxogenomic study provides an in-depth description of the microbial communities in the midgut of *Anopheles gambiae* exposed to *P. falciparum* infection and the links between microbiota and parasitic status by comparing midgut microbiota in *P. falciparum*-positive and *P. falciparum*negative individuals. Authors found significant correlation between the high enterobacterial content and malaria infection. Despite conflicting results on the role of enterobacteria, it has now clearly been established that bacteria present in *Anopheles* populations have a great influence on parasite transmission [18].

In Thailand and Vietnam, malaria is a public health priority with a strong prevalence of this disease in forested regions, in particular along the international borders with Myanmar and Cambodia respectively. In these malaria endemic areas, another parasitic disease occurs, Bancroftian lymphatic filariasis (BLF) for which only limited data are available [2]. Malaria and BLF are mosquito-borne diseases with *Plasmodium* species, especially *P. falciparum*, *P. vivax*, and rural strains of *Wuchereria bancrofti* sharing the same *Anopheles* vector species. In Southeast Asia, *Anopheles* vectors belong to species complexes with different involvement in the transmission of pathogens [19]. Few sibling species of the Dirus and Minimus Complexes and the Maculatus Group are involved in malaria and BLF, but specific role of each sibling species and factors influencing this role have never been studied due to the lack of reliable methods for species identification, now available [20-22]. As mosquito microbiota is one of the factors influencing pathogen transmission, this chapter is presenting the biodiversity of bacteria in the midgut of field-collected adults of 10 *Anopheles* species, topic less studied compared to the large number of studies presenting bacteria in the defense against parasites in laboratory conditions.

#### **1.1. Midgut microbiome of mosquitoes**

Many insects contain large communities of diverse microorganisms that probably exceed the number of cells in the insect itself [23]. More specifically, complex microbiotae have been described in mosquito midgut reporting the presence of numerous Gram-negative rods, including *Serratia marcescens*, *Klebsiella ozaenae*, *Pseudomonas aeruginosa*, *Escherichia coli*, *Enterobacter* spp. [14]. Recently, three metagenomic studies provided a more comprehensive picture of the diversity of midgut microbiota in *Anopheles gambiae*, the main malaria vector in Africa [18, 24, 25]. In wild caught adults of *Anopheles* species, the microbiota showed the common presence of *Pseudomonas* and *Aeromonas* species reported from at least five species among which malaria vectors (Table 1). The following five genera, *Asaia*, *Bacillus*, *Chryseobac‐ terium*, *Klebsiella*, and *Pantoea* have been reported from four field collected *Anopheles* species, while *Serratia* and *Stenotrophomonas* were identified in three species (Table 1). At least three mosquito-specific bacterial species, isolated from the midgut of main malaria vectors of the Gambiae Complex, have been described, such as *Thorsellia anophelis* [26], *Janibacter anophelis* [27] and *Elizabethkingia anophelis* [28]. The first of the three species represents a new genus and species found predominant in the midgut of *Anopheles arabiensis* [29], the same *Anopheles* species in which *J. anophelis* was isolated. The third newly described species is closely related to *Elizabethkingia meningoseptica* as they share 98.6% similarity, and both species have been found in the midgut of *Anopheles gambiae* [11, 28]. The latter species, *E. meningoseptica*, was also isolated from diseased birds, frogs, turtles, cats, being most likely an agent of zoonotic infections, as well as a human meningitis especially in newborn infants [30]. Bacteria of the genus *Asaia* have also been associated with *Anopheles* species, in particular field-collected *An. gambiae*, *An. funestus*, *An. coustani* and *An. maculipennis* (Table 1), as well as a colony of *An. stephensi* in which *Asaia* bacteria was dominant and stably associated [9]. The presence of *Asaia* species in *Anopheles* could serve as candidate for malaria control based on the production of antiparasite molecules in mosquitoes for use in paratransgenic control of malaria [6, 9, 31]. Other bacterial species have been defined as antimalarial agents, especially those producing prodigiosin, a pigment produced by various bacteria, including *S. marcescens* [14].

The number of bacteria not only varied between individuals but also changed markedly during development, depending on both the stage of development and the blood-feeding status of the mosquitoes [31]. The normal midgut microbiota of *Anopheles* mosquitoes need to be further identified [5] as only few studies have reported the microbiota of wild caught malaria vectors (Table 1) [5-7, 9, 11, 12, 17, 24, 26-29, 31-35]. Further investigations of gut microbiota, especially of wild-caught insect vectors, might contribute to understanding the annual and regional variations recorded for vector transmitted diseases [17] and yield novel vector-control strategies [14].

#### **1.2. Exploring bacterial communities by 16S PCR-TTGE**

and *Plasmodium vivax* resulted in only 1% of mosquitoes being infected with oocysts, compared with 71% infection for control mosquitoes without bacteria [17]. A recent meta-taxogenomic study provides an in-depth description of the microbial communities in the midgut of *Anopheles gambiae* exposed to *P. falciparum* infection and the links between microbiota and parasitic status by comparing midgut microbiota in *P. falciparum*-positive and *P. falciparum*negative individuals. Authors found significant correlation between the high enterobacterial content and malaria infection. Despite conflicting results on the role of enterobacteria, it has now clearly been established that bacteria present in *Anopheles* populations have a great

In Thailand and Vietnam, malaria is a public health priority with a strong prevalence of this disease in forested regions, in particular along the international borders with Myanmar and Cambodia respectively. In these malaria endemic areas, another parasitic disease occurs, Bancroftian lymphatic filariasis (BLF) for which only limited data are available [2]. Malaria and BLF are mosquito-borne diseases with *Plasmodium* species, especially *P. falciparum*, *P. vivax*, and rural strains of *Wuchereria bancrofti* sharing the same *Anopheles* vector species. In Southeast Asia, *Anopheles* vectors belong to species complexes with different involvement in the transmission of pathogens [19]. Few sibling species of the Dirus and Minimus Complexes and the Maculatus Group are involved in malaria and BLF, but specific role of each sibling species and factors influencing this role have never been studied due to the lack of reliable methods for species identification, now available [20-22]. As mosquito microbiota is one of the factors influencing pathogen transmission, this chapter is presenting the biodiversity of bacteria in the midgut of field-collected adults of 10 *Anopheles* species, topic less studied compared to the large number of studies presenting bacteria in the defense against parasites

Many insects contain large communities of diverse microorganisms that probably exceed the number of cells in the insect itself [23]. More specifically, complex microbiotae have been described in mosquito midgut reporting the presence of numerous Gram-negative rods, including *Serratia marcescens*, *Klebsiella ozaenae*, *Pseudomonas aeruginosa*, *Escherichia coli*, *Enterobacter* spp. [14]. Recently, three metagenomic studies provided a more comprehensive picture of the diversity of midgut microbiota in *Anopheles gambiae*, the main malaria vector in Africa [18, 24, 25]. In wild caught adults of *Anopheles* species, the microbiota showed the common presence of *Pseudomonas* and *Aeromonas* species reported from at least five species among which malaria vectors (Table 1). The following five genera, *Asaia*, *Bacillus*, *Chryseobac‐ terium*, *Klebsiella*, and *Pantoea* have been reported from four field collected *Anopheles* species, while *Serratia* and *Stenotrophomonas* were identified in three species (Table 1). At least three mosquito-specific bacterial species, isolated from the midgut of main malaria vectors of the Gambiae Complex, have been described, such as *Thorsellia anophelis* [26], *Janibacter anophelis* [27] and *Elizabethkingia anophelis* [28]. The first of the three species represents a new genus and species found predominant in the midgut of *Anopheles arabiensis* [29], the same *Anopheles* species in which *J. anophelis* was isolated. The third newly described species is closely related

influence on parasite transmission [18].

550 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

in laboratory conditions.

**1.1. Midgut microbiome of mosquitoes**

Bacterial communities are classically assessed through culture-dependent methods based on colony isolation on solid medium, sometimes after enrichment by growth in liquid medium. But, it is now obvious that the microbial diversity is poorly represented by the cultured fraction, and culture have been shown to explore less than 1% of the whole bacterial diversity in environment samples [36]. Thanks to sophisticated biotechnological and computational tools of the metagenomics, molecular ecology offers the potential of determining microbial diversity in an ecosystem by assessing the genetic diversity. The complete metagenomic approach will give the total gene content of a community, thus providing data about biodiversity but also function and interactions [37]. For the purpose of biodiversity studies, metagenomics can focus on one common gene shared by all members of the community. The most commonly used culture-independent method relies on amplification and analysis of the 16S rRNA genes in a microbiota [38].

