The complicated induced plant defenses may affect the performance and preference of subsequent herbivores, by affecting the feeding, settling, growth, or oviposition ability of another insect (Karban and Carey, 1984; Leitner et al., 2005). For instance, the plant may become more sensitive to another herbivore after previous attacks by other herbivores. Meanwhile, the phloem-feeding insects were susceptible to the change of nutrient substance and resistant material in the phloem sap (Denno and Roderick, 1992). A substantial number of secondary metabolites in plants induced by pests are extremely detrimental to the growth performance of insects, like terpenoids, phenolic compounds, and alkaloids (Chen et al., 2015). The resistance of insects to the secondary metabolites is due to various biochemical and physiological characteristics of the midgut. The midgut of insects can secrete various digestive enzymes (e.g., proteases, lipases, amylases, sucrases, trehalases) and defense enzymes (superoxide dismutase, catalase, and peroxidase), which occupy a crucial role for insects to combat plant defenses.

The whitefly Bemisia tabaci (Gennadius) is a phloemfeeding pest that causes extensive damage (De Barro et al., 2011; Liu et al., 2012). Due to the wide host range, intense competition exists between the invasive species B. tabaci and other local pests (De Barro et al., 2011; Rao et al., 2011). The carmine spider mite Tetranychus cinnabarinus (Boisduval) is a polyphagous pest that attacks crops, vegetables, and flowers. In particular, the economic yield of cucumbers decreases significantly after heavy spider mite infestations (Sertkaya et al., 2010; Park and Lee, 2016). The spider mite remains a cell-content feeder, which can puncture the host plant leaf epidermis cells and consume the contents of the mesophyll cells. Typically, the mite induces defenses of both the SA and JA pathways (Ament et al., 2004). Moreover, T. evansi down-regulates tomato defensive compounds, and this is correlated with a significantly more excellent performance of herbivores on plants pre-infested by mites of this species (Kant et al., 2008; Alba et al., 2015; Godinho et al., 2016). The mealybug Phenacoccus solenopsis (Tinsley) represent an aggressively invasive species on agricultural and ornamental plants in China. It is an obligate phloem-feeding pest that possesses a specialized stylet and causes minimal damage as it obtains nutrition from plants through the vascular tissue (Will et al., 2013; Huang and Zhang, 2016). All three pests have broad host distributions, and can damage the same crops in the field. Previous studies have shown diverse pests that eat plants often activate different signaling pathways (Zhang et al., 2011). Here we pre-infested cucumbers with T. cinnabarinus and P. solenopsis, insects with different foraging patterns, to explore the impact on B. tabaci.

# MATERIALS AND METHODS

#### Plant and Insects Plant

Cucumber, Cucumis sativus L. (Jinongjiejiegua, Tianjin, China), was the host plant for this experiment. The plants used in the tests were approximately 30 cm in height and had 4–6 true leaves.

#### Insects

The spider mites, T. cinnabarinus, were maintained on broad bean plants (Vicia faba). The mealybugs, P. solenopsis, were cultured on potato plants (Solanum tuberosum). The whiteflies, B. tabaci (MED), were cultured on cucumber plants (Cucumis sativus).

The experiments were conducted at 25 ± 2 ◦C, 65 ± 10% RH and a photoperiod of 16 h:8 h (L:D) with artificial lighting in a greenhouse.

# The Settling and Oviposition of Whitefly

The leaf disks were pre-infested by five 3rd-instar female mealybugs or five red mite females for 12 h. Then, five couples of new emerged whiteflies were placed at the middle of the plastic pipe and allowed to fly to either side. The positions of the two leaf disks were alternated among replicates. Each experiment was replicated 15–30 times. In the experiment, the location of B. tabaci was counted every 12 h. After 72 h post inoculation (hpi), the number of laid eggs deposited on each leaf disk was recorded. In total, there were 15–30 replicates in each treatment. The ratios of the whitefly number of landing on the plant to the total number of releasing in the test were subjected to a t-test (SPSS version 18.0).

# Enzymatic Assay of Defensive Enzymes in Host Plants

The activity of defensive enzymes in plants was tested as follows: (1) T(+), each plant pre-infested by 25 T. cinnabarinus females for 12 h and continue feed for 3 days; (2) P(+), each plant preinfested by 25 P. solenopsis for 12 h and continue feed for 3 days; (3) T(+)+B, after pre-infested for 12 h, the T. cinnabarinus were kept and B. tabaci was infested for 3 days; (4) P(+)+B, after preinfested for 12 h, the P. solenopsis were kept and B. tabaci was infested for 3 days; (5) CK, the plants were uninfected but were kept for the same time and were used as controls.

TABLE 1 | The specific primers for qRT-PCR.

fphys-10-00346 April 2, 2019 Time: 17:28 # 3

Enzymatic assay of PAL was determined according to the procedure of Burrell and Rees (1974). The enzymatic assay of LOX is slightly modified according to the method described by Surrey (1964). This experiment contains 5–8 biological experiments with three technical repetitions.

#### Quantitative Real-Time PCR

We quantified the transcript levels of several genes in the leaves treated. Leaves of the treated and untreated plants were collected, and total RNA was extracted consuming the RNAiso Plus reagent (TaKaRa, Dalian, China). The cDNA was synthesized using the PrimeScript R RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). For the relative quantification of gene expression, the comparative CT method (Livak and Schmittgen, 2012) was used with BIO-RAD CFX96 real-time PCR system (Bio-Rad, United States). The amount of the target was normalized to the endogenous reference gene TUA. The specific primers of all aimed genes for qRT-PCR were showed in **Table 1**. For qRT-PCR, three biological replicates were carried out, and triplicate quantitative assays for each replicate were performed.

#### The Digestive Enzymes, Detoxification Enzymes, and Protective Enzymes in Whiteflies

Here, the detoxification, digestive and protective enzymes in whiteflies were also tested to reveal the effect of induced plant response. The different treatments were as follows: (1) T(+)+B (described above); (2) P(+)+B (described above); (3) Whitefly (the plants that were uninfected but were kept for the same time, and then 25 couples of whiteflies were allowed to feed on the plants as a control). The whiteflies were collected at the end of the experiment and each treatment was repeated 8–10 times.

For the determination of trehalose and sucrase activity, the dinitrosalicylic acid method was adopted (Zhang et al., 2014). For the determination of superoxide dismutase (SOD) activity, the pyrogallol auto-oxidation method was used (Xu et al., 2006). The molybdate colorimetric method was used for determination of catalase (CAT) activity (Cheng and Meng, 1994). The glutathione S-transferase (GST) activity was measured according to Habig and Jakoby (1981). The carboxylesterase (CarE) activities were performed with slight modifications according to Stumpf and Nauen (2002).

#### RESULTS

#### Effects of Mealybug or Spider Mite Infestation on B. tabaci Response

The percentages of B. tabaci that settled on the treatments with T. cinnabarinus were significantly more than those on the control treatments without T. cinnabarinus (12 h, P = 0.008; 24 h, P = 0.002; 36 h, P = 0.003; 48 h, P = 0.009; 60 h, P = 0.007; 72 h, P = 0.007) (**Figure 1**). In contrast, less whiteflies settled on the plants infested with P. solenopsis (12 h, P = 0; 24 h, P = 0.001; 36 h, P = 0; 48 h, P = 0.001; 60 h, P = 0.005; 72 h, P = 0.014) (**Figure 1**). In all, B. tabaci were more likely to choose the leaf disk with T. cinnabarinus treatment than with P. solenopsis treatment. After 3 days, the total laid eggs were significantly higher in the plants with T. cinnabarinus than those on the leaf disk with no treatment (**Figure 2**). In contrast, the total eggs were significantly lower in the plants with P. solenopsis than control (**Figure 2**).

#### The Digestive Enzymes, Detoxification Enzymes, and Protective Enzymes in Whiteflies

According to **Table 2**, we found the trehalose, SOD and CAT activities in B. tabaci from cucumbers infected by mites were higher than those of whiteflies from the uninfected cucumber (P < 0.05). However, the sucrase and GST activities were significantly lower than those of whiteflies reared in cucumbers infected by mites (P < 0.05). The CarE activity of B. tabaci from cucumber fed on by mealybugs was higher than that of whiteflies from cucumbers infected by mites and the control. Similarly, the sucrase activity in whiteflies was significantly lower from cucumber infected by mealybugs than that of whiteflies in the control (P < 0.05).

#### Estimation of Phenylalanine Ammonialyase and Lipoxygenase Activity in Plants

The activity of PAL but not LOX was significantly increased through the induced response in cucumbers fed on by spider mites after 3 days. However, the activities of LOX and PAL in cucumbers were significantly increased by feeding mealybugs. The activities of LOX and PAL in cucumbers were both significantly increased after the whiteflies coexisted with mites for 3 days. Nevertheless, the activities of LOX and PAL in cucumbers were not significantly increased after the whiteflies coexisted with mites for 3 days (**Figure 3**).

#### Changes in Gene Expression

To assess cucumber response to five different combinations of the insect feeding treatments, time course experiments were conducted in which transcript levels of one ETdependent gene (ETR), two JA-dependent genes (LOX2 and

OPR3) and SA-dependent genes (PAL, PR-1, and PR-5) were monitored (**Figure 4**).

Ethylene receptor gene (ETR) is a marker gene of ET signal pathways (Shoresh et al., 2005). The relative expression levels of ETR did not significantly change in the cucumber leaves infested with T. cinnabarinus only or P. solenopsis only after 3 days. However, the transcript levels of ETR in the cucumber leaves induced by T. cinnabarinus were repressed in leaves infested with both T. cinnabarinus and B. tabaci compared to undamaged leaves (P < 0.05) (**Figure 4**).

Lipoxygenase (LOX) is typically a key enzyme in the octadecanoid pathway of the JA biosynthesis (Hu et al., 2009). All treatments apparently upregulated the transcript levels of LOX compared to undamaged leaves. However, the level of LOX induction in leaves infested with T. cinnabarinus was significantly lower than other treatments (**Figure 4**). The oxophytodienoate reductase (OPR3) gene is one marker of the JA upstream genes (Ramírez et al., 2012). The level of the OPR3 gene infested with either P. solenopsis or B. tabaci was significantly higher than other treatments (P < 0.05) (**Figure 4**).

PR-1 and PR-5 are known as two pathogenesis-related genes, which are mainly regulated by the SA signaling pathway (Zhang P.J. et al., 2015). Phenylalanine ammonia lyase (PAL), a ratelimiting enzyme of the phenylpropanoid pathway, is the key enzyme in the biosynthetic pathway of SA and plays an important role in plant development and defense (Hu et al., 2009; Wang et al., 2013; Zhang et al., 2016). The abundance of PAL, PR1, and PR5 transcripts was significantly increased in the cucumber leaves infested with P. solenopsis only after 3 days, but not in the leaves infested with T. cinnabarinus only (P < 0.05; **Figure 4**). In addition, the leaves infested with P. solenopsis and B. tabaci showed a marginally significant increase of the expression of PAL and PR5 in comparison with undamaged leaves. However, the leaves infested with P. solenopsis and B. tabaci witnessed a significant reduction of the expression of PR1 compared to undamaged leaves (**Figure 4**).

# DISCUSSION

From the perspective of temporal and spatial interactions, intraspecific insect interactions affect the population performance, especially because herbivores may affect the

TABLE 2 | The digestive enzymes, detoxification enzymes and protective enzymes in whiteflies.

Paired means ( ± SE) with different letters above bars indicate significant differences in the quantities between control and treatment (ANOVA, P < 0.05).

FIGURE 3 | The activity of two defense enzymes in each treatment on cucumber. Different letters above bars indicate significant differences in the quantities between control and treatment (ANOVA, P < 0.05).

performance of subsequent herbivores on the same plant (Green and Ryan, 1972; Hunter and Price, 1992; Messina et al., 2002; Beale et al., 2006; Xue et al., 2010; Tan and Liu, 2014). Our result showed whiteflies were extremely sensitive to pre-infestation with mealybugs, though there were few previous reports of mealybugs. In contrast, the plants pre-infested with T. cinnabarinus attracted more whiteflies than the plants with no mites. This may depend on the combination of the activation of defensive proteins and volatile organic compounds (Smith and Boyko, 2007; Howe and Jander, 2008), as well as the species of pests and feeding sequences of herbivore insects (Inbar and Gerling, 2008; Erb et al., 2011). Plants known to be impacted by herbivores are capable of producing a series of volatile substances that trigger an indirect plant defense. How plants respond to infestation by multiple herbivores, particularly if these belong to different feeding guilds, is important. The pattern of the plant defense is crucially important for regulating the behavior of herbivorous pests (Walling, 2000). Transcriptomic evidence has demonstrated that chewing insects activate the JA pathway, whereas sucking pests usually elicit the SA pathway.

Next, a cell-content feeder may induce defenses of both the SA and JA pathways (Zhang et al., 2009). In fact, inconsistencies in induced plant defense depend on the species of the pest or the host plant species (Walling, 2000; Diezel et al., 2009). The induced defense pathway is always considered to promote the production of secondary metabolites and is very unfavorable to insects (e.g., affecting pest feeding and digestion) (Walling, 2000; Jormalainen et al., 2001). In addition, the contents of the SA, total phenolic, and tannins were negatively correlated to the fecundity of B. tabaci. Simultaneously, the increase of plant secondary metabolites and the lack of nutrients may affect the feeding preference of B. tabaci.

