The records of the interaction between adults of M. nigripes and seep willow shrubs were taken in a semi-disturbed area adjacent to maize crops, located in the municipality of San Pablo del Monte, Tlaxcala, Mexico (19° 07’ 00” N and 98° 10’ 00” O).

Behavior records were performed by means of direct observation (focal and continuous records) (Altmann, 1984). We conducted a total of twenty- four observational sessions, twelve for females (n = 12) and twelve for males (n = 12). Each session lasted 90 minutes and was carried out from 2012 to 2015 between 11:00 and 13:30 hours. We recorded and described the movements, actions and positions of adult females and males in twenty-four seep willows shrubs randomly chosen. Each description begins when the beetles emerges from the soil and finishes when they began to cut up the leaves with the mandibles.

The sequences of behavioral patterns obtained of the interaction between rose chafers and seep willow were measured and flow charts of these sequences were developed. The percentage of times that a particular action pattern follows another (given the first action pattern), and transitions duration were calculated. The principal behavior patterns were described in an ethogram and patterns-transitions sequences were represented in a kinematic diagram.

A directed sampling was conducted to obtain leaves of the same size and healthy appearance from seep willow shrubs. For each extraction, six leaves with same size and weight were introduced on filter-paper at the bottom of the SPME recipient. Polydimethylsiloxane / Divinylbenzene (PDMS/DVB, 65 ^m) fiber coating was used. The holder with the PDMS/DVB fiber coating was placed on the entrance of the upper section for 60 min, capturing the leaves volatiles. We performed a blank experiment for each extraction. For each of the leaves and blank experiments, we performed ten repetitions (n = 10).

The volatiles isolated by SPME were identified in a coupled-gas chromatography 7890B coupled with a high-resolution mass spectrometer Agilent 7200 (Q-TOF), equipped with a HP-5MS 5% phenyl- methyl-siloxane column, of 30m diameter x 0.25^m of inner diameter. The conditions during this analysis were: 250 °C for the injector temperature, splitless injection mode and helium 1.2 ml/min as the carrying gas. The initial temperature was of 50 °C; it was maintained for 5 minutes until it reached 300 °C at a speed of 10 °C/min.

A spectral comparison of the chromatogram peaks was used to identify the volatile constituents of the leaves. In the mass detector we used chemical ionization with methane to produce ions that were assigned to different chromatogram peaks. Based on the proposed chemical structures from Pherobase database and specialized articles, we calculated de exact mass of the detected compounds and the mass/ charge difference (Am/z) and chemical structures were proposed.

The behavioral patterns shown by females of M. nigripes are shown in Table 1, as well as patterns and transitions sequences are presented in Figure 1. Females emerge from the soil at 11:00 hours and travel in short flights to seep willow shrubs. This movement lasts, on average, 240 secs. Each female settle on the leaf and moves forward and backward for 7 secs, stopping in the superior or posterior leaf margin, positioning itself opposite to the soil and hanging with their legs. Right away, they start moving their abdomen in circles (rotates around its own axis), lifting it in a 45° angle and periodically moving up and down their metatarsus, constantly rubbing them during short periods of time. It has been suggested that this behavior corresponds to the ‘calling’ in this species (Romero-López et al., 2010). In this position or settling on the leaf margin, the females put their mandibles on the leaf margin and starts moving them from right to left for 82 secs obtaining small leaf fragments to ingest. These mandibles movements could be the feeding behavior of M. nigripes. Males emerge from the soil 30 seconds after the females repeating the females’ behavior, except for the abdomen movement.

Table 1

Figure 1.

We obtained fifteen chromatogram peaks from which ten were related to chemical compounds structures. Each chromatogram peak with its retention time (RT), molecular ion exact mass, error valúes (Am/z) and proposed structure are shown in Table 2. Peak 10 with a retention time of 16.82 minutes was the most intensive peak. We found peaks b1, b2, b3 and b4 also in the blank (Table 2, Figure 2).

Table 2

PN= peak number; RT= retention time; Am/z= error values.

Figure 2