The sugarcane aphid, Melanaphis sacchari Zehntner (Hemiptera: Aphididae), is an important insect pest of sorghum and sugarcane crops throughout the United States and Mexico. In 2013, its capacity for crop destruction of these 2 commodities amounted to millions of dollars. Currently, no artificial diets are available that can be used for in vitro screening of potential bioactive substances toxic to this aphid species. The objective of our work, reported herein, was to evaluate liquid artificial diets for use in such in vitro screening bioassays. We evaluated a diet originally developed for the green peach aphid (Myzus persicae Sulzer [Hemiptera: Aphididae]) and another for the whitefly (Bemisia argentifolii Bellow and Perring [Hemiptera: Aleyrodidae]). Melanaphis sacchari did not survive on the M. persicae diet, whereas about 46% survived on the whitefly diet that contained 30% sucrose and 5% yeast extract during the 10 d trial. Further tests then were conducted to determine if sucrose alone at concentrations of 15, 30, and 50% would be a sufficient artificial diet. We found that M. sacchari survival at 10 d post ingestion was greatest (87%) when the diet contained 30% sucrose alone. This solution represented the best diet option for use for in vitro toxicological screening bioassays for the sugarcane aphid.
Melanaphis sacchari Zehntner (Hemiptera: Aphididae), also known as the sugarcane aphid, is an insect pest of African origin that is currently distributed worldwide (Singh et al. 2004). This aphid species adversely affects approximately 20 agricultural crops belonging to the family Gramineae. In addition to causing direct damage to the plant, the aphid also can act as a vector for plant pathogens, including millet red leaf, sugarcane yellow leaf, and sugarcane mosaic viruses (Singh et al. 2004).
In the United States, sugarcane aphid was first reported in 1977 in Florida, and by 1999 had spread into Louisiana (Mead 1978; White et al. 2001). In 1988, Denmark (1988) reported the presence of M. sacchari feeding on sorghum in Florida; at that time it was not considered economically important. In 2013, the aphid appeared in Liberty County, Texas, and eventually spread to 38 counties in 4 states (Bowling et al. 2016). In that same year, the sugarcane aphid was reported from Tamaulipas, Mexico, as well as other northern states in Mexico (Rodríguez-del-Bosque & Teran 2015). By the fall of 2015, M. sacchari had spread to approximately 400 counties in 17 states of the United States. In Mexico, this pest species was found in all of the sorghum producing regions of the country (Rodríguez-del-Bosque & Terán 2015; Bowling et al. 2016).
The rapid expansion and colonization of the sugarcane aphid in 2 of the largest sorghum producers in the world (i.e., the United States and Mexico) present problems regarding its management (Villanueva et al. 2014). If field populations of this pest were to develop resistance to any of the pesticides used for their control, those inherited characteristics could spread rapidly to subsequent generations due to the high rate of sugarcane aphid reproduction (Sarwar 2015). This issue emphasizes the necessity of developing screening assays to identify alternative or novel bioactive substances for this pest. For example, developing in vitro toxicological assays for the sugar cane aphid would be a useful tool to determine insecticidal properties of various secondary plant metabolites and identify promising candidate molecules for further development for this pest (De Geyter et al. 2012; Raga & Sato 2011). Currently, there is no standard artificial diet for M. sacchari to accomplish this task. Because of the importance of the sugarcane aphid to the agricultural regions of the United States and Mexico, development of screening assays is needed urgently. Therefore, the aim of this study was to evaluate various liquid artificial diets for eventual use in M. sacchari in vitro toxicological assays.
Materials and Methods
INSECTS
Native populations of M. sacchari were collected from sorghum crops in the state of Morelos, Mexico (18.7669°N, 98.8970°W) and maintained on sorghum, variety M-550 under greenhouse conditions at 70% RH. This plant variety (Majestic Seeds Co., Hodges, South Carolina, USA) is reported to be tolerant of infestation by M. sacchari. Infested plants were placed in cages (90 cm3) covered with mesh of 650 μm (DM Agromallas S.A. de C.V. Group, Culiacan, Sinaloa, Mexico). In addition, each plant was covered with a fine fabric (cylinder of 40 cm diam x 50 cm tall) to exclude parasitoids and predators, as well as to prevent migration of aphids to adjacent plants.