Numerous investigations indicate that sucking insects activate the SA signaling defenses and inhibit the JA pathway more easily (Kempema et al., 2007; Poelman et al., 2012). For example, B. tabaci activate SA signaling and inhibit JA-dependent responses (Kempema et al., 2007; Zarate et al., 2007; Zhang et al., 2013a). P. solenopsis can limit the expression of JAdepended genes and defensive substances to enhance its nymph adaptive ability on cotton. Specifically, mealybugs could induce the expression of JA-related genes but reduce the production of JA-regulated defensive substances, like gossypol on cotton (Zhang et al., 2011). Due to the particular interaction between JA-SA, mealybugs can enhance the host adaptability of its nymphs (Zhang X. et al., 2015), but not for the performance of B. tabaci. Here, the result showed expression level of LOX was significantly higher in P. solenopsis- infested plants compared to undamaged plants. However, T. cinnabarinus had less ability to induce LOX genes in comparison to P. solenopsis. T. cinnabarinus remain a typical cell-content feeder that may activate JA and SA pathways. Previous research confirmed that insects and mites could suppress the JA pathway to enhance their performance on the same plant, but maybe not for another species (Sarmento et al., 2011; Glas et al., 2014). Here, P. solenopsis significantly upregulated the expression level of the PR genes and PAL gene, a downstream gene regulated by SA. However, T. cinnabarinus appeared to be following the reverse strategy. Although it has previously been reported, that mealybugs and aphids can upregulate PR genes after feeding on hosts (Zhang X. et al., 2015). The direct and substantial roles of PR proteins in defense against various threats (including insects) show diversified mechanisms (Ebrahim et al., 2011), which are not very clear against the phloem-feeding insects. In contrast, JA-regulated genes may perform a crucial role in preventing pest invasions (Rahbé and Febvay, 1993). In our study, a significantly higher level of the LOX gene was observed on leaves pre-infested with mites or mealybug after 3 days. The results suggested the effectiveness of plant defenses elicited by upregulating the level of LOX gene against P. solenopsis' invasion. Taken together, it is important to emphasize that JA/ET-induced defenses carry out an important role in the successful defense against the phloem-feeding pest, like B. tabaci. The P. solenopsis maintains a 'stealthy feeding strategy' that avoids massive cell damage, which may produce insufficient JA levels for gene induction (De Vos et al., 2006). There is antagonism between SA and JA, and the SA signaling pathway may inhibit the JA signaling pathway (Doares et al., 1995).

Previous studies have revealed pests always accumulate SA at the site of oviposition, which may interfere with the accumulation of JA (Bruessow et al., 2010). Therefore, the high content of PR protein not only does not adversely affect B. tabaci, but rather is conducive to reproduction and development (McKenzie et al., 2002). In addition, the leaves infested with P. solenopsis and B. tabaci showed a marginally significant increase of the expression of PAL and PR5 by comparison with the undamaged leaves. However, the leaves infested with T. cinnabarinus and B. tabaci witnessed a significant reduction in the expression of PR1 compared to undamaged leaves. Thus, the high expression of SA regulatory genes did not favor the selection and oviposition of B. tabaci. Another study confirmed that whitefly pre-infestation of the tomato reduced the fecundity of subsequent whiteflies (Cui et al., 2012). Moreover, JA-mediated defenses may in addition cause an effect on egg development (Bruinsma et al., 2007). This suggests that inhibition of plant defense is of importance to phloem feeders, allowing them to effectively utilize the host.

The cross-talk between JA-SA has been demonstrated adequately in the past. Studies have speculated insects can take advantage of this cross-talk to address plant defenses to enhance host adaptability (Zhang et al., 2013b). Like JA, it is equally common for ET signals in plants to be activated by insects. However, ET is not so much a direct stimulus as an indirect regulator, that rarely directly adjust the corresponding plant defense (Dahl and Baldwin, 2007; Diezel et al., 2009). In addition, the ET pathway may equally affect genes regulated by JA in numerous plants (Zhu-Salzman et al., 1998; Royo et al., 1999; Stotz et al., 2000; Winz and Baldwin, 2001). Moreover, the interference between plant hormones allows plants to minimize their own cost of capacity, thereby developing a flexible signaling pathway that is more effective against intruders (Zhang et al., 2009). The ability of plants to regulate signaling pathways may, in turn, provide benefits to other invasive pests (Sarmento et al., 2011; Glas et al., 2014). Comparing to the leaves infested with T. cinnabarinus only, B. tabaci caused a marginally significant reduction of the level of ETR and PR1. However, it facilitated the expression level of LOX, a JA-regulated gene.

Insects equally possess a range of defense strategies to adapt to harsh environments, including the adaptations of detoxifying enzymes, digestive enzymes and protective enzymes (Ahmad et al., 1986; Jongsma et al., 1995; Pearse et al., 2013). B. tabaci, as a sucking pest, will inevitably exchange material with the plant phloem. It is properly known that plants fed by pests can induce the production of secondary metabolites. These substances can stimulate the protective enzymes and detoxification enzymes of B. tabaci, affect the midgut digestive enzymes, and thus their feeding and digestion and absorption capacity (Green and Ryan, 1972; Ahmad et al., 1986). The difference in detoxifying enzymes frequently affects the interspecific competition between B. tabaci and other populations (Julian et al., 2003; Liang et al., 2007; Liu et al., 2008). In this study, the activity of GST in whiteflies was significantly inhibited after feeding by mites, and the activity of CarE increased significantly after co-existence of whiteflies and mealybugs. Previous studies have shown CarE is important for insect resistance and can detoxify a variety of environmental toxicants. Therefore, this study demonstrated that the increase in

CarE activity of B. tabaci may be one of the adaptation measures for it against adverse environmental conditions.

Insects advocate broader tolerance to secondary metabolites, and this tolerance is determined by a variety of biochemical and physiological characteristics of the midgut. As one of the most key components of plant phloem, sucrose can supply energy for insects. Not only that, it can maintain osmotic pressure balance for sucking insects to ensure insects survivein a high osmotic pressure environment (Becker et al., 1996). Trehalose is known as the sugar of life. Its vital function is to synthesize chitinase and participate in the metabolism of insects, therefore providing energy for insect host positioning, courtship, mating, resisting adversity, spawning and other activities (Tang et al., 2012; Qin et al., 2015). Therefore, host adaptability may be reflected by the activity of trehalase and invertase in insects. B. tabaci may respond to the unfavorable situation by increasing trehalase activity. However, the activity of trehalase in whiteflies was inhibited on the plants infected by mealybugs, which may affect the behaviors of B. tabaci, like the oviposition behavior. It was only significantly increased after the pre-infestation of mites. Therefore, it was adequately demonstrated that the presence of mealybugs may cause an undesirable effect on the performance of B. tabaci, like the abilities of digestion and feeding. In addition, trehalase is also involved in feeding regulation and provides energy for insect flight behavior, which may be directly related to the tendency of B. tabaci in the field to select a host (Shi et al., 2016).

SOD can scavenge superoxide anion radicals and prevent damage to the body, but the generated hydrogen peroxide is still harmful to the insect midgut, however, CAT and POD in the body will immediately dispose of it (Tang et al., 2016). The protective enzyme activities of B. tabaci increased significantly after coexisting with T. cinnabarinus. However, this phenomenon does not occur when coexisting with mealybugs.

Because of the broad distribution and overlapping of hosts, all three pests in this study are extremely likely to erupt simultaneously on the consenting host plants in the field. Therefore, exploring the differences in plant defenses induced by different pests helps to reveal an understanding of the rules

#### REFERENCES

and mechanisms of pest invasion in the field. Despite previous reports of competition between mealybugs and whiteflies or spider mites and whiteflies, there has been increasing interest in the interspecies relationships between sap-feeding insects and the interspecific effects between mites and sucking insects (Godinho et al., 2016). In this study, T. cinnabarinus was more likely to activate plant genes regulated by JA, while mealybugs were more likely to activate key genes regulated by SA. Either may be one of the chief factors that affect the resistance of host plants to B. tabaci. Moreover, B. tabaci was more adaptable to plants coexisting with T. cinnabarinus, which may be attributed to the increase in digestive enzyme activity and protective enzyme activity. This result is of great significance to the layout of field crops and pest control or prevention. It additionally provides a firm basis for exploring the functions of the detoxification enzymes GST, trehalase and the protective enzyme systems in the anti-defense of B. tabaci. Through the further study of plant defense mechanisms, it is of great significance to understand the direct interaction of pests as well as the monitoring and control of field pests. These will support us to take effect early warning measures as early as possible and use natural laws to prevent and control natural disasters.

#### AUTHOR CONTRIBUTIONS

QR and HW conceived and designed the experiments. DL, YX, XL, LZ, and JW performed the experiments. DL and QR analyzed the data. DL and QR drafted the manuscript. All authors read and approved the final manuscript.

#### FUNDING

This work was supported by Natural Science Foundation of Zhejiang Province, China (LY14C140003) and Agriculture, Rural Areas, and Farmers Six-Party Science and Technology Cooperation Projects of Zhejiang Province, China (CTZB-F180706LWZ-SNY1).

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host Nicotiana attenuata. IV. Insect-induced ethylene reduces jasmonateinduced nicotine accumulation by regulating putrescine N-methyltransferas. Plant Physiol. 125, 2189–2202. doi: 10.1104/pp.125.4.2189

**Conflict of Interest Statement:** YX was employed by company Zhejiang Chemical Industry Research Institute.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Lin, Xu, Wu, Liu, Zhang, Wang and Rao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

fphys-10-00346 April 2, 2019 Time: 17:28 # 9

# Microorganism-Based Larval Diets Affect Mosquito Development, Size and Nutritional Reserves in the Yellow Fever Mosquito Aedes aegypti (Diptera: Culicidae)

Raquel Santos Souza1 , Flavia Virginio2 , Thaís Irene Souza Riback3 , Lincoln Suesdek4,5 , José Bonomi Barufi6 and Fernando Ariel Genta1,7 \*

1 Laboratório de Bioquímica e Fisiologia de Insetos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil, 2 Laboratório Especial de Coleções Zoológicas, Instituto Butantan, São Paulo, Brazil, 3 World Mosquito Program, Rio de Janeiro, Brazil, 4 Laboratório de Parasitologia, Instituto Butantan, São Paulo, Brazil, 5 Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, São Paulo, Brazil, 6 Laboratório de Ficologia, Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianópolis, Brazil, 7 Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Rio de Janeiro, Brazil

#### Edited by:

Bin Tang, Hangzhou Normal University, China

#### Reviewed by:

Raman Chandrasekar, Kansas State University, United States Qian Han, Hainan University, China Zhen Zou, Institute of Zoology (CAS), China

#### \*Correspondence:

Fernando Ariel Genta<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 12 September 2018 Accepted: 08 February 2019 Published: 09 April 2019

#### Citation:

Souza RS, Virginio F, Riback TIS, Suesdek L, Barufi JB and Genta FA (2019) Microorganism-Based Larval Diets Affect Mosquito Development, Size and Nutritional Reserves in the Yellow Fever Mosquito Aedes aegypti (Diptera: Culicidae). Front. Physiol. 10:152. doi: 10.3389/fphys.2019.00152 Background: Mosquito larvae feed on organic detritus from the environment, particularly microorganisms comprising bacteria, protozoa, and algae as well as crustaceans, plant debris, and insect exuviae. Little attention has been paid to nutritional studies in Aedes aegypti larvae.

Objectives: We investigated the effects of yeast, bacteria and microalgae diets on larval development, pupation time, adult size, emergence, survivorship, lifespan, and wing morphology.

Materials and Methods: Microorganisms (or Tetramin R as control) were offered as the only source of food to recently hatched first instar larvae and their development was followed until the adult stage. Protein, carbohydrate, glycogen, and lipid were analyzed in single larvae to correlate energetic reserve accumulation by larva with the developmental rates and nutritional content observed. FITC-labeled microorganisms were offered to fourth instar larvae, and its ingestion was recorded by fluorescence microscopy and quantitation.

Results and Discussion: Immature stages developed in all diets, however, larvae fed with bacteria and microalgae showed a severe delay in development rates, pupation time, adult emergence and low survivorship. Adult males emerged earlier as expected and had longer survival than females. Diets with better nutritional quality resulted in adults with bigger wings. Asaia sp. and Escherichia coli resulted in better nutrition and developmental parameters and seemed to be the best bacterial candidates to future studies using symbiont-based control. The diet quality was measured and presented different protein and carbohydrate amounts. Bacteria had the lowest protein and carbohydrate rates, yeasts had the highest carbohydrate amount and microalgae

showed the highest protein content. Larvae fed with microalgae seem not to be able to process and store these diets properly. Larvae were shown to be able to process yeast cells and store their energetic components efficiently.

Conclusion: Together, our results point that Ae. aegypti larvae show high plasticity to feed, being able to develop under different microorganism-based diets. The important role of Ae. aegypti in the spread of infectious diseases requires further biological studies in order to understand the vector physiology and thus to manage the larval natural breeding sites aiming a better mosquito control.

Keywords: Aedes aegypti, microorganism, development, nutritional reserves, digestion, yeast, bacteria, algae

#### INTRODUCTION

Mosquitoes are medically the most significant group of insects due to their important role in the widespread of several human infectious diseases including malaria, dengue fever, encephalitis, yellow fever and filariasis (Weaver and Reisen, 2010). The global magnitude of morbidity and mortality caused by arthropod-borne diseases has been a public health emergency of international concern. Early stages of mosquito development are related to aquatic environments, thus understanding the ecological factors involved in the aquatic habitats is essential in order to develop and improve effective strategies of mosquito control.

The biotic and abiotic environmental conditions experienced during the immature stage are determinant for the growth and development of mosquitoes. A considerable number of studies in the early 20th century devoted attention to investigating the food requirements of larvae in order to reduce or eliminate the nutritional supply of these insects in nature (Hinman, 1930). Studies on holometabolous insects suggest that well-nourished larvae become healthier adults (Zeller and Koella, 2016). The biomass accumulation of mosquitoes can be attributed to the efficiency of foraging by larvae and withstanding of starvation (Barrera, 1996).