The 16S rRNA genes are widely used for documentation of the evolutionary history and taxon assignment of individual organisms because they have highly conserved regions for construc‐ tion of universal primers and highly variable regions for identification of individual species [39]. The notion developed by Woese that rRNA genes could identify living organisms by reconstructing phylogenies resulted in the adoption of 16S rRNA gene in microbiology [39]. Its universality and the huge number of sequences stored in databases have established 16S rRNA gene as the "gold standard" not only in microbial phylogeny, systematics, and identi‐ fication but also microbial ecology [40].



*Anopheles* **Total**

[24] [34] 2

**Bacteria genera (species)** *albimanus arabiensis coustani darlingi dureni funestus gambiae maculipennis stephensi*

*xylosoxidans)* [31] [5, 34] <sup>2</sup> *Acidovorax (A. temperans)* [12] 1

*hydrophila)* [24]\* [35] [24] [12, 24] [5] <sup>5</sup> *Anaplasma (A. ovis)* [12] 1

*siamensis)* [24] [24] [9, 24] [9] <sup>4</sup>

*Bordetella* [5] 1

*diminuta)* [31] <sup>1</sup> *Cedecea (C. davisae)* [31] [31] 2

*indologenes)* [24] [24] [24] [34] <sup>4</sup> *Citrobacter (C. freundii)* [34] 1

*Ehrlichia* [12] 1

*chrysanthenum)* [31] [31] <sup>2</sup>

*senegalensis)* [12] [31] <sup>2</sup>

*E. meningoseptica)* [11, 28] <sup>1</sup>

*resinovorum)* [31] <sup>1</sup> *Gluconobacter (G. cerinus)* [31] 1

*limosus)* [12, 27] <sup>1</sup>

*pneumoniae)* [33] [31] [31] [32] <sup>4</sup> *Kluyvera (K. cryocrescens)* [31] 1 *Leuconostoc (L. citreum)* [34] 1 Leminorella (L. grimontii) [34] 1 *Morganella (M. morgani)* [31] 1 *Mycoplasma (M. wenyonii)* [12] 1 Myroides [5] 1

*corynebacterioides)* [12] <sup>1</sup> *Paenibacillus sp.* [12] 1

*stewartii)* [35] [31] [11, 31] [7] <sup>4</sup>

[12] [31] [31] [34] 4

[17] [34] 2

*Achromobacter (A.*

552 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*Acinetobacter sp. (A. hemolyticus, A. radioresistens)*

*Aeromonas sp. (A.*

*Asaia spp. (A. bogorensis, A.*

*Bacillus spp. (B. cereus, B. coagulans, B. megaterium, B. mucoides, B. silvestris, B. simplex, B. thuringensis)*

*Brevundiumonas (B.*

*Chryseobacterium (C.*

*Enterobacter spp. (E. amnigenus, E. cloacae, E.*

*Erwinia (E. ananas, E.*

*Escherichia (E. coli, E.*

*Flavobacterium (F.*

*Klebsiella spp. (K.*

*Nocardia (N.*

*Pantoea (P. agglomerans, P.*

*Elizabethkingia (E. anophelis,*

*Janibacter (J. anophelis, J.*

*sakazaki)*

*Anoph eles* **species**

°, *An. gambiae* s.l. or s.s.; \*, For Osei-Poku et al (2012) [24], genera with low frequency were not considered in this table.

**Table 1.** Bacterial genera isolated from the midgut of wild-caught adults of 9 *Anopheles* species linked to the associated reference numbers.

The complete 16S rRNA gene (1500 bp) gives the accurate affiliation to a species in most cases. In metagenomics, the amplified fragments are shorter, ranging from 200 to 400 bp, but contain nine hypervariable regions (V1-V9) [41], which compensate the lack of information due to the small sequence size by a high rate of mutation. In most studies, the V3 region located in the 5' part of the gene is chosen [42]. However, the phylogenetic information is sometimes insuffi‐ cient to achieve species identification. Depending on the bacterium, sequences provide identification to the genus or family level only. Consequently, the diversity of the community is not described by a list of bacterial species but by a list of operational taxonomic units (OTUs) corresponding to the lower taxonomic level being accurately identified. The 16S rRNA gene, in spite of some recognized pitfalls [43], remains today the most popular marker for studying the specific diversity in a bacterial community. Alternative markers can also be proposed such as *rpoB* [40] but universal *rpoB* PCR primers allowing the exploration of the whole bacterial diversity cannot be designed (Jumas-Bilak E, personal data) and the databases remain poor in *rpo* sequences.

Molecular approaches for assessing biodiversity avoid the bias of cultivability but displayed several pitfalls that should be evaluated and considered for a sound interpretation of the data. Particularly, DNA should be recovered and amplified from all the genotypes in the commun‐ ity, i.e. extraction and PCR should be as universal as possible. Special attention should be given to *Firmicutes* and *Actinobacteria* because they display thick and resistant cell wall. The extraction efficiency should be tested on a wide panel of bacteria to scan a large range of bacterial types. Extraction is generally improved by the use of large-spectrum lytic enzymes and/or by a mechanical grinding [44, 45]. The PCR itself is another cause of limitations in the molecular approaches. It often praises for its detection sensitivity but this sensitivity can fail when complex samples are analyzed. For example, detection thresholds of 103 -104 CFU (colony forming units)/mL are currently described for universal PCR and migration in denaturing gels [44-46]. The detection limit cannot be easily assessed as it depends on both CFU/g count of each OTU and the relative representation of OTUs in the community. Minor populations of less than 1% of total population are generally undetectable for denaturing-gel-based methods used in microbial ecology [45, 47, 48].

In biodiversity studies, the different 16S rRNA genes representative of the community are amplified by PCR and then separated and identified either by cloning and Sanger sequencing or by direct pyro-sequencing [38]. Tools for sequence-specific separation after bulk PCR amplification, such as T-RFLP (Terminal-Restriction Fragment Length Polymorphism) [49], D-HPLC (Denaturing High Performance Liquid Chromatography) [50], CDCE (Constant Denaturing Capillary Electrophoresis) [51], SSCP (Single Stranded Conformation Polymor‐ phism) [52], DGGE (Denaturing Gradient Gel Electrophoresis) [53], TGGE (Temperature Gradient Gel Electrophoresis), [48] and TTGE (Temporal Temperature Gradient Gel Electro‐ phoresis) [47], can also be used. Methods based upon separation in denaturing electrophoresis allow the comparison of microbiotae with low or medium diversity [54]. They easily provide a "fingerprint" of the community diversity and therefore they are suitable for the follow-up of large collection of samples.

PCR-TTGE is a PCR-denaturing gradient gel electrophoresis that allows separation of DNA fragments in a temporal gradient of temperature [47, 55]. PCR amplicons of the same size but with different sequences are separated in the gel. In a denaturing acrylamide gel, DNA denatures in discrete regions called melting domains, each of them displaying a sequence specific melting temperature. When the melting temperature (*Tm*) of the whole amplicon is reached, the DNA is denatured creating branched molecules. This branching reduces DNA mobility in the gel. Therefore, amplicons of the same size but with different nucleotide compositions can be separated based on differences in the behavior of their melting domains. When DNA is extracted and amplified from a complex community, TTGE leads to the separation of the different amplicons and produces a banding pattern characteristic of the community. Counting bands on the TTGE profile provides a diversity score that roughly corresponds to the number of molecular species in the sample. The banding profile can be further analyzed by measuring distance migration of bands and comparing with patterns from known species. This comparison allows the affiliation of band to some representative species. Affiliation of all bands can be achieved by cutting bands from the gel, extracting DNA from bands and sequencing. A method associating migration distances measurement and sequenc‐ ing of selected bands has shown its efficiency in describing bacterial communities of low complexity such as the gut microbiota of neonates [45]. Such an approach is simple enough and cost-effective to survey bacterial communities on a wide range of samples [56].

This chapter presents the bacterial biodiversity in the midguts of malaria vectors from Thailand and Vietnam based on the amplification of the V3 region of the 16S rRNA gene, separation of amplicon by TTGE and sequencing. The bacterial biodiversity among specimens and species in relation to the collection site are discussed.