ARTIFICIAL DIETS
One diet we evaluated was originally developed for Myzus persicae Sulzer (Hemiptera: Aphididae) (Mittler & Koski 1976) and consisted of 15% sucrose (Reasol, Iztapalapa, Mexico City, Mexico); 2.5 g casein hydrolysate (Sigma-Aldrich, 22090-500G, St. Louis, Missouri, USA); 2% yeast extract (MCD Lab, Tlalnepantla, Mexico City Mexico, Mexico); 123 mg MgSO4 7H2O (Jalmek, San Nicolas de los Garza, Nuevo Leon, Mexico); 100 mg ascorbic acid (Sigma-Aldrich, A0278-25G, St. Louis, Missouri, USA); 10 mg niacin (Sigma-Aldrich, N0761-100G, St. Louis, Missouri, USA); 5 mg calcium pantothenate (Sigma-Aldrich, C8731-25G, St. Louis, Missouri, USA); 2.5 mg pyridoxine (Sigma-Aldrich, P5669-5G, St. Louis, Missouri, USA); 2.5 mg thiamine (Sigma-Aldrich, T1270-25G, St. Louis, Missouri, USA); and 1.5 mg K2HPO4 3H2O (Jalmek, San Nicolas de los Garza, Nuevo Leon, Mexico).
The second diet evaluated was originally developed for the whitefly, Bemisia argentifolii (Hemiptera: Aleyrodidae) (Jancovich et al. 1997), and consisted of 30% sucrose and 0.5% yeast extract (MCD Lab., Tlalnepantla, state of Mexico, Mexico) as a nitrogen source. Both diets were prepared with distilled water (adjusted to pH 7.0 with NaOH or HCl) and sterilized at 121 °C for 20 min at 15 psi.
In addition, diets containing 15, 30, and 50% sucrose only (Reasol, Iztapalapa, Mexico City, Mexico) were evaluated. To each of these 3 diets we added 4.4 mL per L of 10% formalin and 7.3 mL per L of 15% choline chloride, to prevent the growth of fungi and bacteria.
FEEDING CHAMBERS
The feeding chamber used in this study was the same as reported by Torres-Quintero et al. (2013) and was fashioned out of two 40 mm ht × 20 mm diam translucent disposable plastic cups (Envases Cuevas, S.A. de C.V, Xalostoc, state of Mexico, Mexico) (Fig. 1). Into 1 cup, 2 mL of diet was pipetted then sealed with Parafilm® (Sigma-Aldrich, BR701605, St. Louis, Missouri, USA). The bottom of the second cup was removed and the top part inverted onto the cup with diet. A strip of parafilm was used to seal the cups together. Aphids were then placed on top of the parafilm of the cup containing diet using a camel's hair brush. Both cups were covered with a piece of fine mesh fabric and affixed firmly with 2 rubber bands (Fig. 1G). Finally, the feeding chamber was inverted and placed on a plastic plate. Humidity in the chamber was maintained by placing a piece of wet cotton beneath it. All materials used to build the feeding chamber were sterilized with UV light prior to construction.
EVALUATION OF SURVIVAL AND FECUNDITY
Fifteen fourth instar M. sacchari were placed in each diet chamber. To evaluate the survival time that aphids could survive without food in the chambers (i.e., negative control), only bottled tap water was provided. Mortality was recorded every 24 h for 10 d and the number of newborn aphids recorded during this period. First instar nymphs were transferred, using a camel's hair brush, to new chambers containing the same diet as adults. Nymphal survival and developmental time were recorded daily. Feeding chambers were maintained at 29 ± 1 °C with a photoperiod of 14:10 h (L:D) at 70 ± 2% RH.
STATISTICS
Preliminary analysis of all mean data was conducted using normality and equal variance tests as reported by Manikandan (2010). Percent data were converted to decimal number for analyses (%/100 = 0.00). Mean number of aphid births and survival data were analyzed by 1-way ANOVA, whereas the pair-wise mean multiple comparison Holm Sidak post hoc test was used to determine differences at P = 0.05 (Reyes et al. 2015). Sigma Plot 11.0 software (Systat Software, Inc., San Jose, California, USA) was used for all statistical analyses. Non-transformed means are presented in figures.