Mosquito populations that develop in containers can be regulated by the availability and amount of food resources in the aquatic habitat (Washburn, 1995). Food deprivation can have several carry-over effects on mosquito life. A longer development time under conditions of food insufficiency has been observed before (Tun-Lin et al., 2000; Arrivillaga and Barrera, 2004; Dominic et al., 2005; Vantaux et al., 2016; Aznar et al., 2018), with mosquito larva that take longer time to reach pupa stage (Telang et al., 2007; Levi et al., 2014; Banerjee et al., 2015). An extended larval stage is generally associated with an increased risk of mortality as a consequence of predation, breeding site instability and human interference (Padmanabha et al., 2011). Beyond development time, the amount of food influences characteristics such as: nutritional reserves (Van Handel and Day, 1989; Briegel, 1990b; Gullan and Cranston, 1999; Arrivillaga and Barrera, 2004), adult emergence (Okech et al., 2007), body size (Grimstad and Walker, 1991; Strickman and Kittayapong, 2003; Jirakanjanakit et al., 2007; Foster et al., 2012; Aznar et al., 2018), response to repellents and insecticides (Xue et al., 1995; Xue and Bernard, 1996), survival (Landry et al., 1988; Dominic and Das, 1996; Sumanochitrapon et al., 1998; Aznar et al., 2018), sexual maturity, fecundity, egg production and longevity of the adult female (Briegel, 1990b; Nasci and Michell, 1994; Naksathit and Scott, 1998; Sumanochitrapon et al., 1998; Reiskind and Lounibos, 2009; Alto et al., 2012; Foster et al., 2012; Takken et al., 2013). The vector competence also could be influenced by the available food resource. Adults that emerge from larvae with low nutritional reserve are smaller (Lehmann et al., 2006) and require more blood feeds to produce eggs (Briegel, 1990b), which may lead to an increase in their vectorial capacity (Muturi et al., 2011). Restricted larval food can extend the time for mosquitoes to become infectious (Shapiro et al., 2016; Vantaux et al., 2016), modulate microbiota (Linenberg et al., 2016) and permissiveness to parasites (Takken et al., 2013; Linenberg et al., 2016), affecting immune traits (Suwanchaichinda and Paskewitz, 1998; Telang et al., 2012).

Previous studies reported that Aedes aegypti size is vulnerable to food amount and population density in immature stages (Jirakanjanakit et al., 2007). Direct measurement of the mosquito body is not satisfactory estimation of size, due to the variation of three-dimensional structures, besides the variation in the dryness of the abdomen. Weight is also an unreliable estimator of body size as it can be influenced by the blood feeding, egg production, among other factors. The mosquito body size may be adequately estimated using the wing length, or even better, using wing centroid size, an isometric and comprehensive estimator of body size (Bookstein, 1992; Siegel et al., 1992; Lounibos, 1994; Carron, 2007; Jirakanjanakit et al., 2007; Strickman and Kittayapong, 2003; Lehmann et al., 2006).

Immature stages of culicids are generally undemanding and have a pliant food behavior, ingesting through different feeding modes (e.g., filtering, suspension feeding, browsing, and interfacial feeding) organic particles in the water and almost everything available in the natural or artificial environments (Walker et al., 1988; Merritt et al., 1992; Clements, 2000). Particulate microorganisms and organic debris are commonly

the main nutritional source of mosquito larvae. Bacteria, viruses, protozoa, fungi (Timmermann and Briegel, 1996; Forattini, 2002) and algae (Merritt et al., 1992; Kivuyo et al., 2014) are some of the organisms that actively contribute to foraging and development during the larval stage. Bacteria seems the most abundant microorganisms present in the larval diet, and may even be the only nutritional source for insect growth and development (Merritt et al., 1992). Pollen particles dispersed in the aquatic environment can also be used as food sources by immature forms (Ye-Ebiyo et al., 2003; Kivuyo et al., 2014; Asmare et al., 2017).

The evolutionary success and extensive dispersal of mosquitoes may have been widely motivated by symbiotic relationships with microorganisms (Ricci et al., 2011a; Coon et al., 2014). Insects harbor numerous symbiont microbial communities, which possibly supplant the number of the cells of the invertebrate (Gusmão et al., 2010). Intracellular symbionts can occur in up to 70% of all insect species, and the intestinal compartment concentrates most of these microorganisms (Gusmão et al., 2010). The contribution of the intestinal microbiota of insects in nutritional ecology is quite relevant due to their impressive biosynthetic and degradative capacity (Douglas, 2009; Kukutla et al., 2014). The insect microbiota plays an important role in the synthesis of vitamins and essential amino acids, steroids and carbohydrates metabolism and promoting the growth and development using the insulin pathway (Shin et al., 2011; Storelli et al., 2011; Douglas, 2014). Besides nutrition, symbionts aid in nitrogen fixation, behavior, reproduction, development and enhance or suppress infections by pathogens (Dillon and Dillon, 2004; Hegde et al., 2015).

Aspects such as digestion, processing, absorption and detoxification of such generalist diets are the result of refined interactions with symbionts and digestive enzymes (Fisk and Shambaugh, 1952; Geering and Freyvogel, 1975; Marinotti and James, 1990; Ho et al., 1992; Souza et al., 2016). It is still unclear as the several microbial nutritional sources may influence the physiology of larval mosquito and which are the main enzymes involved in the digestion of these nutrients.

In this study, we investigated Ae. aegypti larval feeding using a range of microorganisms as a nutritional source. Life parameters including development rates, survival, sex ratio, body size, ingestion rates, quantity and quality of food and nutritional reserve accumulation were reported in this paper. The results suggest that microorganism-based diets can be supported by these insects in laboratory conditions and aim to provide information to laboratory breeding or studies for potential biological larvicides.

#### MATERIALS AND METHODS

#### Mosquito Rearing

The Ae. aegypti specimens eggs used for this study, were originated from eggs of Rockefeller strain gently ceded by Dr. José Bento Pereira Lima - from colonies of the Laboratory of Physiology and Control of Arthropod Vectors (LAFICAVE, - IOC/-FIOCRUZ; Dr. José Bento Pereira Lima). Insects were reared until the adult stage in the Laboratory of Insect Biochemistry and Physiology (LABFISI, IOC/FIOCRUZ) under standard conditions (temperature 26 ± 1 ◦C, relative humidity 80 ± 5% and photoperiod 12:12 h [L: D]). Newly hatched larvae derived from the same egg batch within 2 h of eclosion were fed Tetramin R sprinkled on the distilled water surface until the nutritional trials being performed.

#### Screening of Microorganisms

The nutritional physiology experiments were performed based on the follow microorganisms: Serratia marcescens (SM365), Escherichia coli (D31) and Staphylococcus aureus isolated and cryopreserved in the LABFISI, Saccharomyces cerevisiae (S14) kindly donated by Dr. Pedro Soares de Araújo (Chemistry Institute, University of São Paulo), Asaia sp. (A1), Ochrobactrum intermedium (Om17), Bacillus sp. and Pseudozyma sp. (Pa1) by Dr. Rod J. Dillon (Faculty of Health and Medicine, Lancaster University, United Kingdom), Arthrospira platensis (Spirulina) and Chlorella sp. by Dr. José Bonomi Barufi (Laboratory of Phycology, Federal University of Santa Catarina, Brazil).

#### Preparation of Microorganisms Diets

Aliquots of S. marcescens, E. coli, Bacillus sp, O. intermedium, and S. aureus were inoculated in Luria-Bertani agar plates (LB) and incubated overnight for 24 h at 30◦C. S. cerevisiae and Pseudozyma sp. were inoculated in YEPD agar plates (1% yeast extract, 2% peptone, 2% glucose/dextrose, 2% agar). Growth conditions: S. cerevisiae overnight for 24 h at 30◦C and Pseudozyma sp. 48 h at 30◦C. Asaia sp. were inoculated on GCA agar plates (2% glucose, 0.8% yeast extract, 0.7% CaCO3, 2% agar) and incubated overnight for 72 h at 26◦C (Sant'Anna et al., 2014). Bacteria single colonies were transferred to LB medium, yeast-like fungus to YPD medium and Asaia sp. to GLY medium (glycerol 25 g/l, yeast extract 10 g/l, pH 5.0) in 50 mL polypropylene tubes. All strains were grown according to the incubation temperatures of each strain in a shaking incubator (150 rpm). The microbial suspensions were centrifuged (20 min, 21,000 g, 4◦C) and the supernatant was discarded to fresh mass (FM) measurements. Cells harvested by brief spin were washed with sterile PBS three times, and finally, the bacterial and yeast pellet was resuspended in sterile water and adjusted in a concentration of 800 mg/80 mL (w/v) per strain. Chlorella sp. were inoculated in Bold's Basal Medium (BBM), and A. platensis were inoculated in Spirulina Medium Modified (Andersen, 2005). Chlorella sp. and A. platensis were incubated at 21◦C with a photoperiod of 12:12 h [L: D] in a shaking incubator (100 rpm). They were centrifuged gently (5 min, 5,000 g), before the measure of their biomass. Cells harvested by brief centrifugation were resuspended in their respective medium and were adjusted in a concentration of 150 mg/15 mL (w/v).

#### Microorganisms Viability Trials

To evaluate the capacity of microorganisms used in this study remains alive in the aquatic environment, we observed the viability of these strains on water. Bacteria and yeast were inoculated in 50 mL polypropylene tubes in specific liquid media and growth conditions were described previously (see details in Preparation of Microorganisms Diets). Cells harvested by

centrifugation (20 min, 21,000 g, 4◦C) were resuspended in liquid media or sterile water. Twenty microliter aliquots of each suspension were placed on agar plates and the number of colonies forming units, CFU were recorded after 0, 24, 48, and 120 h. Five biological experiments were performed for statistical analysis.

#### Experimental Nutrition Protocol

Ten diets were compared. Groups of 150 first-instar larvae (L1) were manually counted and transferred to each of three sterile borosilicate glass recipients (22.5 cm × 12.8 cm × 3.59 cm). Under sterile conditions, each container was filled with QSP 250 mL of sterile water or distilled water (density = 0.22 larvae/cm2 of surface area; depth of 39 mm). The same larval density was used in all experiments. The dietary supply was administered only at the L1, 80 mL (corresponding to 800 mg [w/v] and 16 mg/larva) of yeasts and bacterial suspensions and 15 mL (corresponding to 150 mg [w/v] and 3 mg/larva) of microalgae cultures were added to glass recipients. A slurry by mixing the components of Tetramin R in distilled water was prepared to fed standard group. We used 800 mg of Tetramin R resuspended in distilled water QSP to a final volume of 250 mL. Evaporated water was replaced as needed to maintain the initial volume. Three replicates were performed for each dietary experiment.

#### Effects of Diets on Development and Survivorship of Ae. aegypti

For comparison of diet effects, developmental rate and survivorship from eclosion to adult emergence were measured. Larvae were observed daily until pupation, and dead larvae and exuviae were removed. Pupae were collected daily, counted and transferred individually into 15 mL polypropylene tubes covered with mosquito netting and filled with 4 mL of breeding water until adult emergence. The number and the sex of adults emerged were determined. Adults received only cotton wool moistened with distilled water ad libitum. The median time in days for pupation, the emergence of adults (males and females) and adult survivorship were calculated using the number of individuals that reached pupae or the adult stage. We also recorded the proportion of larvae that survived from L1 to the pupal stage, time to metamorphosis (development duration in days, between pupa and adult stage), time to emergence (development duration in days, between L1 and adult stage), adult survival (time from adult emergence to dead), and survivorship full span (using larvae that survived from L1 to dead adult stage). Sex ratio was estimated as the number of males relative to total emerged adults.

#### Wing Length Measurements

To evaluate possible morphological variation in body size of adults reared with microorganism diets, we measured the size of males and females wings separately using standard methods of geometric morphometrics. In this study, we used the wings of adults emerged from larvae fed with standard diet Tetramin R , the yeasts S. cerevisiae and Pseudozyma sp., and the Gramnegative bacteria E. coli and Asaia sp. The wings (both sides) were removed from the thorax of individuals, mounted on Canadian balsam microscope glass slides and processed as reported by Lorenz et al. (2012). Images of the slides were digitized using a digital camera Leica DFC320 coupled to a Leica S6 (40×) stereoscope. To each image were registered coordinates x and y of 18 landmarks (Virginio et al., 2015) using TpsDig software V.2.05 (Rohlf, 2006). The wing size variations were assessed using measurements of the centroid size (CS) (Bookstein, 1992).

# Microorganism Staining and Larval Feeding Behavior

We decided to monitor the ingestion of live microorganisms labeled with fluorescein isothiocyanate (FITC) by Ae. aegypti larvae. The protocol of microorganism staining was performed according to Moraes et al. (2012). FITC-labeled microorganisms were resuspended in 3 mL of sterile water and added to 50 mL polypropylene tubes containing 7 mL of sterile water and 50 fourth instar larvae raised on Tetramin R . After 2 h incubation at 26◦C, 10 larvae were dissected, and single guts were placed in microtubes with 100 µL of sterile NaCl 0.9% (w/v). Samples were homogenized by shaking the tube for 30 s at 25 Hz (MiniBeadBeater; Biospec Products, Bartlesville, OK, United States). The gut fluorescence detection was performed in a FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, United States) on λEx = 495 nm and λEm = 520 nm. Aliquots (10 µL) of microorganisms FITClabeled were mounted on microscope glass slides for fluorescence observation in a Nikon Eclipse E200 (40×), fitted with a B-2A filter (Excitation Filter Wavelengths: 450–490; Dichromatic Mirror Cut-on Wavelength: 500; Barrier Filter Wavelengths: 515). Images were taken with a regular digital camera. Five experiments were performed.

#### Protein and Carbohydrate Contents of Microorganism-Based Diets

Culture samples of 20 mL were centrifuged (7,500 × g, 20 min, 4 ◦C). The supernatant was discarded, and cells were resuspended in 1 mL of water. Aliquots of 10 and 40 µL were withdrawn for protein and sugar measurements, respectively. We assessed total protein content using the bicinchoninic acid method procedure (Smith et al., 1985) and total carbohydrates were measured with the phenol-sulfuric method (Dubois et al., 1956). Eight experiments were performed for each diet.