## **2. Material and methods**

efficiency should be tested on a wide panel of bacteria to scan a large range of bacterial types. Extraction is generally improved by the use of large-spectrum lytic enzymes and/or by a mechanical grinding [44, 45]. The PCR itself is another cause of limitations in the molecular approaches. It often praises for its detection sensitivity but this sensitivity can fail when

forming units)/mL are currently described for universal PCR and migration in denaturing gels [44-46]. The detection limit cannot be easily assessed as it depends on both CFU/g count of each OTU and the relative representation of OTUs in the community. Minor populations of less than 1% of total population are generally undetectable for denaturing-gel-based methods

In biodiversity studies, the different 16S rRNA genes representative of the community are amplified by PCR and then separated and identified either by cloning and Sanger sequencing or by direct pyro-sequencing [38]. Tools for sequence-specific separation after bulk PCR amplification, such as T-RFLP (Terminal-Restriction Fragment Length Polymorphism) [49], D-HPLC (Denaturing High Performance Liquid Chromatography) [50], CDCE (Constant Denaturing Capillary Electrophoresis) [51], SSCP (Single Stranded Conformation Polymor‐ phism) [52], DGGE (Denaturing Gradient Gel Electrophoresis) [53], TGGE (Temperature Gradient Gel Electrophoresis), [48] and TTGE (Temporal Temperature Gradient Gel Electro‐ phoresis) [47], can also be used. Methods based upon separation in denaturing electrophoresis allow the comparison of microbiotae with low or medium diversity [54]. They easily provide a "fingerprint" of the community diversity and therefore they are suitable for the follow-up

PCR-TTGE is a PCR-denaturing gradient gel electrophoresis that allows separation of DNA fragments in a temporal gradient of temperature [47, 55]. PCR amplicons of the same size but with different sequences are separated in the gel. In a denaturing acrylamide gel, DNA denatures in discrete regions called melting domains, each of them displaying a sequence specific melting temperature. When the melting temperature (*Tm*) of the whole amplicon is reached, the DNA is denatured creating branched molecules. This branching reduces DNA mobility in the gel. Therefore, amplicons of the same size but with different nucleotide compositions can be separated based on differences in the behavior of their melting domains. When DNA is extracted and amplified from a complex community, TTGE leads to the separation of the different amplicons and produces a banding pattern characteristic of the community. Counting bands on the TTGE profile provides a diversity score that roughly corresponds to the number of molecular species in the sample. The banding profile can be further analyzed by measuring distance migration of bands and comparing with patterns from known species. This comparison allows the affiliation of band to some representative species. Affiliation of all bands can be achieved by cutting bands from the gel, extracting DNA from bands and sequencing. A method associating migration distances measurement and sequenc‐ ing of selected bands has shown its efficiency in describing bacterial communities of low complexity such as the gut microbiota of neonates [45]. Such an approach is simple enough

and cost-effective to survey bacterial communities on a wide range of samples [56].


CFU (colony

complex samples are analyzed. For example, detection thresholds of 103

used in microbial ecology [45, 47, 48].

554 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

of large collection of samples.

#### **2.1. Mosquito collections and species identification**

In Thailand, populations of *Anopheles* mosquitoes were collected from three different sites located in malaria endemic area along the Thai-Myanmar border (Figure 1). One study site is in Pu Teuy, a village located in Sai Yok District, Kanchanaburi Province, western Thailand (14° 17'N, 99° 01'E). The rural site is located in mountainous terrain mostly surrounded by forest. The main water body near the collection site is a narrow, slow running stream, bordered with native vegetation [57]. This stream represents the main larval habitat for *An. minimus* s.l. [58]. A total of 1,330 malaria cases were reported in 2011 in the Sai Yok District with a prevalence of 389 cases of *P. falciparum* (44.7%) and 481 cases of *P. vivax* (55.3%) with a mortality rate per 100,000 inhabitants of 0.71 [59]. The second site located in Mae Sod District, Tak Province, is in the northern part of Thailand (16° 67'N, 98° 68'E). This is a forested area associated to agricultural fields and small streams. In 2011, 1,876 malaria cases were reported in this district with 187 cases of *P. falciparum* (28.3%) and 473 cases of *P. vivax* (71.7%). The mortality rate per 100,000 was of 0.56 [59]. The third site in Sop Moei District is the most southern district of Mae Hong Son Province (17° 86'N, 97° 96'E). This mountainous province is located north of Tak Province with a high malaria transmission occurring from June to August, during the rainy season [60]. In 2011, 1,643 malaria cases were found in this district due to *P. falciparum* with 419 cases (45.0%) and *P. vivax* with 511 cases (55.0%) and a mortality rate per 100,000 of 0.41 [59].

The specimens from Vietnam were collected from six sites located in Dak Ngo Commune, Tuy Duc District, Dak Nong Province (11°59'N, 107°42'E - central Highlands) where 848 malaria cases were reported in 2011, of which, 322 cases (54.9%) were caused by *P. falciparum*, 209 cases (35.6%) by *P. vivax* and 56 cases (9.5%) were mixed infections [61]. This province was named in 2004 after integrating parts from northern area of Binh Phuoc Province and southern area of Dak Lak Province. The average temperature in this province is around 24° C with the rainy season ranging from May to October and the dry season from November to April. The climate is favorable for agriculture, especially coffee, pepper and rubber plantations. Crops of coffee, pepper or cashew nuts were normally cultivated around houses. Villages were surrounded by cassava, corn and rice fields and located in the fringe forest. Every year, during harvest period, workers from neighbourhood come to work in the field, which generate high population movements in this area.

*Anopheles* mosquitoes were morphologically sorted by taxon before using specific AS-PCR assays for species identification within the complex or the group [20-22]. Each individual was split in two pieces, head-thorax for species identification and abdomen for midgut bacteria analysis.

**Figure 1.** Map of Southeast Asia showing the locations of three provinces in Thailand (blue dots) and the province in Vietnam (red dot) where the mosquito collections were implemented.

#### **2.2. DNA extraction**

Mosquitoes stored at -20°C were surface rinsed twice in purified water prepared for injectable solution, and abdomen was thoroughly disrupted using a tissue crusher device in 150 µl of TE buffer. DNA was extracted using the Master Pure Gram Positive DNA purification kit as recommended by the supplier (Epicentre Biotechnologics, Madison, USA).

#### **2.3. PCR**

The V2–V3 region of the 16S rRNA gene of bacteria in the samples was amplified using the primers HDA1/HDA2 [45]; HDA1: 5'-ACTC CTA CGG GAG GCA GCA GT-3', HDA2: 5'-GTA TTA CCG CGG CTG CTG GCA-3'. A 40-bp clamp, named GC (5'-CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG G-3') flanked the 5' extremity of HDA1 [47] in order to form HDA1-GC. PCR was performed using an Eppendorf thermal cycler® (Eppen‐ dorf, Le Pecq, France) and 0.5 ml tubes. The reaction mixture (50 µl) contained 2.5 units of Taq DNA Polymerase (FastStart High fidelity PCR system, Roche, Meylan, France), 0.2 mM of each primer and 1 µl of DNA in the appropriate reaction buffer. Amplification was 95°C for 2 min, 35 cycles of 95°C for 1 min, 62°C for 30 s, 72°C for 1 min and 7 min at 72°C for final extension. To avoid contamination, solutions were prepared with sterile DNA-free water and preparation of the mastermix, addition of template DNA and gel electrophoresis of PCR products were carried out in separate rooms. PCR amplification was checked by DNA electrophoresis in 1.5% agarose gels containing ethidium bromide and visualized under ultraviolet light.

#### **2.4. TTGE migration**

**Figure 1.** Map of Southeast Asia showing the locations of three provinces in Thailand (blue dots) and the province in

Mosquitoes stored at -20°C were surface rinsed twice in purified water prepared for injectable solution, and abdomen was thoroughly disrupted using a tissue crusher device in 150 µl of TE buffer. DNA was extracted using the Master Pure Gram Positive DNA purification kit as

The V2–V3 region of the 16S rRNA gene of bacteria in the samples was amplified using the primers HDA1/HDA2 [45]; HDA1: 5'-ACTC CTA CGG GAG GCA GCA GT-3', HDA2: 5'-GTA TTA CCG CGG CTG CTG GCA-3'. A 40-bp clamp, named GC (5'-CGC CCG GGG CGC GCC

recommended by the supplier (Epicentre Biotechnologics, Madison, USA).

