Study area and sampling design

The study was conducted in the municipality of Erongarícuaro, which is part of the Basin of Patzcuaro Lake in Michoacan state. The region has a subhumid temperate climate with summer rains (Cw). The temperature range is between 5 and 26°C with an average annual rainfall of 1 040.8 mm (^{INEGI, 2009}). It has a vegetation type of pine and oak forest (^{Díaz-Barriga et al., 1988}).

Three sites with plots for maize production were selected. The first site is located in the town of Ex Hacienda Charahuén (19° 31’ 23”-19° 31’ 13” north latitude and 101° 42’ 32”-101° 42’ 19” west longitude), while the remaining two are in the locality of Napízaro (19° 35’ 94”-19° 35’ 54” N and 101° 41’ 85”-101° 41’ 51” west longitude and 19° 36’ 10”-19° 36’ 00” north latitude and 101° 41’ 93”-101° 41’ 56” west longitude). In all three sites the presence of grasshoppers was observed in the fallow period (May 2015), when most of its species are in the egg phase. The observations were confirmed through interviews with the producers.

In each site two plots with an area of 9×200 m were arbitrarily selected, one with agricultural management of MC of maize and its monoculture marginal vegetation (VMM) and another of PC composed of maize, beans, zucchini and its polyculture marginal vegetation (VMP). Three replicates of each type of agricultural management were carried out, with their respective marginal vegetation, giving a total of six plots, none of which were applied with insecticide.

Grasshoppers were collected once per month during the growing season (July to December, 2015). The collections were made in transects covering the plot longitudinally, one for the interior of the MC (MC-I), another for the PC (PC-I) and another for the marginal vegetation of each one (VMM and VMP). The collections were carried out the third week of each month, with a striking network of 33 cm in diameter, between 10 and 13 h, by 200 strokes of net per transect to a height of 1.50 m. The collected grasshoppers were introduced alive into one-liter plastic containers and transferred to the laboratory. One specimen of each morphospecies was fixed in 70% alcohol and identified to species level based on criteria of ^{Mariño et al. (2011)}. Some specimens were identified to subfamily level, due to the absence of adult male specimens. The findings were corroborated by the Colección Nacional de Insectos del Instituto de Biología of the UNAM.

Isolation and identification of HEP

Grasshoppers collected in the field were confined in one liter containers at a density of ten specimens per container at room temperature. They were feed with commercial oat flakes and lettuce disinfected by immersion in 5% sodium hypochlorite solution for 20 min and then rinsed with distilled water. This procedure was performed for up to two weeks. The dead individuals were placed in individual wet chambers, consisting of a 3.5×4.5 cm plastic container with a lid and a filter paper in the bottom, moistened every 2 d with 100 μL of distilled water for 10 d, in order to induce sporulation of HEP.

The isolation of HEP was carried out in a laminar flow hood and consisted of the scraping of conidia present on the cadaver and its seeding in potato dextrose agar medium (PDA) in Petri dishes, which were incubated at 27 °C for four to six days. During this period, mycelial growth was observed every 48 h. The pure cultures were obtained by performing re-insultation of the fungus from the original culture (^{Monzón, 2001}). The isolates obtained will be integrated into the HEP collection of the Centro Nacional de Referencia de Control Biológico (CNRCB).

The identification of grasshopper HEP was performed by macro and microscopic observation of the growth morphology and fungus structures (mycelium, conidia and conidiophores). Dichotomous keys (^{Samson et al., 1988}) were used to identify HEP genres. HEP richness and the number of infected grasshoppers were recorded.

A Bray-Curtis matrix was constructed with data similarity measures of the cumulative abundance of the six samples of each species using the Biodiversity Pro version 2 package (^{McAleece, 1997}), in order to obtain the ecological distances expressing the differences in the structure and composition of the grasshopper community among plots (^{Bray and Curtis, 1957}) as well as an array of geographical distances between plots transformed log+1 to homogenize the extreme distances (^{Su et al., 2004}; ^{Manly, 2006}). With these two matrices, the ^{Mantel (1967)} test was applied with 10 000 permutations, using the XLSTAT statistical software (^{Addinsoft, 2009}) for Excel, to explore the spatial independence of the samples and use them as replicas in the statistical analyzes.

To test the effect of treatments on the abundance of grasshopper species and on abundance of the dominant species, a mixed linear model was used using the statistical software R version 3.2.3. (^{Pinheiro and Bates, 2000}). The model was adjusted using the “lme” function of the “nlme” library for R (^{Pinheiro et al., 2007}). As predictors there were included the agricultural management, time, both linear term (T) as square (T²) and their interaction. The observed species richness and abundance of the dominant species in the marginal vegetation were included as covariates (S) in wealth and abundance models, respectively. The site and plot were included as random factors, in order to model the spatial association of treatments and the different collections over time, respectively. The model’s equations for richness and abundance were:

Where: S= covariables: grasshopper species richness and abundance of the dominant species in the marginal vegetation of monoculture and polyculture; T= time.