#### Measurement of the Energy Reserves in Single Individuals

Fourth instar newly molted larvae were individually weighed and immediately frozen for analysis of nutritional reserves. We quantified protein, total carbohydrate, glycogen, and lipids in single individual fourth instar entire larvae, gut and rest of the body for comparison. Larvae were reared as reported in item "Experimental Nutritional Protocol" and dissected as described in "Microorganism Staining and Larval Feeding Behavior." The biochemical analysis was performed accordingly the Van Handel (1985a) method adapted by Foray et al. (2012). Protein content was measured as Bradford (1976) method using ovalbumin as a standard. Carbohydrates and glycogen were detected by an anthrone procedure using glucose as a standard (Van Handel, 1985a). Total lipid was determined in chloroform-methanol solvent solution by vanillin–phosphoric acid reaction (Van Handel, 1985b, 1988) using Glyceryl trioleate as standard (Van Handel and Lum, 1961). The assays were performed in 96-well microplates. Ten experiments were performed for each diet.

#### Statistical Analysis

fphys-10-00152 April 8, 2019 Time: 17:47 # 5

For all experiments, measurements were described using mean ± SEM. Developmental parameters (larval development, pupation, emergence, and survivorship) were analyzed using the GraphPad InStat v.3.01 (San Diego, CA, United States) and Excel R . The correlation among non-parametric variables was performed using the Log-rank (Mantel-Cox), Wilcoxon and Fisher tests. Tests for normality of the sample distribution were assessed by D'Agostino-Pearson omnibus test. Microorganism's viability significance and measurement of protein and carbohydrate content on diets were examined with a T-test. Analysis of variance (ANOVA 1) was used in ingestion of live microorganisms labeled with FITC and measurement of the energy reserves in the whole larva. The wings morphometric statistical analyses were managed with the software MorphoJ (Klingenberg, 2011), and GraphPad InStat v.3.01 (San Diego, CA, United States). The normality and homoscedasticity of samples distribution were assessed by Shapiro–Wilks with the software Past3. In populations that had a Gaussian distribution, the parametric T-test was used based on means. In populations that did not, the non-parametric Mann–Whitney test was used based on medians.

#### RESULTS

To verify if all the strains used in our experiments were viable on aquatic conditions we tested the viability of the microorganism cells in water. CFU counts revealed that re-suspension in water does not affect the viability and number of cells (p > 0.05, paired T-test, n = 15, **Figure 1**). S. cerevisiae, S. marcescens, Bacillus sp., and O. intermedium remained viable after being incubated in water for 48 h, and Pseudozyma sp., E. coli, Asaia sp., and S. aureus were viable until 24 h (**Figure 1**). These data demonstrate that it is possible to expose larvae to live cells and that these microbial cells might be used as a nutritional source.

#### Estimates of Development and Survivorship for Ae. aegypti Reared Using Exclusive Microorganism-Based Diets

To assess the development and survivorship until the adult stage of Ae. aegypti reared exclusively with microorganism-based nourishment at immature stages, we tested four strains of Gramnegative bacteria were used: S. marcescens (SM365), E. coli (D31), Asaia sp. (A1), and O. intermedium; two strains of Gram-positive bacteria, Bacillus sp. and S. aureus; the yeasts: S. cerevisiae (S14) and Pseudozyma sp. (Pa1), a genus of microalga Chlorella sp., and a species of cyanobacteria (blue–green algae), A. platensis (Spirulina). The biological life attributes measured for the Ae. aegypti Rockefeller strain under controlled laboratory conditions are presented from **Supplementary Tables S1–S6** and **Tables 1**–**5**, and summarized as follows.

The developmental time from L1 to pupae differed significantly (p < 0.0001) between the diets. Larvae of the standard group fed with Tetramin R developed in 5.3 ± 0.04 days. Pseudozyma sp. and S. cerevisiae (mean 6.5 ± 0.12; 8.1 ± 0.05 days) developed faster than the larvae on other microorganism-based diets. Larvae fed with the Chlorella sp. take longer to develop until pupation with a mean time of 61.5 ± 2.09 days until pupa (**Supplementary Table S1**). S. marcescens (42.0 ± 3.0 days) and Bacillus sp. (34.0 ± 0.0 days) showed the longest time to achieve pupal stage compared with the other bacterial diets.

The next biological parameter evaluated was the duration of the metamorphosis period of larvae in adult mosquitoes. No significant differences (p > 0,05) were detected in diets that used Asaia sp. (2.0 ± 0.04 days; p = 0.6804), O. intermedium (2.0 ± 0.0 days; p = 0.3652), Chlorella sp. (1.9 ± 0.07 days; p = 0.0978) and A. platensis (1.9 ± 0.09; p = 0.1042) compared to the standard group fed with Tetramin R (2.09 ± 0.02 days) (**Supplementary Table S2**). Time from L1 to adult emergence differed significantly (p < 0.0001) among each diet. Time until the adult stage was higher to Chlorella sp. (**Supplementary Table S3**).

The survival of adults maintained only with water was evaluated once a day until confirmation of the death of all insects. The diets containing E. coli (6.2 ± 0.3 days) and Asaia sp. (5.5 ± 7.52 days) showed the closest survival rates compared to the standard diet (8.9 ± 0.1 days). Bacillus sp. (2.0 ± 0.0 days), S. aureus (2.3 ± 6.1 days), and O. intermedium (2.5 ± 2.1 days) revealed the lowest survival rates (**Supplementary Table S4**). The full lifespan from L1 to adult death is significantly (p < 0.0001) different between diets (**Supplementary Table S5**).

Biological parameters were analyzed separately by gender to disclose possible sex-specific effects in development rates and survivorship. The development time of L1 to pupae differed significantly in females and males (p < 0.001; **Table 1**) from different diets. Larvae fed with yeast diet developed faster in both genders (mean 7.2 ± 0.2; 8.4 ± 0.06 days for females; 6.1 ± 0.1; 7.9 ± 0.07 days for males) than larvae on bacteria and microalgae diets. Male development time until pupa is shortest than female larvae in all diets used (**Table 1**). No significant differences (p = 0.7867) were detected for female adult metamorphosis on diets containing Asaia sp. However, a significant (p < 0.0001) effect was observed among all the other diets compared to the standard group fed with Tetramin R (**Table 2**).

Male development time until adult metamorphosis differed significantly (p < 0.0001) solely on yeasts diets, and no significant differences were observed among the other diets (**Table 3**). Female average development time until adult emergence was longest than males. In both genders a significant difference (p < 0.0001) was detected when compared microorganismbased diets with the group fed with the standard diet Tetramin (**Table 3**). The survival of adults (males or females) differs significantly (p < 0.0001) between diets. The average survival time of each female adult varied from 2.6 ± 0.93 to

NA, not available. Differences were analyzed with Log-rank and Wilcoxon.

5.1 ± 0.73 days across the different dietary supply. Females fed with A. platensis (5.1 ± 0.73 days), Asaia sp. (4.8 ± 0.39 days) and E. coli (4.6 ± 0.40 days) exhibited an elongated average survival (**Table 4**).

The adult survival pattern observed in males differed partially from females. The average observed in the survival span for males varied from 2.1 ± 0.39 to 7.0 ± 0.44 days. Males fed with E. coli (7.0 ± 0.44) and Asaia sp. (6.1 ± 0.24 days) displayed the same longest survival observed for females. Additionally, males fed with Pseudozyma sp. (5.6 ± 0.19 days) and S. cerevisiae (5.5 ± 0.28 days) also presented a long survival (**Table 4**). The full lifespan of males and females from L1 larvae to pupation differ significantly among diets (p < 0.0001). The average life span varied from 13.0 ± 0.23 to 73.5 ± 4.74 days for females and 14.0 ± 0.17 to 65.0 ± 2.32 days for males (**Table 5**). The overall adult sex ratio was more male-biased (**Supplementary Table S6**). The development time of L1 to pupae, adult emergence, survivorship, and lifespan of all dietary studied have been depicted graphically in **Figures 2**–**5**.

Yeast diets revealed a similar development time to standard diet Tetramin R . The diets of bacteria and microalgae, in the opposite, presented a lethargic larval development (**Figures 2**, **3**). Regarding adult mortality, E. coli and Asaia sp. presented the highest survival mean in days, surpassing even the yeast diets (**Figure 4**). The full lifespan was extended in immature stages fed with bacteria and microalgae diets. These results suggest a possible badly nourishment which could breed smaller larvae with difficulty to attain the critical mass that is necessary

#### TABLE 2 | Average metamorphosis time for females and males in days.

NA: Not available. Differences were analyzed with Log-rank and Wilcoxon.

TABLE 3 | Average emergence time for females and males in days.

NA, not available. Differences were analyzed with Log-rank and Wilcoxon.

#### TABLE 4 | Adult average survival time in females and males.

NA, not available. Differences were analyzed with Log-rank and Wilcoxon.

#### TABLE 5 | The full lifespan of females and males in days.

fphys-10-00152 April 8, 2019 Time: 17:47 # 8

NA, not available. Differences were analyzed with Log-rank and Wilcoxon.

for metamorphosis (**Figure 5**; Telang et al., 2007). All the compiled biological development data were detailed in the **Supplementary Spreadsheet S1**.

Concerning the wing centroid size analysis, which is herein used as a predictor of body size, the majority of samples showed a normal distribution (p > 0.05, **Supplementary Tables S7, S8**). In general terms, the comparisons between microorganismbased diets and the standard group (with normal and nonnormal distribution, respectively), showed a significant difference of sizes in both genders and wing sides (p < 0.05). The isometric size of males and females reared in Tetramin R showed larger adults than all the other diets (**Figure 6**). On average, among the experimental diets (excluding the standard group), the largest females were reared in S. cerevisiae, followed by Pseudozyma sp., Asaia sp., and E. coli diet. The males were largest in S. cerevisiae, Asaia sp., Pseudozyma sp., and E. coli diet, respectively (**Figure 6**).

#### Nutritional Evaluation of Diets, Energetic Reserve Accumulation Measurement and Feeding Behavior of Ae. aegypti Larvae Reared Exclusively With Microorganisms

Nutritional quality influences the physiology of Ae. aegypti immature stages directly. To evaluate the impact of quality of two important macronutrients on larval breeding, we quantified the protein and carbohydrate contents present in the dietary supplies used. Differences in the nutritional composition of the

FIGURE 3 | The impact of microorganism-based diets on adult emergence. A representative emergence curve is comparing larvae fed with Tetramin (Dark blue line, left y-axis) and larvae fed with microbial cells (red line, right y-axis). The dietary supply was administered only at L1. Dead individuals were removed daily.

microorganism diets were detected and may have influenced the immature stages development time (**Table 6**). S. cerevisiae and Pseudozyma sp. diets have higher amounts of protein (116 ± 6.1 mg; 75 ± 6.9 mg) and carbohydrate (180 ± 9.2 mg; 98 ± 3.8 mg) than the bacterial diets (7.0 ± 0.6 mg to 15.4 ± 1.4 mg and 0.3 ± 0.1 mg to 1.6 ± 0.02 mg). Yeast diets, on the other hand, have less protein than A. platensis (341 ± 34 mg) and Chlorella sp. (300 ± 17.5 mg) diets. Conversely, the content of carbohydrates was higher on diets with yeasts (180 ± 9.2 mg; 98 ± 3.8 mg) compared to the diets based in A. platensis (35 ± 4 mg) and Chlorella sp. (26 ± 1.5 mg).

Four major energetic components (protein, carbohydrate, glycogen, and lipids) and the body weight were measured in individual fourth instar entire larvae and compared with the contents recovered from their guts and rest of body tissues. The results are summarized in **Table 7**. The absolute values of the

nutritional reserves differed significantly (p < 0.0001) among individuals compared with the standard group Tetramin R . Larvae fed with S. cerevisiae, Pseudozyma sp, E. coli, and Asaia sp. accumulate the highest amounts of protein (194 ± 18 mg to 89 ± 3 mg), carbohydrates (103 ± 1.3 mg to 16 ± 0.3 mg), glycogen (12 ± 0.8 to 3 ± 0.4 mg) and lipids (71 ± 4.9 to 27 ± 0.8 mg). The nutritional reserve accumulation in A. platensis, Chlorella sp., S. aureus, S. marcescens, Bacillus sp., and O. intermedium showed the lowest amounts of energetic components (protein, carbohydrate, glycogen, and lipid) and these results are in agreement with the longer developmental rates observed in the experiments above (**Supplementary Tables S1–S5** and **Tables 1**–**5**). Mean body weight differed significantly with larval diet (**Table 7**). Only S. cerevisiae and Pseudozyma sp. did not differ significantly compared with the standard diet.

To further investigate the rates of ingestion of the different microorganisms by larvae, the consumption rate of each diet was measured by fluorescence in the gut of individual larvae after 2 h of incubation with FITC-labeled cells. Larvae fed with S. cerevisiae, Pseudozyma sp., and E. coli consumed more food than larvae fed with the other diets after 2 h (**Figure 7**). Our results showed that Ae. aegypti larvae actively consumed all the microorganisms used, but with some preference for the microorganisms above (**Supplementary Figures S1, S2**).

#### DISCUSSION

The nutrition environment experienced by larvae strongly influences the physiology and behavior of mosquitoes. The current work evaluated the impact of nourishment from live microbes in the development and survival of Ae. aegypti. The organisms selected for feeding were strains of Gram-negative bacteria S. marcescens (SM365), E. coli (D31), Asaia sp. (A1), and O. intermedium (Om17), Gram-positive bacteria Bacillus sp. and S. aureus, yeast-like fungi S. cerevisiae (S14), and Pseudozyma sp. (Pa1), the cyanobacteria (blue–green algae) A. platensis (also known as Spirulina), and the marine microalga Chlorella sp. All microorganisms used in this paper showed stability in water until 120 h. Therefore, we decided to perform the feeding experiments by dispersing the strains directly in rearing water under sterile conditions.