Results
Melanaphis sacchari did not survive on the M. persicae diet, whereas an overall cumulative average of about 46% survived on the sucrose/yeast (whitefly) diet during the 10 d trial (Table 1). Percent survival of aphids from all 3 sucrose-only diets ranged from about 60% to slightly < 100% (Fig. 2). The negative control (water only) resulted in complete aphid mortality on the third day (Table 1). Overall, the 30% and 50% sucrose-alone diets produced the best survival results at the end of the study. Although survival rates for 30% and 50% sucrose were not significantly different, the 30% diet exhibited higher mean daily survival values with lower standard deviations (Fig. 2). Approximately 20% of newborn nymphs remained alive at 48 h after transfer from the original feeding chamber.
Fig. 1.
Construction of the feeding chamber: (A) rubber bands, (B) parafilm, (C) bottom removed from 1 plastic cup, (D) plastic cup with diet, (E) parafilm strip, (F) fine fabric mesh, (G) completed chamber.
Table 1.
Cumulative mean (± SE) survival of Melanaphis sacchari during a 10 d feeding evaluation of 15, 30, and 50% sucrose and 30% sucrose supplemented with 0.5% yeast diets.
Fecundity was greatest for all diets during the first 4 d of evaluation and represented > 80% of the total, then steadily decreased after this period (Table 2, Fig. 3). Daily abundance of newborn aphids differed according to diet (Table 2). For the whitefly diet with 30% sucrose supplemented with 0.5% yeast extract, a total of 66 aphids were born during the evaluation period. A total of 80 aphids were born from adults that fed on the 15% sucrose diet, 134 aphids from the 30% sucrose diet, and 108 aphids from the 50% sucrose diet.
Discussion
The use of artificial diets has played a very important role in facilitating the development of novel bioassay screening procedures to identify bioactive substances for possible development as insecticides. In the family Aphididae, several diets have already been developed and used in toxicological, physiological, and behavioral research, as well as in studies of endosymbiosis, virus transmission, mortality, and nutrition (Hunter & Hsu 1999). However, no dietary studies for M. sacchari have been reported in this regard. Generally, in vitro feeding assays usually require a minimum evaluation time that varies between 2 and 7 d (Liu et al. 2002). Moreover, a 10 d evaluation period has been considered sufficient time in which to screen natural toxins, entomopathogens, or synthetic chemicals for insecticidal properties for aphids (Sabtharishi & Naveen 2017; Torres-Quintero et al. 2016; Han et al. 2014). We found that the 30% sucrose diet (herein designated as the “SA” [sugarcane aphid] diet) yielded an 87.4% survival rate of aphids up to 10 d. We expect that this artificial diet will provide researchers a simple in vitro method for screening chemicals or toxins for biocidal activity for M. sacchari.
Fig. 2.
Daily cumulative mean (± SE) survival of Melanaphis sacchari during a 10 d feeding evaluation of diets containing only 15, 30, 50% sucrose, and a 30% sucrose diet supplemented with 0.5% yeast extract. aMeans with a different letter are significantly different, Holm Sidak multiple comparison test (P = 0.05).
Fig. 3.
Mean (± SE) number of aphids born daily during a 10 d evaluation of diets containing only 15, 30, 50% sucrose, and a 30% sucrose diet supplemented with 0.5% yeast extract. aMeans with a different letter are significantly different, Holm Sidak multiple comparison test (P = 0.05).
Table 2.
Daily birth average (± SE) and percent fecundity during the first 4 d of evaluation of diets containing only 15, 30, 50% sucrose, and a 30% sucrose diet supplemented with 0.5% yeast extract.
Acknowledgments
The authors gratefully acknowledge CONACYT (National Council of Science and Technology) for the scholarship granted to Erubiel Toledo-Hernández for his doctorate degree (CVU 419143). This work is part of the Ph.D. thesis; appreciation is extended to Lucrecia Barboza for her critical reading of the manuscript and 2 anonymous reviewers who improved previous drafts.