Vietnam (red dot) where the mosquito collections were implemented.

556 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**2.2. DNA extraction**

**2.3. PCR**

TTGE was performed using the DCode universal mutation detection system (Bio-Rad Laboratories, Marne-la-Coquette, France) in gels that were 16 cm × 16 cm by 1 mm. The gels (40 ml) were composed of 8% (wt/vol) bisacrylamide (37.5:1), 7 M urea, 40 µl of N,N,N',N' tetramethylethylenediamine (TEMED), and 40 mg ammonium persulfate (APS). Gels were run with 1X Tris–acetate–EDTA buffer at pH 8.4. The 5 µl of DNA was loaded on gel with 5 µl of in-house dye marker (saccharose 50%, Bromophenol Blue 0.1%) using capillary tips. Denatur‐ ing electrophoresis was performed at 46 V with a temperature ramp from 63°C to 70°C during 16 h (0.4°C/h) after a pre-migration of 15 min at 20 V and 63°C. Gels were stained with ethidium bromide solution (5 µg/ml) for 20 minutes, washed with de-ionized water, viewed using a UV transillumination system (Vilbert-Lourmat, France) and photographed.

#### **2.5. TTGE band sequencing and OTU affiliation**

TTGE bands were excised and the DNA was eluted with 50 µl of elution buffer (EB) of the Qiaquick PCR purification kit (Qiagen, Courtabeuf, France) overnight at 37°C before PCR amplification with HDA1/HDA2 used without GC clamp. The reaction conditions were identical to those described above. PCR products were sequenced on an ABI 3730xl sequencer (Cogenics, Meylan, France). Each sequencing chromatograph was visually inspected and corrected. The sequences were analyzed by comparison with Genbank (http:// www.ncbi.nlm.nih.gov/) and RDPII databases (http://rdp.cme.msu.edu/) using Basic Local Alignment Search Tool (BLAST) and Seqmatch programs, respectively. The reference sequence with the highest percentage was used for OTU affiliation. A sequence was affiliated to a species-level OTU when the percent of sequence similarity was above 99.0%, as previously proposed [62]. This value is over the recognized cut-off value for the delineation of species [63], but warrants high stringency for species-level OTU affiliation. Below 99.0%, the sequence is affiliated to the genus of the reference sequence with the highest percentage. When several species reference sequences match equally, affiliation was done to the genus level. For example, sequence with 99.5% in similarity to the species *Aeromonas caviae* and *Aeromonas hydrophila* was only assigned to the genus *Aeromonas*. Low cut-off is not defined for the genus delineation since affiliation to a higher taxonomic rank such as family or order will be done considering the taxonomic frame of the clade using Greengenes database [64]. On each TTGE gel, about 50% of the bands were sequenced, the others being affiliated to an OTU by comparison of their migration distance with that of sequenced bands.

#### **2.6. Phylogeny**

The sequences for phylogenetic analysis were selected in the GenBank database using BLAST program and taxonomy browser (http://www.ncbi.nlm.nih.gov). The sequences were then quality checked using SEQMATCH program in the 16S rDNA-specialized database, RDPII (http://rdp.cme.msu.edu). Sequences were aligned using the ClustalX program [65], and the alignment was manually corrected to exclude gaps and ambiguously aligned regions. Maxi‐ mum-likelihood (ML) phylogenetic analysis was performed using PhyML v2.4.6 [66], the model being General Time Reversible plus gamma distribution plus invariable site. ML bootstrap support was computed on 100 reiterations using PhyML.

## **3. Results**

#### **3.1.** *Anopheles* **species**

Among the 175 specimens of *Anopheles* collected in Thailand and Vietnam, a total of 10 species were identifiedincludingsixspeciesper countryofwhichtwo,*An.maculatus*and*An.dirus*,were common to both countries (Table 2). Eight species out of 10 belong to a group or a complex of which the sibling species were identified using the appropriate PCR assay (see Material and Methods). The Maculatus Group was represented by two species, *An. maculatus* and *An. sawadwongporni*, the latter collected in Thailand only. Within the Dirus Complex, three species were identified, *An. dirus*, *An. baimaii* and *An. scanloni*, the latter two were also collected in Thailand, as well as two species of the Minimus Complex, *An. minimus* and *An. harrisoni*. Three additional species were collected in Vietnam, *An. gigas* belonging to the Gigas Complex, *An. barbumbrosus*, and *An. crawfordi*. Among the 10 collected species, the former seven species of the MaculatusGroup,Dirus andMinimusComplexes aredefinedas importantmalariavectors and the latterthree specieshavenotbeenreportedasbeinginvolvedinmalaria transmission[19, 67].

#### **3.2. PCR-TTGE profiles and diversity index in midgut bacterial communities of** *Anopheles*

The midgut microbiota of 175 specimens of *Anopheles* mosquitoes was investigated by 16S rRNA gene PCR-TTGE anchored in the V3 hypervariable region. A representative gel is given in Figure 2. TTGE profiles were obtained for 144 samples, 31 samples (17.7%) giving no amplification in PCR or a faint PCR signals leading to non-detectable TTGE profiles. Negative results suggested a low bacterial inoculum rather than a total absence of bacteria in the corresponding samples. Most negative samples came from Vietnam mosquitoes (n=26), compared to Thailand (n=5), and seemed to be unrelated to the *Anopheles* species. Finally, V3 16S PCR-TTGE approach led to the description of a microbial community for about 80% of the specimens analyzed and therefore appeared as an efficient tool to investigate midgut bacterial diversity in a large population of mosquitoes.

50% of the bands were sequenced, the others being affiliated to an OTU by comparison of their

The sequences for phylogenetic analysis were selected in the GenBank database using BLAST program and taxonomy browser (http://www.ncbi.nlm.nih.gov). The sequences were then quality checked using SEQMATCH program in the 16S rDNA-specialized database, RDPII (http://rdp.cme.msu.edu). Sequences were aligned using the ClustalX program [65], and the alignment was manually corrected to exclude gaps and ambiguously aligned regions. Maxi‐ mum-likelihood (ML) phylogenetic analysis was performed using PhyML v2.4.6 [66], the model being General Time Reversible plus gamma distribution plus invariable site. ML

Among the 175 specimens of *Anopheles* collected in Thailand and Vietnam, a total of 10 species were identifiedincludingsixspeciesper countryofwhichtwo,*An.maculatus*and*An.dirus*,were common to both countries (Table 2). Eight species out of 10 belong to a group or a complex of which the sibling species were identified using the appropriate PCR assay (see Material and Methods). The Maculatus Group was represented by two species, *An. maculatus* and *An. sawadwongporni*, the latter collected in Thailand only. Within the Dirus Complex, three species were identified, *An. dirus*, *An. baimaii* and *An. scanloni*, the latter two were also collected in Thailand, as well as two species of the Minimus Complex, *An. minimus* and *An. harrisoni*. Three additional species were collected in Vietnam, *An. gigas* belonging to the Gigas Complex, *An. barbumbrosus*, and *An. crawfordi*. Among the 10 collected species, the former seven species of the MaculatusGroup,Dirus andMinimusComplexes aredefinedas importantmalariavectors and the latterthree specieshavenotbeenreportedasbeinginvolvedinmalaria transmission[19, 67].

**3.2. PCR-TTGE profiles and diversity index in midgut bacterial communities of** *Anopheles*

The midgut microbiota of 175 specimens of *Anopheles* mosquitoes was investigated by 16S rRNA gene PCR-TTGE anchored in the V3 hypervariable region. A representative gel is given in Figure 2. TTGE profiles were obtained for 144 samples, 31 samples (17.7%) giving no amplification in PCR or a faint PCR signals leading to non-detectable TTGE profiles. Negative results suggested a low bacterial inoculum rather than a total absence of bacteria in the corresponding samples. Most negative samples came from Vietnam mosquitoes (n=26), compared to Thailand (n=5), and seemed to be unrelated to the *Anopheles* species. Finally, V3 16S PCR-TTGE approach led to the description of a microbial community for about 80% of the specimens analyzed and therefore appeared as an efficient tool to investigate midgut bacterial

migration distance with that of sequenced bands.

558 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

bootstrap support was computed on 100 reiterations using PhyML.

**2.6. Phylogeny**

**3. Results**

**3.1.** *Anopheles* **species**

diversity in a large population of mosquitoes.