#### General Developmental Parameters in A. aegypti Raised on Microorganisms-Based Diets

All diets showed important differences in developmental and survival rates when compared individually to the standard group Tetramin R . Larva fed with yeast take less time to achieve pupation than all the other microorganism-based diets tested (**Figure 2**). Parameters as adult emergence (**Figure 3**), survival (**Figure 4**) and full lifespan (**Figure 5**) showed a slight delay in developmental rates when compared to the standard group Tetramin R . Due to the satisfactory developmental rates in all life parameters tested, and the rapid growth speed of cultures in lowcost media, S. cerevisiae, and Pseudozyma sp. seem to be suitable candidates for diets in the mass rearing of mosquitoes or the regular laboratory breeding (Imam et al., 2014). Trager (1935a,b) studies pioneered larval nutrition, demonstrating that Ae. aegypti larvae can reach adulthood being fed only with yeast powder. Confirming the results obtained by Trager, 1935a showed that autoclaved yeasts suspended in CaCl2 0.01% is sufficient for the

sp. and S. cerevisiae). Vertical lines: individuals. Asterisks: Non-normal distribution.

between control diet (Tetramin R

) and Gram-negative bacteria (Asaia sp. and E. coli). (E–H) Comparison between control diet [Tetramin (R)] and Yeasts (Pseudozyma

TABLE 6 | Protein and carbohydrate contents in microorganism-based diets.

(ANOVA 1; ∗∗∗∗p < 0.001).

full larval lifespan of Ae. aegypti. Souza et al. (2016) conducted a study based on feeding larvae with a specific diet containing live or dead S. cerevisiae cells only. The data obtained in this paper corroborated the works described previously, that highlighted the ability of Ae. aegypti larvae to feed and digest living yeast cells through the enzyme beta-1,3-glucanase. Yeast and fungi can also be suitability as a paratransgenic vehicles or integrated pest management (IPM) tools.

Interestingly, the symbiont Wickerhamomyces anomalus (Saccharomycetales) can be found in the gut and reproductive organs of some mosquito vector species. This symbiont can be easily cultured in cell-free media and seem to be a good candidate for the expression of effector molecules in the gut of mosquito vectors (Ricci et al., 2011b). Murphy et al. (2016) demonstrated that genetically modified S. cerevisiae could be used as biopesticide through oral delivery of species-specific dsRNA. This application as biopesticide decreases larval survivorship, reduces locomotor activity and reproductive fitness in the insect pest Drosophila suzukii. This yeast biopesticide approach could be adapted to a large number of species once many interactions have been observed between sylvatic yeasts and insect species as Diptera, Coleoptera, and Hymenoptera (Gonzalez, 2014; Abrieux and Chiu, 2016). The authors also postulate that biopesticide design may be favored in the managing of an insect pest that both consumes yeast as food and has systemic RNAi.

Studies of biological control agents based on entomopathogenic fungi have been reported in several vector mosquitoes. The potential of Metarhizium anisopliae fungus was tested in Anopheles gambiae, Ae. aegypti, Aedes albopictus, Culex quinquefasciatus (Alves et al., 2002; Scholte et al., 2007; de Paula et al., 2008; Pereira et al., 2009; Fang et al., 2011). Geneticengineered M. anisopliae inhibited Plasmodium sp. development within the mosquito and prevented malaria infection in Anopheles (Fang et al., 2011). In Ae. aegypti, A. albopictus, and C. quinquefasciatus, the full lifespan of M. anisopliaecontaminated mosquitoes was significantly reduced and showed high mortality rates compared to uninfected mosquitoes (Alves et al., 2002; Scholte et al., 2007; de Paula et al., 2008; Pereira et al., 2009). Recently, two strains of M. anisopliae were tested against Ae. aegypti and besides the increase in mortality, the fungus also reduced egg laying (Jemberie et al., 2018). The fungi: Lagenidium giganteum and Leptolegnia chapmanii were also tested as promising biological control agents for use against Ae. aegypti adults (McCray et al., 1973; McInnis and Zattau, 1982). The development of innovative strategies using yeast has, therefore, potential as an eco-productive alternative for the management of mosquito-borne diseases.

Bacteria are considered the most common microorganism present in the nourishment of mosquito larvae (Laird, 1956, 1988; Christophers, 1960). Previous studies reported that bacteria could be used as a unique food requisite to mosquito growth (Hinman, 1932; Rozeboom, 1935). Larvae fed with S. marcescens, Bacillus sp, S. aureus, O. intermedium, S. aureus, E. coli, and Asaia sp. showed a severe delay to achieve pupal stage when compared to the standard group Tetramin R (**Supplementary Table S1**). Other biological parameters as metamorphosis (**Supplementary Table S2**), emergence (**Figure 3**), survival (**Figure 4**) and full lifespan also strongly affected (**Figure 5**). Studies obtained by Dickson et al. (2017) provide the concept that larval exposure to different bacterial communities during larval development can drive variation in Ae. aegypti adult traits. Therefore, the results observed here are similar to other studies reported. Noteworthy, E. coli and Asaia sp. take result in shorter developmental times into pupa than the other bacterial diets (**Figure 2**). The survival span for larvae fed with E. coli and Asaia sp. were superior to those observed in diets using S. cerevisiae and Pseudozyma sp. E. coli and Asaia sp. seem to be the best bacterial models for laboratory-reared larvae up until now.

Food stress was expected to reduce survival, however, larva fed with Asaia sp. and E. coli had higher adult survival (**Supplementary Table S4**). There are different ways to interpret these results. Nutritional stress can increase the life-span by a hormetic model (Mattson, 2008). Larvae fed with E. coli and Asaia sp. might be presenting a stress-induced response, hormesis, that can be an overcompensation to environmental, nutritional stress (Calabrese, 2001). Hormesis induces cellular protective mechanisms through an increased in gene expression, working as a key regulator of many cellular defenses that allow survival in response to stress (Lin et al., 2000; Motta et al., 2004; Heilbronn et al., 2005). Enhanced levels of heat shock proteins (HSPs) and antioxidants to cellular maintenance are also considered as part of the hormetic mechanism (Gems and Partridge, 2008; Mattson, 2008). The beneficial effects of hormesis on survival and longevity have been described for years, and our results might thus exemplify a beneficial carry-over effect of the hormetic stress on larval development, reinforcing the Ae. aegypti phenotypic plasticity in limiting environments (Aznar et al., 2018). Previous studies had already shown that larvae reared in restricted diets might be associated with prolonged life in Ae. aegypti (Joy et al., 2010; Zeller and Koella, 2016) and Anopheles sp. (Vantaux et al., 2016).

The positive correlations between the presence of microbiota and larval development might be another way to explain the results observed in larvae fed with Asaia sp. and E. coli. The acetic acid bacterium genus Asaia have been shown to be stably associated with larvae and adults of anophelines and Ae. aegypti (Favia et al., 2007; Damiani et al., 2010; Ricci et al.,

NutritionalamountsofsolubleandtotalinA.larvaeraisedindifferentdiets,and

fphys-10-00152 April 8, 2019 Time: 17:47 # 13

2012a; Rossi et al., 2015). Mitraka et al. (2013) showed that a diet supplemented with Asaia in the Anopheles gambiae larval environment had a significant boost in developmental rate and Chouaia et al. (2012) observed a delayed larval development in Anopheles mosquitoes deprived of Asaia bacterial symbionts. Besides the effects on development rates, a possible mutual exclusion or a competition between Asaia and Wolbachia may contribute to explain the inability of Wolbachia to colonize the female reproductive organs of anophelines, inhibiting its vertical transmission and explaining the absence of Wolbachia infection in Ae. aegypti and in the majority of natural populations of Anopheles mosquitoes (Rossi et al., 2015). These results drive us to believe that Asaia may play a significant role in mosquito larval development. Although the molecular nature of the developmental improvement caused by the Asaia symbiont needs to be identified, these bacteria can be considerate as candidate paratransgenic vehicle for the control of mosquito-borne diseases (Favia et al., 2008; Ricci et al., 2012b; Mitraka et al., 2013).

Coon et al. (2017) showed that each mosquito species including Ae. aegypti contains a simple bacterial community and that the composition of bacterial gut communities can also be strongly influenced by diet. Their results also showed that axenic larvae could not develop, but several community members and E. coli can rescue the larval development. Using E. coli K-12 as a model for studies of molecular interactions that underlie bacteria-dependent growth of larvae into adults, Coon et al. (2017) unveiled one of the molecular mechanisms involved in mosquito development. They showed that bacteria through the cytochrome b oxidase gene mediate a reduction of oxygen levels in the digestive tract of larvae, working as a signal for ecdysone-induced molting. Thereby, E. coli plays an essential role in mosquito development and may have important implications to be used in symbiont-based control techniques for disabling the growth of larvae into mosquito adults.

S. marcescens, Bacillus sp., O. intermedium, and S. aureus had a lower adult rate survival contrary to results described in diets with Asaia sp. and E. coli (**Supplementary Table S4**). The effect of a restricted diet in mosquito larva is a rather speculative issue. Previous studies reported that dietary restrictions can lead to longer development rates (Olivo et al., 1979; Tun-Lin et al., 2000; Arrivillaga and Barrera, 2004; Couret et al., 2014; Levi et al., 2014; Aznar et al., 2018), with larvae extending time to achieve the pupal stage (Chambers and Klowden, 1990; Telang et al., 2007; Foster et al., 2012; Banerjee et al., 2015). Some individuals with slower growth rates try to counterbalance this deficit through compensatory growth (Wilson and Osbourn, 1960). This ecological factor allows that once the same nutritionally deprived individuals have the opportunity to acquire more food, speed up or slow down the growth rates to employ the food resources in the maintenance of important biologicals traits as reproduction and survival (Dmitriew, 2011; Dmitriew and Rowe, 2011; Zeller and Koella, 2016).

Bacillus sp. and S. marcescens presented only two adults (**Supplementary Table S1**). S. marcescens is recognized by its entomopathogenic properties in some conditions (Flyg et al., 1980; Flyg and Xanthopoulos, 1983). However, in this study, it does not appear to be associated with infections in Ae. aegypti larvae. In the present study, larvae fed with bacterial diets, including S. marcescens, remained in the third instar for several weeks. Other studies have been shown that withstand to starvation could be measured by the time spent in the third larval instar without adequate nourishment (Wigglesworth,

1942). The duration of development from the L1 larval stage to adult mosquito is faster when food is abundant (Tun-Lin et al., 2000); thus, the prolongation in third instar larvae observed in our results may indicate a state of malnutrition. We did not observe the presence of melanized-killed larvae or any specific phenotype more related to pathogenicity. However, previous studies have already reported that food restriction alters immunological traits in Ae. aegypti (Grimstad and Walker, 1991; Alto et al., 2005, 2008; Muturi et al., 2011; Telang et al., 2012). More studies are necessary to evaluate the influence that the diets used in this study exert on the immune system of Ae. aegypti larvae.

Lowering of growth rates may be a response to dietary stress and adaptive behavior in calorie-depleted environments (Arendt, 1997; Badyaev, 2005). It is possible that Ae. aegypti larvae raised under bacterial diets suffer nutritional depletion, resulting in developmental delays. In natural environments, mosquitoes are commonly found with reduced sizes and low energy reserves. This remarkable capacity of withstanding starvation situations and recovery later in more favorable conditions can justify the great success for the establishment of these insects in the environment (Barrera, 1996; Barrera and Medialdea, 1996; Zeller and Koella, 2016). Therefore, our data show that Ae. aegypti larva can develop in all bacterial diets tested, even at a higher ecological cost.

In natural conditions, the biomass of algae is the major content of mosquito larvae guts (Hinman, 1930; Garros et al., 2008). Thus, algae seem to play an important role in the development and survival of larvae. Ae. aegypti larvae were not able to develop in microalgae diets with the standard concentration established for the other diets (16 mg/larva), so we adjusted the values offered to lower concentrations (3 mg/larvae). The development failure observed in the standard concentration (data not shown) might have been caused by several factors among them the junction between a controlled photoperiod environment as well as a low larval density. Together these factors might have accelerated the exponential microalgae growth triggering a toxicological response by larvae which impaired their development.

Larvae fed with the microalgae Chlorella sp or with A. platensis (3 mg/larvae) showed a delay in development similar to the observed in bacterial diets, but with shorter adult survivals (**Supplementary Table S4**). Kivuyo et al. (2014) evaluated larval survival and the development rate of the aquatic stage using green filamentous algae and dry powdered filamentous algae as diet. The survivorship and pupation rates in algae food diet had the worst performance between all foods assessed. Some species of algae seem to be resistant to digestion and are discarded entirely after passage through the larval gut (Laird, 1988). However, there is no evidence that Chlorella sp. and A. platensis belong to this specific group.

Interestingly, a genus of Chlorella was used as a larvicide against Ae. aegypti by Borovsky et al. (2016). Chlorella desiccata was engineered to express an insect peptide hormone, the trypsin modulating oostatic factor (TMOF), that is recognized by Ae. aegypti ovaries and controls the translation of the gut's trypsin mRNA. Feeding mosquito larvae with transformed C. desiccata cells kill by starvation 60% of Ae. aegypti larvae in 4 days (Borovsky et al., 2016). A. platensis (Spirulina) have never been used in nourishment experiments with mosquito larvae before. Our results showed that Chlorella and A. platensis could be used as a food source by mosquito larvae, but with slower developmental rates. Their rapid grow under heterotrophic culture, with a relatively low cost, make these microalgae interesting candidates for future methods of vector control through genetic engineering (Dawson et al., 1997; Madkour et al., 2012).