In both cases the response variable was transformed with log (response+1) in order to meet the assumptions of variance homogeneity and residues normality. Finally, the association of the frequency of HEP infection isolated in grasshoppers with the treatments in which they were collected was reviewed, using a Chi square test.

CS composition

2920 grasshoppers belonging to two families of four subfamilies, ten genera and 17 species were collected. 97.8% of all individuals collected belonged to a single species, Sphenarium purpurascens purpurascens Charpentier (Table 1).

Tratamientos: MC-I= interior del monocultivo, PC-I= interior del policultivo. Covariables: VMM= vegetación marginal del monocultivo y VMP= vegetación marginal del policultivo. Datos acumulados de muestreos con redes de golpeo realizados entre julio y diciembre de 2015.

Table 1 Grasshoppers species and their abundance in two types of agricultural management and vegetation of the plots margins in Erongarícuaro, Michoacán.  

The apparent dominance of S. p. purpurascens in the study sites implies that this is the most economically important species for mazie producers in the region. Mantel’s test showed that the structure and composition of the CS in the plots were not correlated with geographical distances between them (R= -0.178, p= 0.348) so they were considered as independent samples for comparative analysis (Table 2).

PC= policultivo; MC= monocultivo.

Table 2 Similarity matrix of ecological distance between plots in Erongarícuaro, Michoacán. 

Evaluation of the management system on the richness of grasshopper species

Species richness changed significantly in the T being the quadratic term that best described the change (p< 0.05). The agricultural management had no significant effect on overall species richness (p> 0.05) nor modified the temporal trend of richness (p> 0.05). Species richness in the marginal vegetation did not explain species richness in plots (p> 0.05, Table 3). The variation explained by the fixed effects of the model was 29%. The random effects (site/plot) did not have an additional explanatory capacity.

Modelo lineal mixto tratamientos: MC-I= interior del monocultivo; PC-I= interior del policultivo. S: covariables T= tiempo; T2= tiempo cuadrático; gl= grados de libertad; F= de distribución F de Fisher. ^*= p< 0.05; ^**= p< 0.01.

Table 3 Grasshoppers richness in two types of agricultural management and vegetation of the plots margins in Erongarícuaro, Michoacán. 

Abundance of S. p. purpurascens

Abundance of S. purpurascens purpurascens changed significantly in the T. The quadratic term was the one that best described this change (p< 0.05). The agricultural management had no significant effect on the abundance of S. p. purpurascens, or alter the temporal trend of abundance (p> 0.05, Table 4). The abundance of this species in the VMM and VMP sites and the abundance in the MC-I and PC-I (p< 0.05, Figure 1 and Table 4). The variation explained by the fixed effects of the model was 57%. The random effects did not have an additional explanatory capacity.

Tratamiento: MC-I0= interior del monocultivo; PC-I= interior del policultivo S= covariables (vegetación marginal del monocultivo y vegetación marginal del policultivo). T= tiempo; T2= tiempo cuadrático. gl= grados de libertad; F=distribución de Fisher; *= p< 0.05; **= p< 0.01.

Table 4 Comparison of the abundance of Sphenarium pupurascens purpurascens two types of agricultural management and the vegetation of plots margins in Erongarícuaro, Michoacán. 

Figure 1 Average monthly abundance of S. p. purpurascens in two types of agricultural management and margins plots vegetation in Erongarícuaro, Michoacán,2015. N= 72. Treatments: MC-I= monocultures interior; PC-I= polyculture interior. Covariables: VMM= marginal vegetation of monoculture; VMP=marginal vegetation of polyculture  

The PC-I did not hinder the attack of S. p. purpurascens, so no low abundances were reported. In fact, in September, the higher abundance was recorded in this type of agricultural management than in the MC-I. This insect is considered polyphagous (^{Cano-Santana and Oyama, 1994}; ^{Tamayo-Mejía, 2009}), and according to the results of this study, plant diversity increased its abundance, since VMM and PMV showed higher values than those recorded in MC-I and PC-I. S. p. purpuascens abundance in margins vegetation influenced the abundance found in the MC-I and PC-I (Table 4).

^{Cerritos and Cano-Santana (2008)} reported that the density of S. purpurascens eggs was 14.9 times higher at the edges of the alfalfa plots than within the plots. This contrasts with ^{Andow (1991)} that a particular pest will find less possible hosts in a more diverse habitat and, therefore, pest insects are less harmful in polycultures compared to monocultures. However, insects with a broad spectrum of hosts usually do not reduce their population by crop diversity (^{Andow, 1991}). Therefore, other strategies should be used for their control.