**Figure 2.** Representative TTGE analysis of V3 16S rRNA gene PCR products amplified from midgut samples of Anophe‐ *les* mosquitoes from Thailand. Each lane corresponded to a specimen microbiota.

A raw diversity index that globally reflects the bacterial diversity in a sample is classically evaluated by counting the bands in TTGE profiles. At a first glance, the number of bands on TTGE profiles (Figure 2) ranged from 1 to 10 suggesting that the bacterial diversity per specimen ranged from 1 to 10 OTUs. However, sequencing showed that bands with different distance of migration could belong to the same OTU. This atypical phenomenon was observed for bacteria displaying sequence heterogeneity among their 16S rRNA gene copies. For instance, members of the genus *Acinetobacter* as well as most members of the genera affiliated to the family *Enterobacteriaceae* displayed a high level of 16S rRNA gene heterogeneity leading to complex banding patterns in V3 16S PCR-TTGE. Considering that *Acinetobacter* and *Enterobacteriaceae* were prevalent in our samples, the raw diversity index drastically overesti‐ mated the bacterial diversity. Therefore, a refined diversity index was calculated after affiliation of each band to an OTU by sequencing or by comparative approach (see Material and Methods).

The refined diversity index showed a low bacterial diversity per specimen with an average of 1.5 OTU per specimen. Most positive samples displayed a diversity index of 1 or 2 (Figure 3). Five OTUs is the maximal biodiversity per specimen observed in our population of *Anophe‐ les* mosquitoes. Figure 3 showed that the number of OTUs per specimen differed slightly between populations from different origin, with an average of 1.7 and 1.3 OTU per specimen in Thailand and Vietnam, respectively. Considering mosquito species, the average diversity varied between 0 for *An. sawadwongporni* and 3 for *An. harrisoni* (Table 2).


**Table 2.** Bacterial genera detected in midgut of *Anopheles* species caught in Thailand (blue) and Vietnam (red) with the number of specimens carrying each genus. Diversity index links to *Anopheles* species and origin is given at the bottom of the table. Genera described for the first time in *Anopheles* are marked with asterisk. Vertical lines delineated, from left to right, both countries with their respective number of *Anopheles* specimens, and groupings of *Anopheles* species, such as the Maculatus Group, the Minimus and Dirus Complexes, and the non-vector species including the Gigas Complex and 2 additional species.

**Figure 3.** Distribution of the *Anopheles* mosquito populations from Thailand and Vietnam according to their refined diversity index

#### **3.3. Bacterial diversity in the whole population of** *Anopheles* **mosquitoes**

**COUNTRY**

**Vietnam (n=100)**

*Aeromonas* 8 *Asaia* 2 4

*Chromobacterium\** 1

*Citrobacter* 1

*Cronobacter\** 5 *Diaphorobacter\** 1

*Escherichia* 1 *Gluconacetobacter\** 2 *Kluyvera* 3

*Pantoea* 1

*Shewanella\** 1

including the Gigas Complex and 2 additional species.

*An. maculatus (n=28)*

*An. maculatus (n=11)*

*An. sawadwongporni (n=1)*

*Cellvibrio\** 1

*Chryseobacterium* 1 1 1

*Diplorickettsia\** 1 1

*Enterobacter* 1 11 1 1 2

*Enhydrobacter\** 1

*Microbacterium\** 1

*Nitrincola\** 3

*Psychrobacter\** 1 *Raoultella\** 2 9 2

*Stenotrophomonas* 2

*Moraxella\** 1 1 2

*Pseudomonas* 8 1 8 1 1 2

*Sphingomonas\** 20 16 8 1 1 *Staphylococcus\** 1 1

**Diversity Index 1.7 1.3 1.3 1.8 0 1.5 3 0.6 1.3 1.2 1.7 1.4 0.6 0.3**

**Table 2.** Bacterial genera detected in midgut of *Anopheles* species caught in Thailand (blue) and Vietnam (red) with the number of specimens carrying each genus. Diversity index links to *Anopheles* species and origin is given at the bottom of the table. Genera described for the first time in *Anopheles* are marked with asterisk. Vertical lines delineated, from left to right, both countries with their respective number of *Anopheles* specimens, and groupings of *Anopheles* species, such as the Maculatus Group, the Minimus and Dirus Complexes, and the non-vector species

*Riemerella\** 1 *Serratia* 8 6 1 2 3

*Elizabethkingia* 4 1

*Corynebacterium\** 1

*An. minimus (n= 37)*

*Acinetobacter* 14 10 10 1 1 11 18 3 5

*Bacillus* 1 1 1

**Thailand (n=75)**

560 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

**VECTORS**

*Maculatus Minimus Dirus*

*An. harrisoni (n=13)*

*An. baimaii (n=4)*

*An. scanloni (n=3)*

*An. dirus (n=6)*

*An. dirus (n=23)*

*An. gigas (n=24)*

*An. barbumbrosus (n=13)*

**NON VECTORS**

*An. crawfordi (n=12)*

16S rRNA gene PCR-TTGE is focused on hypervariable region V3 produced sequences of about 200 bp, which are generally not informative enough for species affiliation. Consequently, we presented here the bacterial diversity to the genus level. However, probable species affiliation willbeproposedforseveralgenerawhenthephylogeneticsignaloftheV3regionwassignificant.

Contrasting with the low diversity per specimen, OTU diversity in the whole population was high with the detection of 31 different bacterial genera (Table 2) distributed in four phyla, *Proteobacteria*, *Bacteroidetes/Chlorobi*, *Firmicutes* and *Actinobacteria*. *Proteobacteria* largely dominated the midgut microbiota of *Anopheles* mosquitoes with 232 OTUs in the population studied. Their diversity encompassed *Alpha, Beta-* and *Gamma* superclasses of *Proteobacteria*.

The gamma-proteobacterial genera *Acinetobacter*, *Pseudomonas*, *Enterobacter*, *Serratia* and *Raoul‐ tella* were widely detected in our populations. A total of 40% of specimens and 70% of *Anopheles* species were colonized by members of the genus *Acinetobacter*, which therefore could be con‐ sidered as a 'core genus' of the midgut microbiota of *Anopheles*. The sequences affiliated to the genus *Acinetobacter* were identified to the species level by a phylogenetic approach (Figure 4). The *Anopheles* midgut microbiota included 6 main species, *Acinetobacter baumannii, Acinetobact‐ er calcoaceticus, Acinetobacter johnsonii, Acinetobacter soli*, *Acinetobacter guillouiae* and *Acinetobact‐ er junii*, the two latter being more represented. The genus *Acinetobacter* belongs to the order *Pseudomonadales* in gamma-proteobacteria together with *Pseudomonas* (*Pseudomonas fluorescens* and *Pseudomonas alcaligenes*), *Moraxella, Enhydrobacter*, *Psychrobacter* and *Cellvibrio*. *Enterobac‐ teriales* was the second main order of gamma-proteobacteria represented in the midgut micro‐ biota of *Anopheles*. In enterobacteria, the species affiliation could not be achieved since genera are very close together in 16S rRNA gene phylogeny, particularly for *Enterobacter* and its rela‐ tives *Cronobacter* and *Pantoea*. Members of gamma-proteobacteria of the orders *Legionellales* (*Diplorickettsia*), *Oceanospirillales* (*Nitrincola*), *Alteromonadales* (*Shewanella*), *Xanthomonadales* (*Stenotrophomonas*) and *Aeromonadales* (*Aeromonas*) were also detected showing the very wide diversity of gamma-proteobacteria in the midgut microbiota of *Anopheles*.

**Figure 4.** Maximum Likelihood phylogenetic tree of the genus *Acinetobacter.* Lineages of strains detected in the mi‐ crobiota of *Anopheles* mosquitoes are in color, blue for Thailand, red for Vietnam. Bootstrap percentages (>50 %) af‐ ter 100 resamplings are shown. Bar: 0.5 % sequence divergence.

The diversity was lower in alpha- and beta-proteobacteria. However, the genus *Sphingomo‐ nas* that belonged to *Alphaproteobacteria*, was the second main genus detected in this study (26% of the *Anopheles* species colonized) mostly represented by sequences affiliated or related to the species *Sphingomonas aromaticivorans* and *Sphingomonas glacialis*. Acetic-acid bacteria (*Asaia* and *Gluconacetobacter*) belonged to *Alphaproteobacteria* and were sporadically represented as well as *Chromobacterium* and *Diaphorobacter*, the two members of *Betaproteobacteria*.