#### Sex-Related Parameters in A. aegypti Raised on Microorganism-Based Diets

The biological parameters evaluated in Ae. aegypti were analyzed separately for males and females in all diets (**Tables 1**–**5** and **Supplementary Table S6**). The results showed that, as already described in the literature, males take less time to reach adulthood and survive longer. When mosquitoes are fed only water as adults, the only nutritional resources available lie in reserve acquired during immature stages. The transference of reserves from the larval nutrition to the mosquito adult stage using only microbial cells as nourishment has never been investigated before. Thus, it is possible that males show more withstand to starvation during the larval stage and a lower nutritional threshold for pupation than females. These traits might have influenced the better developmental rates observed in males and increased their survival. The measured lifespan of adult males and females fed with nectar is approximately 9 weeks and around 12–17 weeks for females that were also blood fed (Putnam and Shannon, 1934). We were not able to find available data in the literature to compare the mortality rates of males and females under laboratory conditions without any nutritional stimulus. Aznar et al. (2018) reported that treatments with scarcity or excess of food might preferentially influence the proportions and survival of females over males. They have shown that females exhibit a larger extension of development time in response to food deprivation than males, and relate this result to a male fitness advantage, ability of an individual to survive, reproduce and spread genes, this advantage is obtained when males emerge early and can copulate with non-mated females. The longer development time on females is similar to the observed under a competitive environment (Bedhomme et al., 2003). Under variated conditions, it is expected that females take more time to develop and spend more time to enhance their fitness abilities, achieving larger body sizes and thus boost their fecundity (Bedhomme et al., 2003; Wormington and Juliano, 2014). Previous field studies already reported a faster development in a male over female mosquito larvae, that showed a slower development and a higher mortality rate (Yates, 1979).

Imbalances in sex ratio (males: females) were observed in all diets tested (**Supplementary Table S6**). This male-biased sex ratio may result from underfeeding. Some studies support a sex-related difference in larval nutrient metabolism, possibly due to the earlier ecdysteroid peak in Ae. aegypti male during pupal-adult development (Brust, 1967; Whisenston et al., 1989;

Chambers and Klowden, 1990; Puggioli et al., 2013; Balestrino et al., 2014). In field studies, distortion in sex ratio is frequent and associated to the slow development of female larvae and differential response of the sexes to egg hatching stimuli (Yates, 1979; Shroyer and Craig, 1981; Sims and Munstermann, 1983; Frank et al., 1985; Lounibos and Escher, 2008). Other studies observed a density-dependent alteration and a sexspecific response to a critical day period time, trough feedback mechanisms that are dependent on density or mortality by selective sexual predation (Chambers, 1985; Frank et al., 1985; Alto et al., 2012). The highest proportion of males observed in this study seem favorable to future studies using the Sterile Insect Technique (SIT). A greater male pupae production is important to SIT mass rearing and determine the number of males that can be selected for release in the natural environment (Puggioli et al., 2016).

In this study, it was also possible to realize that microbial diets (yeast and Gram-negative bacteria) influenced the wing size of Ae. aegypti adults, and presumably the whole body size, of both genders. The correlation of wing size with food concentration is interesting, mainly because the "centroid size" (Bookstein, 1992) is considered a more informative estimator of body size than the traditional size measurements (Jirakanjanakit and Dujardin, 2005; Jirakanjanakit et al., 2007). As previously mentioned, we quantified the wing centroid size as a readout of the adult size. In general, the wings of the mosquitoes fed with Tetramin R in their immature stages were larger than those that were fed with microorganism-based diets. Also, in all comparisons, females showed larger wings than males. Sexual dimorphism is present in wing shape and size of Ae. aegypti (Virginio et al., 2015; Sánchez and Liria, 2017). However, independently of the inherent wing sexual dimorphism, and some asymmetry, we recorded some patterns of differentiation. In males, the group fed with E. coli showed lower sizes than the Asaia sp. group, which in turn was more similar to Tetramin R . In females, Asaia sp. and E. coli were lower than Tetramin R , although Asaia sp. has been closer to the Tetramin R diet. On the yeast diets, males of Pseudozyma sp. and S. cerevisiae showed similar scores, and females of the Pseudozyma sp. were lower than S. cerevisiae, both being lower than the Tetramin R score. Among all the microorganism-based diets tested, S. cerevisiae showed the wing sizes that are closest to the Tetramin R diet. Jong et al. (2017), showed significant shorter wings in A. albopictus under sub-optimal food availability. Jirakanjanakit et al. (2007) and Aznar et al. (2018) reported that low food concentration in Ae. aegypti immature stages could alter mosquito size. Those results support this study, suggesting a strong influence of microbial food composition on mosquito body size.

# Nutritional Analysis of Microorganism-Based Diets and Nutrient Acquisition by Larvae

The differences reported in the developmental time and adult size with the microbial diets are consistent with other studies on environment stress (Badyaev, 2005). Response to stress conditions such as nutritional limitation (Bubliy et al., 2000), extreme temperatures (Sisodia and Singh, 2009) and larval crowding (Imasheva and Bubliy, 2003) in Drosophila have been studied and related to genetic modifications. Schneider et al. (2011) demonstrated a genotype variation in Ae. aegypti adult size in response to larval food depletion, reporting wide phenotypic plasticity and adaptive behavior to changing environments. Aznar et al. (2018), suggested that the variety in food stress conditions in the natural habitat can increase genetic modifications in Ae. aegypti. This genetic variety, as mutation and recombination rates, is usually hidden under regular food conditions and facilitates the development of novel adaptations to adverse environments (Badyaev, 2005).

The next step was to analyze the nutritional quality through the measurement of the main energetic components of diets. Our goal was to observe if these developmental and size variations are a result of malnourishment. The nutritional requirements of mosquitoes are divided into two majority classes: macronutrients (energetic nutrients) and micronutrients (nonenergetic nutrients) (Singh and Brown, 1957; Foster, 1995; Arrese and Soulages, 2010; Canavoso et al., 2011; Rivera-Pérez et al., 2017). Aquatic environments with abundant nutritional richness supply all the energy necessary to mosquito larvae metamorphosis. Carbohydrates and proteins are among the main nutritional requirements of Ae. aegypti (Singh and Brown, 1957).

The nutritional values of the diets were evaluated through protein and total sugar quantification (**Table 6**). Chlorella sp and A. platensis showed higher protein amounts than the other diets, including the standard diet Tetramin R . These results were already expected once both species are rich in proteins and can be employed even for human consumption (Mühling, 2000; Guccione et al., 2014; Bleakley and Hayes, 2017). Yeast diets also presented robust protein values. S. cerevisiae has more protein than Pseudozyma sp. Souza et al. (2016) showed that S. cerevisiae contains higher protein amounts than the standard diet Cat food. However, Tetramin R seems to be more nutritive and present a superior protein amount. Bacillussp., S. marcescens, S. aureus, and O. intermedium showed lower protein values than E. coli and Asaia sp. These results coincide with the developmental pattern observed (**Supplementary Tables S1–S5**). As previously mentioned, protein and amino acids consumption are directly related to growth and development in mosquitoes (Golberg and De Meillon, 1948; Merritt et al., 1992). Scarce consumption of proteins during the immature stages might interfere in life cycle duration, adult emergence, body size and fecundity (Singh and Brown, 1957; Dadd, 1977). Thus, our results are in line with other studies, showing that badly nourishment leads to delayed larval development and adults with low energetic reserves (Timmermann and Briegel, 1999; Arrivillaga and Barrera, 2004; Telang et al., 2007; Banerjee et al., 2015; Zeller and Koella, 2016).

The sugar amounts in S. cerevisiae and Pseudozyma sp. were higher than in the other diets. S. cerevisiae did not show significant differences (p < 0,005) when compared to the standard diet (**Table 6**). All the bacterial diets had low sugar contents and E. coli and Asaia sp. presented the

best values, reinforcing our hypothesis that both bacteria are better sources for larval feeding. Chlorella sp. and A. platensis presented high sugar values. However, in contrast to the protein amounts, their sugar content is lower than the yeast or the standard diet. Carbohydrate is directly associated with pupation (Chambers and Klowden, 1990; Telang et al., 2007). Carbohydrate storage is associated to pupal commitment and larval growth (Sneller and Dadd, 1977; Van Handel, 1988; Telang et al., 2007), and the low levels observed in our results suggest that larvae have not sufficient sugar to achieve the pupal stage in these diets, resulting in developmental delay. Although the microalgae species had a significant amount of protein and sugar, the development time in these diets was prolonged, leading us to believe that Ae. aegypti larvae may not assimilate nutrients adequately from these organisms.

Besides the nutritional quality measurements, we analyzed the nutrient reserves of Ae. aegypti larvae. We have undertaken a comparative study in the larval development rates and their storage of protein, free carbohydrates, glycogen and lipids concerning different dietary conditions. There is a positive correlation between body mass and caloric reserves stored during the larval stage, triggering endocrine responses that lead to insect molting (Chambers and Klowden, 1990). Insects must achieve a minimum weight in the immature stage in order for continuing their development (Nijhout and Williams, 1974; Nijhout, 1975; Lounibos, 1979; Safranek and Williams, 1984; Chambers and Klowden, 1990; Davidowitz et al., 2003; Lan and Grier, 2004; Mirth et al., 2005). In Ae. aegypti larvae, the metamorphic capacity depends on nutritional reserves and needs a minimum critical mass that is estimated between 2.7 and 3.2 mg (Telang et al., 2007). Critical mass is defined as the mass that results in 50% of starved larvae achieving the pupal stage. That usually occurs 24 h after the transformation into the final fourth instar, in optimal conditions (Chambers and Klowden, 1990; Davidowitz et al., 2003; Lan and Grier, 2004; Telang et al., 2007). In this minimum period these larvae require to acquire food so that at least 50% of them pupate and emerge as adults, and at this age, the ecdysteroid production begins to rise (Telang et al., 2007). Nutrient intake and energetic accumulation are important factors to metamorphosis. Most larvae that are starved after reaching their critical weight will molt, but if larvae are starved before they have achieved the critical weight, metamorphosis is delayed, or they eventually die without initiating this process (Chambers and Klowden, 1990; Davidowitz et al., 2003; Telang and Wells, 2004).

Insects fed exclusively with bacteria and microalgae seem unable to accumulate sufficient nutrients during the larval stage to reach the minimum critical mass required for pupation in regular time (**Table 7**). Restrictive food amount in Ae. aegypti larvae delay pupation, but larvae remain able to pupate even in underfeeding conditions if sufficient energetic accumulation has been done in previous larval stages (Telang et al., 2007). Thus, the delay observed in our results in the metamorphosis rates might afford to larvae an additional period to feed, grow, and meet the critical mass to achieve the pupal stage. In all diets tested, the energetic larval contents were lower than in larvae fed with the standard diet Tetramin R . These results were expected, once the insect physiological parameters were strongly affected (**Supplementary Tables S1–S5**). Additional evidence for developmental delays comes from studies in Manduca sexta. The underfeeding in M. sexta during the last larval stage resulted in death or a delay in metamorphosis (Nijhout and Williams, 1974).

The protein storage in larvae fed with bacteria or algae showed the lowest proteins levels and Asaia sp. and E. coli showed better levels than the others including Chlorella sp. (54 ± 19.1 mg) and A. platensis. Although microalgae have a high protein content, Ae. aegypti seems not able to store this protein satisfactorily. The energetic reserves in gut and rest of body of all diets tested were proportional to values obtained in whole larvae, and the rest of the body showed higher contents than the gut (**Table 7**). Yeast-fed larvae showed the highest levels of protein, suggesting that Ae. aegypti larvae can process and store this protein efficiently. In immature stages, proteins play an important role in metabolic processes as well for adults of several insect species (Hagen et al., 1984; Zucoloto, 1988). In immature stages ingestion of proteinaceous food is essential for growth, survival, nutritional reserve for the pupation and utilization in the adult stage, principally for egg production (Chan et al., 1990). Our results are in line with the decline in body size observed in Ae. aegypti, Culex pipiens, Anopheles albimanus, and Anopheles gambiae caused by different feeding regimes (Timmermann and Briegel, 1999).

Carbohydrates content in bacterial and microalgae diets showed significant storage differences (p < 0.0001). Larvae fed with S. cerevisiae and Pseudozyma sp. presented carbohydrates reserves similar to the standard diet, showing no significant differences (p > 0.05). As expected, E. coli and Asaia sp. had better carbohydrates storage amounts than other bacteria and microalgae, reinforcing the potential of these diets. Carbohydrate is necessary for optimal growth and developmental rates in larvae, and nutritional environment with low levels of sugar leads to a substantially delayed growth (Sneller and Dadd, 1977). Previous studies with M. sexta suggested the dependence of sugar ingestion to reaching the pupa stage and the requirement to both dietary sugar and protein to growth until the adults (MacWhinnie et al., 2005). Carbohydrate role in Ae. aegypti larvae development is associated with pupal commitment and an inverse relation between hemolymph trehalose levels, and juvenile hormone titers have been described (Jones et al., 1981; Chambers and Klowden, 1990; Telang et al., 2007). Therefore, our data confirm that sugar and other digestible carbohydrates are required for adult stage maintenance and larval development.

The larvae glycogen storage showed significant differences (p = 0.0001) in all diets tested. Bacterial diets resulted in low glycogen contents, and Asaia sp. and E. coli showed higher rates than other bacteria diets. Larvae fed with S. cerevisiae and Pseudozyma sp. showed better glycogen levels, confirming the excellent storage capacity of Ae. aegypti in yeast diets. Past studies reported a critical threshold of larval glycogen as a stimulant to metamorphosis through a drastic drop in juvenile hormone titer and a concomitant

increase in ecdysone that trigger molting (Chambers and Klowden, 1990; Timmermann and Briegel, 1999). Telang et al. (2007) reported that the timing of ecdysteroid release is not critical to start the larval-pupal molt for Ae. aegypti larvae, however, both the ecdysteroid titer and the nutritional condition of fourth instars are determinant factors in initiating the metamorphic molt. Earlier studies indicated that larval nutrient reserves (protein, lipid, and glycogen) are important for egg production and the endocrine regulation of egg development in Ae. aegypti and Ochlerotatus atropalpus (Telang et al., 2006). High levels of glycogen and protein overtake a threshold set in the insect nervous system that activates ovarian ecdysteroid production and inhibits juvenile hormone biosynthesis by the corpora allata, which together enable vitellogenesis and egg production. Without sufficient threshold levels, the corpora allata increase the juvenile hormone levels secretion, decreasing ovarian ecdysteroid production and as consequence egg maturation is delayed (Telang et al., 2006).