The maximum abundance of S. p. purpurascens of PC-1 the VMM and VMP was recorded in August except the MC-I which increased in September and subsequently decreased. ^{Cano-Santana (1994)} reported that the highest densities of S. purpurascens were recorded in early July on abrupt and flat sites s of the Reserve Pedregal de San Angel in México City in 1990 and 1991. In 1991 it was observed that after achieving the máximum density, the population declined drastically in subsequent months.

High humidity combined with low temperatures could have caused high mortality of first instars of S. purpurascens.^{Tamayo-Mejía (2009)} reported that the 1 and 2 nymph stages of grasshopper Melanoplus differentialis (Thomas) and S. purpurascens showed a 50% mortality in field, caused by biotic and abiotic factors. Based on the results of this paper and those mentioned above it is inferred that the dynamics of populations of S. p. purpurascens is affected by environmental conditions and herbaceous vegetation of the study sites (^{Cano-Santana, 1994}; ^{Cerritos and Cano-Santana, 2008}; ^{Tamayo-Mejía, 2009}).

It is necessary a precise study of the interaction between S. p. purpurascens and weeds found in the plot margins, since it may be useful for manipulation of the composition of weeds species in crop margins to function as trap vegetation. It is important to carry out assessments in plants that have demonstrated repellent properties and manipulate its presence within the crops, in order to push away S. p. purpurascens from the main crop. Among the species that can be evaluated are include castorbean (Ricinus communis L.), garlic (Allium sativum L.) and marigolds (Tagetes erecta L.) (^{Grainge and Ahmed, 1988}; ^{Rodríguez and Nieto, 1997}; ^{Vidal et al., 2008}). This would allow having areas that concentrate a greater diversity of plants with a specific purpose within the agroecosystem, in order to minimize the damage that the grasshopper causes to the main crop.

In this paper the abundance of S. p. purpurascens in the VMM and the VMP determined the abundance of the species within the plots regardless of the type of agricultural management used (MC-I or PC-I). Besides the integrated pest management, there is another alternative to regulate S. purpurascens populations, consisting of harvesting to use it as a food source. At present this insect is sold in markets in the State of Mexico and the Central Valleys of Oaxaca, where it forms part of the regional cuisine (^{Marcos et al., 2015}).

^{Cerritos and Cano-Santana (2008)} compared the impact of malathion insecticide application with the capture of S. purpurascens for human consumption on the density of ootheques m^-2 within the alfalfa plot and its margins in two years. The ootheques density was lower in plots where malathion was applied with respect to those of mechanical control through harvesting. However, the density of ootheques in the mechanical control was lower compared to the control. The eggs density at the plots margins was 14.9 times higher than those found within the plots. The oviposition areas of female S. p. purpurascens were found most frequently in the edges of the plots, because in these areas the soil is less compact (^{Cerritos and Cano-Santana, 2008}). The mechanical control method is a viable alternative to regulate populations of S. p. purpurascens, plus it is a profitable product for the above regions (^{Cerritos and Cano-Santana, 2008}).

HEP isolated in S. p. purpurascens

Of 2920 individuals collected, 12 species of S. p. purpurascens were found infected by HEP, which corresponds to 0.41% of the total (Table 5). All HEP were isolated from adult individuals, but two nymphs on the site of the VMM infected with Metarhizium sp. Sorokin. 75% of infected grasshoppers had Metarhizium sp. and the remaining 25% were found infected by Beauveria sp. (Balsamo) Vuillemin. 16.7% of the grasshoppers infected with HEP were collected in the MC-I, 41.7% in the PC-I, 16.6% in the VMM and finally 25% in the VMP. Generally speaking, the fungus with the highest infection frequency of grasshoppers in the region was Metarhizium sp., Finding an association between the proportion of infected grasshoppers with this HEP on the PC-I, compared to those who were found infected in the VMP (x ² = 3.84, p= 0.04). In the case of the other sites no significant association (p> 0.05) was found (Table 5).

MC-I= interior del monocultivo; PC-I= interior del policultivo; VMM= vegetación de los márgenes del monocultivo; VMP= vegetación de los márgenes del policultivo.

Table 5 Sphenarium purpurascens purpurascens infected with entomopathogenic fungi in maize agroecosystems of the Lake of Patzcuaro Basin region.  

HEP found infecting S. p. purpurascens, had minimal presence in maize agroecosystems. It is known that Metarhizium and Beauveria show low levels of natural infection in CS compared to E. grylli, which it is a species that was expected to find in this research, as is has been documented infecting different populations of grasshoppers in México and in other countries in the Americas (^{Sánchez et al., 2011}; ^{Uribe-González y Santiago-Basilio, 2012}). However, in this paper there was an association between Metarhizium sp. and PC-I compared to the VMP, so that the infection of Metarhizium seems to be favored by plant diversity, since in these systems, the microclimate changes mainly by increasing relative humidity, favoring the presence of HEP (^{Van Huis, 1997}).