Beside *Proteobacteria*, the phyla *Bacteroidetes/Chlorobi*, *Actinobacteria* and *Firmicutes* were represented by only few genera: 15, 9 and 5 respectively. *Chryseobacterium*, *Elizabethkingia* and *Riemerella*, which colonized only 8 mosquitoes, belonged to *Bacteroidetes/Chlorobi* and class *Flavobacteriia*. Sequences affiliated to the genus *Elizabethkingia* could not be related with certainty to *Elizabethkingia anophelis*, because the V3 region did not discriminate between this *Anopheles-*specific species and the human pathogen *Elizabethkingia meningoseptica*. *Bacillus* and *Staphylococcus* (*Firmicutes*), *Corynebacterium* and *Microbacterium* (*Actinobacteria*) were the sole Gram-positive genera found in the population of *Anopheles* mosquitoes. The most related species were *Bacillus cereus*, *Corynebacterium freiburgense* and *Microbacterium trichothecenolyti‐ cum*. The *Staphylococcus* sequences found in two mosquitoes were identical to those of a strain isolated in the midgut of the ladybug *Harmonia axyridis* and were linked to the species *Staphylococcus sciuri*.

#### **3.4. Bacterial associations and relationship**

tives *Cronobacter* and *Pantoea*. Members of gamma-proteobacteria of the orders *Legionellales* (*Diplorickettsia*), *Oceanospirillales* (*Nitrincola*), *Alteromonadales* (*Shewanella*), *Xanthomonadales* (*Stenotrophomonas*) and *Aeromonadales* (*Aeromonas*) were also detected showing the very wide

**Figure 4.** Maximum Likelihood phylogenetic tree of the genus *Acinetobacter.* Lineages of strains detected in the mi‐ crobiota of *Anopheles* mosquitoes are in color, blue for Thailand, red for Vietnam. Bootstrap percentages (>50 %) af‐

The diversity was lower in alpha- and beta-proteobacteria. However, the genus *Sphingomo‐ nas* that belonged to *Alphaproteobacteria*, was the second main genus detected in this study (26% of the *Anopheles* species colonized) mostly represented by sequences affiliated or related to the species *Sphingomonas aromaticivorans* and *Sphingomonas glacialis*. Acetic-acid bacteria (*Asaia* and *Gluconacetobacter*) belonged to *Alphaproteobacteria* and were sporadically represented as well

as *Chromobacterium* and *Diaphorobacter*, the two members of *Betaproteobacteria*.

ter 100 resamplings are shown. Bar: 0.5 % sequence divergence.

diversity of gamma-proteobacteria in the midgut microbiota of *Anopheles*.

562 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

*Acinetobacter* spp. was present in all mosquito specimens except in *An. maculatus* and *An. dirus* from Thailand. Specimens of these two *Anopheles* species were mainly colonized by *Pseudomonas* and *Serratia* (Table 2). When the microbiota of each specimen is considered (data not shown), the pair *Pseudomonas* / *Serratia* never co-habited with *Acinetobacter* in the same midgut. *Pseudomonas* strains associated with *Serratia* were related to the species *P. fluorescens* whereas *P. alcaligenes* was never associated with *Serratia* and inhabited midguts colonized with *Acinetobacter*. These results suggested that the association *P. fluorescens / Serratia* might specifically inhibit the colonization of *Anopheles* midgut by *Acinetobacter*.

Negative relationships between *Sphingomonas* and enterobacteria were also suggested in Table 2 for mosquitoes from Vietnam. Considering each specimen, we always observed the absence of enterobacteria when *Sphingomonas* colonized the midgut (data not shown).

#### **3.5. Comparison of bacterial diversity in the midgut of** *Anopheles* **from Thailand and Vietnam**

Table 2 and Figure 5 showed the differential distribution of bacterial genera according to the geographic origin of mosquitoes. Eight genera were shared between specimens from Thailand and Vietnam and corresponded to genera with high prevalence such as *Enterobacter*, *Serratia*, *Pseudomonas* and *Acinetobacter*. In Thailand, each of these four genera colonized more than 10% of specimens, each of the genera *Raoultella*, *Cronobacter*, *Aeromonas*, *Elizabethkingia* and *Asaia* colonized 3 to 10% of the specimens, and 10 other genera colonized 2% or less of the specimens (Figure 5A).

Except for the core genus *Acinetobacter*, main genera found in Thailand were not prevalent in specimens from Vietnam, *Enterobacter*, *Serratia*, *Pseudomonas, Raoultella*, and *Asaia* colonizing each 2% or less of the Vietnam specimens (Figure 5B). *Cronobacter*, *Aeromonas* and *Elizabeth‐ kingia* were not detected in *Anopheles* mosquitoes from Vietnam. Except for the genus *Acine‐ tobacter* again (40%), the more prevalent genera in specimens from Vietnam appeared originspecific. Indeed, *Sphingomonas* and *Moraxella* present in *Anopheles* from Vietnam at 36% and 3% respectively, were not detected in mosquitoes from Thailand (Figure 5B). When the species forming the genus *Acinetobacter* were considered, we observed again an origin-specific distribution with *A. junii* and *A. johnsonii* dominating the microbiota of mosquitoes from Vietnam but absent from the Thailand samples. Gut microbiota of mosquitoes from Thailand displayed a wider *Acinetobacter* diversity with four species represented, *A. baumannii*, *A*. *calcoaceticus*, *A. soli* and *A. guillouiae* (Figure 4). In the same phylogenetic clade of *Acinetobact‐ er*, bacterial lineages from Thailand mosquitoes differed from bacterial lineages of Vietnam mosquitoes. For instance, the lineages *A. baumannii* and *A. junii* belonged to the same clade in the 16S rRNA gene tree but inside this clade, each lineage was origin-specific (Figure 4).

**Figure 5.** Repartition in genera of OTU assigned bands obtained by PCR-TTGE from 175 specimens of *Anopheles* mos‐ quitoes from Thailand (A) and from Vietnam (B).

Considering bacterial taxa higher than the genus, the microbiotae of *Anopheles* from Thailand and Vietnam were both dominated by *Pseudomonadales* (Figure 6) due to the general high prevalence of *Acinetobacter*. *Enterobacteriaceae* largely dominated the microbiota of *Anopheles* from Thailand but contributed little to bacterial diversity in *Anopheles* from Vietnam. <sup>18</sup>

forming the genus *Acinetobacter* were considered, we observed again an origin-specific distribution with *A. junii* and *A. johnsonii* dominating the microbiota of mosquitoes from Vietnam but absent from the Thailand samples. Gut microbiota of mosquitoes from Thailand displayed a wider *Acinetobacter* diversity with four species represented, *A. baumannii*, *A*. *calcoaceticus*, *A. soli* and *A. guillouiae* (Figure 4). In the same phylogenetic clade of *Acinetobact‐ er*, bacterial lineages from Thailand mosquitoes differed from bacterial lineages of Vietnam mosquitoes. For instance, the lineages *A. baumannii* and *A. junii* belonged to the same clade in the 16S rRNA gene tree but inside this clade, each lineage was origin-specific (Figure 4).

**Figure 5.** Repartition in genera of OTU assigned bands obtained by PCR-TTGE from 175 specimens of *Anopheles* mos‐

(A)

564 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

(B)

quitoes from Thailand (A) and from Vietnam (B).

**Figure 6.** Repartition in significative high-level bacterial taxa of OTU from 175 specimens of *Anopheles* mosquitoes from Thailand (A) and from Vietnam (B). GP for *Gammaproteobacteria*.

This low prevalence of enterobacteria in the midgut of *Anopheles* from Vietnam was particu‐ larly noteworthy (Fig. 6B). In opposite, *Sphingomonadales* was the major high-level taxon in Vietnam specimens but absent from Thailand specimens. Therefore, the ratio *Enterobacteria‐ ceae/ Sphingomonadales* appeared as a signature differentiating Thailand and Vietnam *Anopheles* specimens. Other signatures, which should be confirmed with more specimens, were *Betapro‐ teobacteria* and *Actinobacteria* in specimens from Thailand and Vietnam, respectively.