The low glycogen reserve in Ae. aegypti larvae fed with bacteria and microalgae might have affected the insect neuroendocrine system, resulting in the delaying of pupation time, due to a lack of the minimum glycogen threshold required for a successful molt (Nijhout and Wheeler, 1982; Chambers and Klowden, 1990; Lan and Grier, 2004; Noriega, 2004; Telang and Wells, 2004; Margam et al., 2006; Telang et al., 2006, 2007). Glycogen storage seems to be a larval strategy valid to withstand starvation when the larva is waiting for additional nutrition during the last larval instar, under badly nourishment conditions (Timmermann and Briegel, 1999). This strategy might be used by larvae fed with E. coli and Asaia sp., which showed higher glycogen levels and prolonged their survival.

Levels of lipids were lowest in larvae fed with S. marcescens and Bacillus sp. These diets showed the worst developmental rates (**Supplementary Table S1**) with a severe delay in all biological parameters evaluated (**Supplementary Tables S1– S5**). Although, the other experimental diets have also shown significant and substantial differences (p < 0.05) in the lipid contents. Lipid reserve influence pupal commitment, and the endocrine regulation of egg development in autogenous and anautogenous female mosquitoes (Briegel, 1990b; Foster, 1995; Ziegler and Ibrahim, 2001; Briegel et al., 2002; Zhou et al., 2004; Telang et al., 2006). Under favorable nutritional conditions, lipids start accumulating after glycogen has reached a plateau (Van Handel, 1984). Environments with nutritional stress lead to drastic reductions in whole-body lipid, protein and carbohydrate contents (Briegel, 1990b). Due to the diets poor in nutrients (**Table 6**), Ae. aegypti larvae storage lesser amounts of energetic components (**Table 7**), affecting the developmental rates directly. The metamorphic capacity clearly depends on all four nutrient reserves in different ways.

Nutrients accumulated by larvae are correlated with adult emergence and body size (Telang et al., 2007; Zeller and Koella, 2016). Nutrition, temperature and larval density also influence growth, development, energy reserves, egg production, longevity of adult females, immunity, vector capacity and insecticide-resistance (Stearns and Koella, 1986; Briegel, 1990a,b; Kitthawee et al., 1992; Lyimo et al., 1992; Juliano and Stoffregen, 1994; Ameneshewa and Service, 1996; Telang et al., 2006, 2007; Murrell and Juliano, 2008; Reiskind and Lounibos, 2009; Arrese and Soulages, 2010; Muturi et al., 2011; Kulma et al., 2013; Murray et al., 2013). Larvae fed with S. cerevisiae and Pseudozyma sp. were not affected in body weight, what is expected as these diets showed good developmental rates, high nutritional quality, and efficient energetic accumulation. The fresh mass of larva fed with bacteria and microalgae were significantly lower (p < 0.05) when compared to controls. In natural environments, it is common to find mosquitoes with reduced sizes and low energy reserves (Zeller and Koella, 2016). Telang et al. (2007) showed that nutritional stress leads to smaller adults with reduced hemocyte numbers. Muturi et al. (2011) reported that nutritional stress during larval development cause changes in phenotype and immunity of mosquitoes and increased susceptibility of these adults to pathogens. Therefore, the nutritional quality of larvae in diets with microorganisms may affect not only the entire life history of Ae. aegypti, but also their size, immunity, and vector competence.

#### Larval Preference for Ingestion of Microorganisms

Our results showed that Ae. aegypti larvae consumed all microorganisms used at different rates (**Figure 7**). The guts of larvae fed with S. cerevisiae, Pseudozyma sp., and E. coli presented high mean fluorescent intensity after 2 h of feeding. The lower consumption rates in other diets might suggest a possible food preference. Despite that, more studies must be performed, increasing the feeding exposure time. We have not found studies involving food preference in Ae. aegypti larvae, hence exploring this subject is necessary for a better understanding of larval physiology.

# CONCLUSION

Our results provide new knowledge into the effect of microorganism-based diets in different larval biological parameters as developmental rates, pupation time, emergence, survival, lifespan, and wing size. Larvae fed with bacteria and microalgae shown lethargic development and low survival, due to bad nourishment and low energetic reserve accumulation. Asaia sp. and E. coli seem the best bacterial models for future studies aiming the development of symbiont-based methods for mosquito control. Larvae fed with yeasts showed developmental rates that are similar to the standard diet Tetramin R , being nutritional rich and providing high energetic storage. S. cerevisiae and Pseudozyma sp. seem suitable candidates to improve mosquito laboratory breeding and a low-cost diet to mosquito mass rearing. Ae. aegypti larvae ingested all the tested microorganisms 2 h after addition of them to the water. Therefore, Ae. aegypti larvae showed very high plasticity about feeding, being able to develop under different microbial diets.

#### AUTHOR CONTRIBUTIONS

fphys-10-00152 April 8, 2019 Time: 17:47 # 19

RS and FV performed the experiments and analyzed the data. RS, FV, TR, LS, JB, and FG conceived the study. All authors wrote and revised the manuscript.

#### FUNDING

JB, LS, and FG are fellows of CNPq. LS, FV, JB, and FG are members of their respective departments. RS is a Ph.D. student of Oswaldo Cruz Institute and received a grant from CAPES.

#### REFERENCES

# ACKNOWLEDGMENTS

We thank Fernanda Almeida for technical support in microscopy and Dr. José Bento Pereira Lima for supplying Aedes aegypti eggs.

# SUPPLEMENTARY MATERIAL

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Souza, Virginio, Riback, Suesdek, Barufi and Genta. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Dietary Stress From Plant Secondary Metabolites Contributes to Grasshopper (Oedaleus asiaticus) Migration or Plague by Regulating Insect Insulin-Like Signaling Pathway

Shuang Li1† , Xunbing Huang2 \* † , Mark Richard McNeill3 , Wen Liu2 , Xiongbing Tu1,4 , Jingchuan Ma1,4, Shenjin Lv2 and Zehua Zhang1,4 \*

#### Edited by:

Su Wang, Beijing Academy of Agriculture and Forestry Sciences, China

#### Reviewed by:

Yifan Zhai, Shandong Academy of Agricultural Sciences, China Qiong Rao, Zhejiang Agriculture and Forestry University, China

#### \*Correspondence:

Xunbing Huang<EMAIL_ADDRESS>Zehua Zhang<EMAIL_ADDRESS>†These authors have contributed equally to this work

#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 22 July 2018 Accepted: 15 April 2019 Published: 03 May 2019

#### Citation:

Li S, Huang X, McNeill MR, Liu W, Tu X, Ma J, Lv S and Zhang Z (2019) Dietary Stress From Plant Secondary Metabolites Contributes to Grasshopper (Oedaleus asiaticus) Migration or Plague by Regulating Insect Insulin-Like Signaling Pathway. Front. Physiol. 10:531. doi: 10.3389/fphys.2019.00531 1 State Key Laboratory of Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China, 2 College of Agriculture and Forestry Science, Linyi University, Linyi, China, 3 Canterbury Agriculture and Science Centre, AgResearch, Christchurch, New Zealand, 4 Scientific Observation and Experimental Station of Pests in Xilin Gol Rangeland, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Xilinhot, China

Diets essentially affect the ecological distribution of insects, and may contribute to or even accelerate pest plague outbreaks. The grasshopper, Oedaleus asiaticus B-Bienko (OA), is a persistent pest occurring in northern Asian grasslands. Migration and plague of this grasshopper is tightly related to two specific food plants, Stipa krylovii Roshev and Leymus chinensis (Trin.) Tzvel. However, how these diets regulate and contribute to plague is not clearly understood. Ecological studies have shown that L. chinensis is detrimental to OA growth due to the presence of high secondary metabolites, and that S. krylovii is beneficial because of the low levels of secondary metabolites. Moreover, in field habitats consisting mainly of these two grasses, OA density has negative correlation to high secondary metabolites and a positive correlation to nutrition content for high energy demand. These two grasses act as a 'push-pull,' thus enabling the grasshopper plague. Molecular analysis showed that gene expression and protein phosphorylation level of the IGF → FOXO cascade in the insulin-like signaling pathway (ILP) of OA negatively correlated to dietary secondary metabolites. High secondary metabolites in L. chinensis down-regulates the ILP pathway that generally is detrimental to insect survival and growth, and benefits insect detoxification with high energy cost. The changed ILP could explain the poor growth of grasshoppers and fewer distributions in the presence of L. chinensis. Plants can substantially affect grasshopper gene expression, protein function, growth, and ecological distribution. Down-regulation of grasshopper ILP due to diet stress caused by high secondary metabolites containing plants, such as L. chinensis, results in poor grasshopper growth and consequently drives grasshopper migration to preferable diet, such as S. krylovii, thus contributing to grasshopper plague outbreaks.

Keywords: grasshopper, diet stress, gene, plague, plant secondary metabolites

# INTRODUCTION

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Nearly half of all insect species are herbivores (Gatehouse, 2002; Wu and Baldwin, 2010), that have co-evolved with plants for 350 million years (Kessler and Baldwin, 2002). Phytophagous insects have specific adaptability to various host plants (Howe and Jander, 2008; Rominger et al., 2009; Unsicker et al., 2010), which also determine their ecological distribution and population dynamics (Whitman, 1990; Franzke et al., 2010). Such phytophagous insect-host plant relationships are examples of co-adaptation, co-evolution, and co-speciation (Scriber, 2002; Powell et al., 2006; Zhang and Fielding, 2011).

Plants have evolved various defense mechanisms against phytophagous insect (Padul et al., 2012). Such defenses can be broadly classified into two categories: constitutive defenses, including physiological barrier and nutritional hurdle; and inducible defenses, including secondary metabolites and protein inhibitors (PIs) (Zavala et al., 2004; Wu and Baldwin, 2010; Padul et al., 2012). Both types are achieved through similar means but differ in that constitutive defenses are present before herbivore attacks, while induced defenses are activated only when attacks occur (Wu and Baldwin, 2010; Padul et al., 2012). The regulatory elements in networks that modulate herbivory induced responses in plants mainly include jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) (Baldwin, 1998; Wu and Baldwin, 2010; Kessler and Baldwin, 2002). Plant defense mechanisms are also categorized into direct and indirect responses according to their role and function (Wu and Baldwin, 2010). Indirect defenses include volatile organic compounds produced when the plant is subject to herbivory that attract predators and parasitoids of the insect (Dicke and Loon, 2000; Despland and Simpson, 2005; Despres et al., 2007). Plant chemistry, especially those associated with secondary metabolites, is an important component of the phenotype that mediates plant-insect interactions (Mendelsohn and Balick, 1995; Despres et al., 2007). In general, insect dietary stress predominantly originates from high levels of plant secondary metabolites (Behmer, 2009; Despres et al., 2007; Wetzel et al., 2016).

Conversely, insect herbivores also evolved various detoxification mechanisms, mainly including avoidance, excretion, sequestration, metabolic resistance, and target mutation, which allow them to consume and develop on toxic plants producing high levels of secondary metabolites (Despres et al., 2007). Such insect feeding continues the selective pressure on plants to develop increased or novel chemical defenses (Musser et al., 2002; Becerra, 2003; Dussourd, 2003; Helmus and Dussourd, 2005). Insect herbivores' response to diet stress are well-documented in many insects, mainly focusing on insect behavioral, physiological, chemical, genetic, ecological, and evolutionary mechanisms (Raubenheimer and Simpson, 2004; Giri et al., 2006; Dicke and Baldwin, 2010; Ibanez et al., 2013). Some herbivorous species are strongly attractive or indispensable for some specific plant species (Schutz et al., 1997). Existence of those plants may contribute to, or even accelerate, insect population outbreaks (Powell et al., 2006). For example, Phragmites australis (Cav.) Trin. provides an optimal food source (Zhu, 2004; Ji et al., 2007), which could significantly benefit Locusta migratoria manilensis (Meyen) population growth.

Many genes and related pathways of insects, such as the insect insulin-like signaling pathway (ILP), play important roles in specific insect-diet relationship, and contribute to the variation of insect performance (Bishop and Guarente, 2007; Taguchi and White, 2008; Ragland et al., 2015). The insect ILP is considered to act as a sensor of the dietary status and to stimulate the progression of anabolic events when the status is positive (Taguchi and White, 2008; Badisco et al., 2013). It plays a crucial role in a number of fundamental and interrelated physiological processes, including insect growth, energy metabolism, and detoxification (Claeys et al., 2002; Wu and Brown, 2006; Kawada et al., 2010; Fujisawa and Hayakawa, 2012). Many studies in different metazoan species have indeed demonstrated that not only the insulin-related peptides are evolutionarily conserved, but also the components of their signaling pathway, such as IGF/INSR/IRS/PI3K/PDK/AKT/FOXO (**Figure 1**), which play an important role in insulin resistance, are also conserved (Sim and Denlinger, 2008; Badisco et al., 2013). Insulin signaling can be delivered by phosphorylation or dephosphorization of proteins, such as INSR/IRS/AKT/FOXO (Kramer et al., 2008; Hedrick, 2009). ILP changes associated with diet stress can influence insect growth (Kawada et al., 2010; Fujisawa and Hayakawa, 2012; Badisco et al., 2013), which can potentially affect pest distribution or even plague outbreaks. However, the role of the ILP signaling pathway in regulating pest plague outbreaks is poorly understood.

FIGURE 1 | IGF-PI3K-AKT-FOXO pathway of insulin signaling. The insulin-like signaling system includes different well-defined ligands, such as insulin-like growth factor (IGF), which regulate the activity of the homologous insulin receptor INSR. Insulin receptor substrate (IRS) proteins act as messenger molecule-activated receptors to signaling, and which is an important step in insulin's action. Phosphoinositide 3-kinase (PI3K), 3-phosphoinositide-dependent protein kinase (PDK), and protein kinase B (AKT), three major nodes downstream of IRS, and have been implicated in many of the metabolic actions of insulin. The forkhead transcription factor (FOXO) regulates transcription of genes involved in stress resistance, xenobiotic detoxification and DNA repair. FOXO is negatively regulated by insulin-like signaling when the PI3K → AKT cascade stimulates phosphorylation of FOXO and promotes its secretion from the nucleus and inactivation in the cytosol.

Oedaleus asiaticus Bey-Bienko is a specialist grass-feeder, with preference for Poaceae species, particularly Stipa krylovii Roshev (Poaceae) (Zhang et al., 2013; Qin et al., 2017). It is a member of the subfamily Oedipodinae (Orthoptera: Acrididae: Oedipodinae), and a dominant grasshopper of northern Asian grasslands, generally distributed in Inner Mongolia of north China (Cease et al., 2012; Zhang et al., 2014). Outbreaks of O. asiaticus have often lead to significant loss in grasses and economic disruption (Liu et al., 2013). From routine surveys of plant and grasshoppers composition in Stipa (S. krylovii) and Leymus (Leymus chinensis) (Trin.) Tzvelev (Poales: Poaceae) grasslands (Han et al., 2008), we found that O. asiaticus was mainly confined to the former (Huang et al., 2016, 2017a). In addition, we also found that the presence of plant secondary metabolites in L. chinensis can have a negative impact on O. asiaticus growth parameters (Huang et al., 2017a), while acting as a catalyst to drive grasshopper migration and plague outbreak. In the present study, we investigated how diet stress influences insect growth, distribution, and the ILP, to decipher the relationship between diet stress and pest outbreaks. We also discuss the role of diet stress in driving pest plague outbreaks, and how this information provides new insights into pest management.

# MATERIALS AND METHODS

#### Ethics Statement

Insects (O. asiaticus) were collected from the Xilin Gol grassland from 2011 to 2017. It is a common agricultural pest and not in the "List of Protected Animals in China." No permits were required for the described field studies.

#### Study Area

The research grassland (E115◦ 130–117◦ 060 , N43◦ 020–44◦ 520 ) is located in the Xilin Gol League, Inner Mongolia, northeast China. This grassland is a region representative of the Eurasian steppe grassland and characterized by L. chinensis- and S. krylovii dominated plant communities (Han et al., 2008). The aboveground biomass of these two plants accounts for more than 80% of the total community production (Huang et al., 2016). Plants in this grassland cover only 30 to 40% of the ground area with the remainder being bare ground for rapid steppe degradation in part driven by livestock over-grazing (Chen and Wang, 2000; Han et al., 2008). O. asiaticus is the dominante grasshopper species (Guo et al., 2006), and generally hatches between late-May and late-June, reaching adulthood between early to late July (Huang et al., 2016). This grasshopper mainly feeds on Poaceae species, particularly S. krylovii (Wu et al., 2012; Zhang et al., 2013).

#### Large-Scale Survey of Plant Biomass and O. asiaticus Density on Grazed Grassland

We examined the relationship between above-ground plant biomass composition and O. asiaticus density in this grazed grassland area, in mid-July for each year from 2011 to 2017. Those areas were mainly dominated by S. krylovii and L. chinensis, with rare distribution of other plant species. Each year, we selected eight 1 km2 sample plots (∼10 km apart). In each plot, we selected five 1-m2 quadrats (∼50 m apart) randomly. Grass S. krylovii and L. chinensis plants within each quadrat were cut to ground level and placed separately into envelopes. Those collected grass were dried at 90◦C for 24 h, and weighed to provide the relative mean above ground biomass (g DM/m2 ) of the two plant species for each plot.

We estimated the relative density of the grasshopper O. asiaticus in each plot in mid-July for each year 2011 to 2017, using the same method described in our previous published paper (Huang et al., 2016). We averaged the four samples in each plot, to derive a relative O. asiaticus density (number of individuals per 100 sweep-nets) for each of the eight plots in each year.

Sampling produced eight means (one for each of the eight 1 km2 plots) for both grass species and O. asiaticus relative densities for each year from 2011 to 2017.

#### Cage Study of O. asiaticus Growth in Grassland

To study O. asiaticus growth for different host plant species, a field cage study was carried out on S. krylovii and L. chinensis grasslands during late June of both 2016 and 2017. In each of those two grasslands, all of the other plants were removed to assure that only one host plant remained. We constructed 10 screen cages (1 m × 1 m × 1 m) using iron rod frames covered with 1 mm2 cloth mesh. In each plant species, five cages were used as the biological replicates. All of the visible spiders and other natural enemies in the field cages were removed carefully before adding female 4th instar O. asiaticus.

We collected 4th instar O. asiaticus nymphs by sweep net from the grassland mainly containing these two grasses on 21 June, 2016 and 2017. Those collected individuals were then temporarily maintained in metal-frame cages and starved for 24 h. Then, female 4th instar O. asiaticus nymphs (total 160 individuals) were assigned to the 10 field cages (16 individuals per cage) randomly. Those experimental individuals were selected to be as uniform in size as possible, with fresh body mass weighed and verified by ANOVA to confirm there were no significant differences in the weight of O. asiaticus nymphs amongst the four treatments. Besides, we killed another cohort of 30 O. asiaticus 4th instar females by chloroform and dried them at 90◦C for 24 h. Those dried grasshoppers were individually weighed (mg), and a mean dry mass determined to serve as the baseline data. Once grasshoppers were assigned to every cage, we checked field cages to monitor survival every day. In each cage, grasshoppers were able to feed ad libitum on grass. The plant biomass in the cages could provide sufficient food to allow development through to adults. When all of the surviving individuals became female adults, they were also killed and dried using the same method above to get adult dry mass (mg). Adult body dry mass was used to calculate grasshopper increased body mass (mg) by subtracting the 4th instar body dry mass. Grasshopper survival rate (%) was calculated by the number of adult individuals / number of initial individuals (n = 16). Grasshopper development time (days) was calculated by the following formula: DT = Pn i=1 i ∗Ni Nt , where i is the number of days from 4th instar to adult; Ni is the number of individuals with the development time corresponding to the value of "i"; and Nt is the number of adult individuals (Huang et al., 2016). Grasshopper growth rate (mg/day) was calculated by increased body dry mass / development time, and overall performance calculated from growth rate × survival rate (Cease et al., 2012).

#### Grass Chemical Traits

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In 2017, we cut the remaining plants from each cage at ground level after removing the adults. Each species were placed in a separate plastic container and used to detect the starch, nitrogen, and lipid content by Iodine-starch colorimetric method, Kjeldahl method, and Soxhlet extraction method, respectively. We then estimated crude protein content of each plant sample by 6.25 × nitrogen content (Bawa and Yadav, 1986). We also detected plant secondary compounds (tannins, phenols, alkaloids, terpenoids, and flavonoids) content in each sample by high performance liquid chromatography (HPLC), using the same method described in previous published papers (Ossipov et al., 1995; Griffin et al., 1999; Naczk, 2004; Friedman et al., 2006; Guo, 2007).

#### Gene Expression of ILP

We investigated seven genes of O. asiaticus ILP signaling pathway (IGF, INSR, IRS, PI3K, PDK, AKT, and FOXO) to compare their relative expression when exposed to different plant species. Unigene sequences were acquired from our previous transcriptome profiles (RSA accession number SRP072969) to design gene-specific primers (**Supplementary Table S1**). We randomly collected one adult sample from each replicate of the two treatments (10 samples). The relative expression of these genes was analyzed by qRT-PCR, using the same method described in our previous published paper (Huang et al., 2017b). Then, relative gene expression was normalized by the internal standard (actin), and calculated using the 2−11CT Method. Expression values were adjusted by setting the expression of O. asiaticus feeding on S. krylovii to be one for each gene. All qRT-PCRs for each gene with 10 samples (five biological replicates for each treatment) used 3 technical replicates per experiment.

#### Protein Phosphorylation Analysis

We used the rapid ELISA-Based Measurement to detect the protein phosphorylation of IRS, INSR, AKT, and FOXO in the insect ILP. Grasshopper samples were homogenized in 1 ml PBS, and the resulting suspension subjected to ultrasonication to further break the cell membranes. After that, we centrifuged the homogenates for 15 min at 5000 rpm, collected the supernatants and stored at −20◦C until required for further analysis.

We prepared all of the reagents (**Supplementary Table S2**) and brought all of the reagents and samples to room temperature (18◦C−25◦C). After 30 min at room temperature, we added 50 µl standard to each standard well, 50 µl sample to each sample well and 50 µl sample diluent to each blank/control well. Then, 100 µl of HRP-conjugate reagent was added to each well, and covered with an adhesive strip and incubated for 60 min at 37◦C. The Microtiter Plate was washed 4 times using Wash Buffer (undiluted), and then 50 µl Chromogen Solution A and 50 µl Chromogen Solution B was sequentially added to each well. This was then gently mixed and then incubated for 15 min at 37◦C in dark, after which 50 µl Stop Solution was added to each well. A change in the color of the solution from blue to yellow was expected. If the color in the wells was green or the color change did not appear uniform, the plate was gently taped to ensure thorough mixing. The Optical Density (O.D.) at 450 nm was read using a Microelisa Stripplate reader within 15 min of adding the Stop Solution. Then, we constructed the standard curve of each protein, and calculated the amount of phosphorylated protein in each sample.

#### Data Analysis

Student's t test was used to compare grasshopper growth variables (body size, survival rate, development time, growth rate, and overall performance), protein phosphorylation level, and relative gene expression. Linear regression was used to analyze the growth, distribution, ILP gene expression, and phosphorylation to grass chemical content in O. asiaticus. We used SAS version 8.0 for these analyses.

# RESULTS

#### Chemical Traits of Stipa krylovii and Leymus chinensis Grasses

The main nutrition and secondary metabolites were different in the two plant species (**Figures 2**, **3**). The highest starch content (t = 3.805, df = 8, P = 0.005) was present in S. krylovii, and the highest crude protein content (t = 4.085, df = 8, P = 0.004) was present in L. chinensis (**Figure 2**). The sum of the three nutritive substances (crude protein, lipid, and starch) was not significantly different between the two species.

For the five secondary metabolites, L. chinensis had higher levels of alkaloids (t = 9.440, df = 8, P < 0.001), tannins (t = 8.534, df = 8, P < 0.001), and terpenoids (t = 8.149, df = 8, P < 0.001) than S. krylovii. The total amount of all five secondary

metabolites was highest in L. chinensis (t = 11.099, df = 8, P < 0.001) (**Figure 3**).

#### Relationship Between O. asiaticus Performance and Grass Chemical Composition

The mean survival rate (**Figure 4A**) (2016: t = 7.732, df = 8, P < 0.001; 2017: t = 6.641, df = 8, P < 0.001), developmental time (t = 4.647, df = 8, P = 0.002; t = 5.077, df = 8, P = 0.001) (**Figure 4B**), adult fresh mass (t = 10.521, df = 8, P < 0.001; t = 13.311, df = 8, P < 0.001) (**Figure 4C**), growth rate (t = 7.838, df = 8, P < 0.001; t = 13.311, df = 8, P < 0.001) (**Figure 4D**), and overall performance (t = 7.732; df = 8; P < 0.001; t = 5.949; df = 8; P < 0.001) (**Figure 4E**) of O. asiaticus were significantly lower in insects that fed on L. chinensis, compared to those that

fed on S. krylovi. Feeding on L. chinensis provided less benefit for O. asiaticus growth and development. There was a significant negative linear relationship between grasshoppers and total plant secondary metabolites (**Figure 5**; R 2 = 0.893, F = 76.095, P < 0.001). Based on these results, we concluded that S. krylovi with less secondary metabolites resulted in better grasshopper growth. Conversely, high levels of secondary metabolites in L. chinensis, created a higher level of dietary stress, which lowered growth of O. asiaticus.

#### Relationship Between O. asiaticus Density and Grass Chemical Traits in Field Habitat

Survey results of 7 years showed that the relative density of O. asiaticus exhibited a significant positive relationship to S. krylovii above-ground biomass (**Figure 6**, linear correlation: y = 0.1886x + 0.7506, R 2 = 0.365, N = 56, F = 31.006, and

FIGURE 5 | Relationship between grasshopper overall performance and plant chemical composition. Red circles indicate total nutrition, and blue triangles indicate total secondary metabolites.

FIGURE 6 | Relationship between the relative density of O. asiaticus (mean number of individuals per 100 sweep-nets) and mean above-ground biomass (g/m2 ) of S. krylovii (red circles) and L. chinensis (blue circles). Data combined from measurements recorded from 2011 – 2017 (N = 8 means per year), with 2011–2014 values from our published data.

P < 0.001). In contrast, the relative density of O. asiaticus was significant negative related to L. chinensis above-ground biomass (**Figure 6**, power correlation: y = 13.393x−0.523 , R 2 = 0.283, N = 56, F = 21.298, and P < 0.001).

We also used above plant chemical data to evaluate total chemical traits of surveyed grassland. There was significant multiple linear relationship between grasshopper density and variable nutrition (x1) and secondary metabolites (x2) (**Figure 7**; y = 1.11x1 − 49.435x2 + 6.687, R 2 = 0.366, F = 15.285, and P < 0.001). From the standardization regression coefficient (x1 = 0.638, x2 = 0.624), we concluded that the content of secondary metabolites had the largest effect (negative) on grasshopper density.

# Change in O. asiaticus ILP Due to Diet Stress

#### Gene Expression

qRT-PCR to determine the relative expression of seven genes in ILP signaling pathway indicated that the genes IGF (t = 8.472, df = 8, P < 0.001), INSR (t = 17.851, df = 8, P < 0.001), IRS (t = 14.951, df = 8, P < 0.001), PI3K (t = 14.951, df = 8, P < 0.001), PDK (t = 8.944, df = 8, P < 0.001), AKT (t = 10.633, df = 8, P < 0.001), and FOXO (t = 6.529, df = 8, P < 0.001) were significantly down-regulated in O. asiaticus that fed on L. chinensis (**Figure 8**).  Based on this evidence, we concluded that S. krylovi with less secondary metabolites determined the high gene expression involved in grasshopper ILP. But, L. chinensis