#### **3.6. Links between microbiota composition and** *Anopheles* **species or species complexes**

Table 2 showed the distribution of bacterial genera according to the species of *Anopheles*. To evaluate the link between bacteria and host species, we first compared the microbiotae of the same mosquito species but from different origins. Specimens of *An. maculatus* gave a good model for this comparison because it was enough represented in both geographic sites (Figure 7). The two groups of microbiotae differed clearly, in particular considering the origin-specific signature, i.e. the ratio *Enterobacteriaceae / Sphingomonadales* (Figure 7). Therefore, the case of *An. maculatus* indicated that the microbiota composition was influenced by sampling geo‐ graphic sites rather than *Anopheles* species. Comparison of the microbiotae between *An. dirus* from Thailand and Vietnam resulted in the same conclusion (Table 2).

**Figure 7.** Comparison of the microbiota of *An. maculatus* caught in Thailand (n=11) (A) and in Vietnam (n=28) (B).

Sibling species within a group or a complex have been linked to the microbial content of the midgut. As previously observed for the species *An. maculatus*, the corresponding complex displayed a non-specific microbiota but its bacterial colonization is influenced by the geographic origin. Similar situation was observed in the Dirus Complex for which the shared bacterial genera were *Acinetobacter* and *Enterobacter*, as well as for *An. gigas* of the Gigas Complex with shared bacteria belonging to the dominating genera *Acinetobacter* and *Enterobacter*.

In the Minimus Complex, *An. minimus* and *An. harrisoni* were colonized by 18 different bacterial genera but only three were shared by both species. Two shared genera corresponded to bacteria widely represented in the whole population, *Acinetobacter* and *Pseudomonas*, while the third one, *Raoultella*, seemed to be more specific, represented in two and nine specimens of *An. minimus* and *An. harrisoni* respectively, showing its higher association to the latter species.

## **4. Discussion**

**3.6. Links between microbiota composition and** *Anopheles* **species or species complexes**

*Raoultella 6%*

from Thailand and Vietnam resulted in the same conclusion (Table 2).

*Pseudomonas 44%*

(A)

566 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

(B)

*Acinetobacter 38%*

*Asaia 2%*

*Moraxella 3%*

*Bacillus 3%*

**Figure 7.** Comparison of the microbiota of *An. maculatus* caught in Thailand (n=11) (A) and in Vietnam (n=28) (B).

Sibling species within a group or a complex have been linked to the microbial content of the midgut. As previously observed for the species *An. maculatus*, the corresponding

*Enterobacter 6%*

Table 2 showed the distribution of bacterial genera according to the species of *Anopheles*. To evaluate the link between bacteria and host species, we first compared the microbiotae of the same mosquito species but from different origins. Specimens of *An. maculatus* gave a good model for this comparison because it was enough represented in both geographic sites (Figure 7). The two groups of microbiotae differed clearly, in particular considering the origin-specific signature, i.e. the ratio *Enterobacteriaceae / Sphingomonadales* (Figure 7). Therefore, the case of *An. maculatus* indicated that the microbiota composition was influenced by sampling geo‐ graphic sites rather than *Anopheles* species. Comparison of the microbiotae between *An. dirus*

> *Serratia 44%*

*Sphingomonas 54%*

To our knowledge, this study describes the midgut microbiotae of the largest population of field-collected *Anopheles* species with 10 species (Table 2) when the literature shows 9 analyzed species, *An. gambiae* being the most studied species of all (Table 1). Thereby, 16S rRNA gene PCR-TTGE focused on hypervariable region V3 proves its efficiency to study microbiota of *Anopheles* mosquitoes. This method, that presents a relative low resolution, is efficient to follow bacterial communities with low to moderate diversities. This limit is due to the number of bands that can be separated within the length of the gel. Optimization of TTGE conditions allows separation of bands by a minimum of 0.1 mm over all the gel length. Therefore, TTGE would be difficult to interpret if the diversity exceeds 25 to 30 OTUs [45]. Microbiotae of *Anopheles* displays TTGE profiles that do not exceed 10 bands but the profiles have been interpreted with difficulties due to heterogeneities in rRNA genes for most bacteria in the mosquito midgut ecosystem. At the genomic level, rRNA genes are generally organized in multigene families [68] in which sequences show low variability within species, subspecies or genome [69]. However, intra-genomic heterogeneity in the form of nucleotide differences between 16S rRNA gene copies are described in relation to fine-tuning of the ribosome function to optimize bacterial niche fitness [70]. In PCR-TTGE, heterogeneities lead to multiple bands for a single OTU and then to an overestimation of OTU diversity. This pitfall has been avoided here by band sequencing that led to the definition of a refined diversity index drastically lowered in comparison with the raw diversity index. The level of ribosomal heterogeneity in bacteria genome from midgut of mosquitoes suggested adaptation processes in a rather instable niche.

With the development of high-output sequencing, twenty-one century metagenomics consider fingerprint approaches as obsolete. However, these methods remain of great interest to give a snapshot of microbiota in large populations of hosts. Thereby, we described herein the midgut microbiotae of 175 specimens of 10 *Anopheles* species with a sequencing effort of less than 150 reads compared to 5 millions of reads estimated for the same study by pyrosequenc‐ ing. A pyrosequencing study of the midgut microbiota of *An. gambiae*(30 laboratory breed and two field-collected mosquitoes) has been recently published [18]. Authors described bacteria belonging to 26 phyla, among which, five represented more than 99% of the total microbiota: *Proteobacteria*, *Bacteroidetes*, *Actinobacteria*, *Firmicutes*, and *Fusobacteria*. Except the latter, four phyla corresponded to those described in this study, suggesting that PCR-TTGE explored the majority of bacterial populations in the microbiota. Among 147 OTUs detected by pyrose‐ quencing, only 28 genera had an abundance of >1% in at least one mosquito midgut [18]. This is in accordance with our results describing 31 bacterial genera in the microbiota of fieldcollected *Anopheles*. Fourteen of the 31 genera have been previously detected in diverse studies on field-collected *Anopheles*(Table 1). Then, we would like to highlight the fact that 17 bacterial genera were described herein for the first time (Table 2), 6 (32%) and 12 (60%) from the populations of Thailand and Vietnam respectively, suggesting that the bacterial diversity associated to midgut of *Anopheles* remains underestimated. It is noteworthy that twice as many new genera were found in specimens from Vietnam compared to Thailand. Newly described genera were scarcely represented in few specimens except for *Sphingomonas* found in 46 specimens belonging to five species from Vietnam and *Raoultella* found in 13 specimens belonging to 3 species from Thailand and Vietnam. Of note the genus *Sphingomonas* has been detected by pyrosequencing in the midgut of a population of *An. gambiae* maintained in standard insectary conditions [18].

The gut microbiota of mosquitoes presented a large inter-specimen variability but was dominated by few genera, *Acinetobacter*, *Pseudomonas*, *Enterobacter*, *Serratia*, *Raoultella* and *Sphingomonas*. Among them, *Acinetobacter* was considered as a mosquito midgut core genus because it was detected in most specimens. *Acinetobacter*, *Pseudomonas* and *Sphingomonas* also belong to the *An. gambiae* midgut core microbiota defined by Boissière et al. [18]. *Asaia* was found in all samples by pyrosequencing but its relative abundance showed great variations ranging from 0.04 to 98.95% according the *An. gambiae* specimen [18]. We detected *Asaia* in only 6 specimens of *An. maculatus* and *An. harrisoni*. Again, our result compared to pyrose‐ quencing data suggested that PCR-TTGE failed to detect minority and/or low loaded popu‐ lations. This low resolution is certainly a limit but we also see it as a benefit because the majority taxa detected by TTGE probably corresponds to true colonizers of the midgut and not to transient or contaminant bacteria.

*Anopheles*-associated bacterial species recently described were not detected or identified with confidence in this study. Members of the genus *Elizabethkingia* detected in *Anopheles* from Thailand could not be identified as *E. anophelis* owing to its relatedness in 16S rRNA gene sequence with *E. meningosepticum*. Of note, *Thorsellia anophelis* has been detected in mosquitoes used in the optimization step of this study but not in mosquitoes included in the study population.

In spite of the large inter-specimen variability, sub-populations from different geographic origins exhibit drastically different midgut microbiotae. High prevalence of *Enterobacteriacea* and absence of *Sphingomonas* spp. characterize microbiotae of *Anopheles* caught in Thailand whereas *Anopheles* in Vietnam displayed high prevalence of *Sphingomonas* and low rate of enterobacteria. Similar differences in enterobacteria prevalence have been described in *An. gambiae* originating from two sampling sites in Cameroon [18]. Composition of the midgut microbiota seems unrelated to *Anopheles* species, except for *Raoultella* and *An. harrisoni* but their relationship needs to be confirmed on additional specimens. Some positive and negative associations of bacteria suggested complex interactions in the microbiota. The most striking result was the pair *P. fluorescens* / *Serratia* which never co-habited with *Acinetobacter*. *Pseudo‐ monas fluorescens* is well known as a great anti-microbial and bacteriocin-like substances producer [71] exhibiting negative effect on diverse Gram-negative bacteria and biofilm formation [72]. The bacteriocins are narrow-spectrum toxins that typically kill bacteria related to the producing strain as is the case for *P. fluorescens* and *Acinetobacter*, which both belong to the order *Pseudomonadales*. Moreover, bacteriocins can play an important role in the fitness of a strain by killing or inhibiting bacterial co-inhabitants that compete for the limited resources probably found in the midgut environment [71]. Similar antagonism was observed between *Sphingomonas* and enterobacteria in mosquitoes from Vietnam. *Sphingomonas* is a sparsely known genus but antimicrobial activities against *Candida* have been described recently [73]. Culture of the natural isolates of *P. fluorescens*, *Serratia*, *Acinetobacter* and *Sphingomonas* should confirm these potential antagonisms and give insights about their mechanism.

Antagonism against enterobacteria is of particular interest because it has been suggested that mosquitoes harboring *Enterobacteriacae* are more likely to be infected by *P. falciparum* [18]. In our collection, *An. minimus* specimen KAN-27 from Pu Teuy, Kanchanaburi was infected by *P. falciparum* and displayed a microbiota containing exclusively enterobacteria that belonged to four genera, *Pantoea, Enterobacter, Cronobacter* and *Escherichia*. This specimen displayed the highest enterobacterial diversity of the *Anopheles* collection and the core genus *Acinetobacter* was not detected. Identification of the *Enterobacter* species in our samples will be the next step with the search for *Enterobacter* (Esp\_Z), which was reported to inhibit *P. falciparum* develop‐ ment in *An. gambiae* [15]. As the microbiota might have an impact on pathogen development in *Anopheles* mosquitoes and disease transmission, more studies need to be done for better understanding the role of some specific bacteria in wild mosquito populations before devel‐ oping potential method of control.

## **5. Conclusion**

belonging to 26 phyla, among which, five represented more than 99% of the total microbiota: *Proteobacteria*, *Bacteroidetes*, *Actinobacteria*, *Firmicutes*, and *Fusobacteria*. Except the latter, four phyla corresponded to those described in this study, suggesting that PCR-TTGE explored the majority of bacterial populations in the microbiota. Among 147 OTUs detected by pyrose‐ quencing, only 28 genera had an abundance of >1% in at least one mosquito midgut [18]. This is in accordance with our results describing 31 bacterial genera in the microbiota of fieldcollected *Anopheles*. Fourteen of the 31 genera have been previously detected in diverse studies on field-collected *Anopheles*(Table 1). Then, we would like to highlight the fact that 17 bacterial genera were described herein for the first time (Table 2), 6 (32%) and 12 (60%) from the populations of Thailand and Vietnam respectively, suggesting that the bacterial diversity associated to midgut of *Anopheles* remains underestimated. It is noteworthy that twice as many new genera were found in specimens from Vietnam compared to Thailand. Newly described genera were scarcely represented in few specimens except for *Sphingomonas* found in 46 specimens belonging to five species from Vietnam and *Raoultella* found in 13 specimens belonging to 3 species from Thailand and Vietnam. Of note the genus *Sphingomonas* has been detected by pyrosequencing in the midgut of a population of *An. gambiae* maintained in

The gut microbiota of mosquitoes presented a large inter-specimen variability but was dominated by few genera, *Acinetobacter*, *Pseudomonas*, *Enterobacter*, *Serratia*, *Raoultella* and *Sphingomonas*. Among them, *Acinetobacter* was considered as a mosquito midgut core genus because it was detected in most specimens. *Acinetobacter*, *Pseudomonas* and *Sphingomonas* also belong to the *An. gambiae* midgut core microbiota defined by Boissière et al. [18]. *Asaia* was found in all samples by pyrosequencing but its relative abundance showed great variations ranging from 0.04 to 98.95% according the *An. gambiae* specimen [18]. We detected *Asaia* in only 6 specimens of *An. maculatus* and *An. harrisoni*. Again, our result compared to pyrose‐ quencing data suggested that PCR-TTGE failed to detect minority and/or low loaded popu‐ lations. This low resolution is certainly a limit but we also see it as a benefit because the majority taxa detected by TTGE probably corresponds to true colonizers of the midgut and not to

*Anopheles*-associated bacterial species recently described were not detected or identified with confidence in this study. Members of the genus *Elizabethkingia* detected in *Anopheles* from Thailand could not be identified as *E. anophelis* owing to its relatedness in 16S rRNA gene sequence with *E. meningosepticum*. Of note, *Thorsellia anophelis* has been detected in mosquitoes used in the optimization step of this study but not in mosquitoes included in the study

In spite of the large inter-specimen variability, sub-populations from different geographic origins exhibit drastically different midgut microbiotae. High prevalence of *Enterobacteriacea* and absence of *Sphingomonas* spp. characterize microbiotae of *Anopheles* caught in Thailand whereas *Anopheles* in Vietnam displayed high prevalence of *Sphingomonas* and low rate of enterobacteria. Similar differences in enterobacteria prevalence have been described in *An. gambiae* originating from two sampling sites in Cameroon [18]. Composition of the midgut microbiota seems unrelated to *Anopheles* species, except for *Raoultella* and *An. harrisoni* but their

standard insectary conditions [18].

568 *Anopheles* Anopheles mosquitoes - New insights into malaria vectors mosquitoes - New insights into malaria vectors

transient or contaminant bacteria.

population.

Based on the analysis of the midgut microbiota of 10 field-caught *Anopheles* species from Thailand and Vietnam, we described 17 bacterial genera for the first time in *Anopheles* mosquitoes, suggesting that the bacterial diversity associated to midgut of *Anopheles* remains underestimated. Low bacterial diversity ranging from one to three per specimen was found which contrasted with a high OTU diversity in the whole *Anopheles* population that presented 31 different bacterial genera distributed in four phyla, *Proteobacteria, Bacteroidetes/Chlorobi, Firmicutes,* and *Acinetobacteria*. More specifically, the association of *Pseudomonas* and *Serratia* never co-habited with *Acinetobacter* in the same mosquito midgut. The same presence/absence was observed between *Sphingomonas* and enterobacteria. Midgut microbiota was drastically different for the *Anopheles* from Thailand compared to those from Vietnam showing the importance of the geographic origin. The ratio *Enterobacteriaceae / Sphingomonadales* appeared as a signature differentiating the *Anopheles* specimens from Thailand and Vietnam.

## **Acknowledgements**

We are thankful to Naritsara Malaithong and Wanapa Ritthison, students of the Department of Entomology (Kasetsart University, Bangkok, Thailand) for their involvement in the optimization step of this study. This study was funded by the French Ministry of Foreign Affairs in the framework of the "Partenariat Hubert Curien" - PHC Thai project 20627SD.

## **Author details**

Sylvie Manguin1\*, Chung Thuy Ngo1,2, Krajana Tainchum3 , Waraporn Juntarajumnong3 , Theeraphap Chareonviriyaphap3 , Anne-Laure Michon4 and Estelle Jumas-Bilak4

\*Address all correspondence to: sylvie.manguin@ird.fr

1 Institut de Recherche pour le Développement (IRD), Faculté de Pharmacie, Montpellier, France

2 National Institute of Veterinary Research, Ha Noi, Viet Nam

3 Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand

4 University Montpellier, Equipe Pathogènes et Environnements, Faculté de Pharmacie, Montpellier, France

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**Author details**

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Sylvie Manguin1\*, Chung Thuy Ngo1,2, Krajana Tainchum3

\*Address all correspondence to: sylvie.manguin@ird.fr

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We are thankful to Naritsara Malaithong and Wanapa Ritthison, students of the Department of Entomology (Kasetsart University, Bangkok, Thailand) for their involvement in the optimization step of this study. This study was funded by the French Ministry of Foreign Affairs in the framework of the "Partenariat Hubert Curien" - PHC Thai project 20627SD.

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